TY - JOUR A1 - El Mouali, Youssef A1 - Gerovac, Milan A1 - Mineikaitė, Raminta A1 - Vogel, Jörg T1 - In vivo targets of Salmonella FinO include a FinP-like small RNA controlling copy number of a cohabitating plasmid JF - Nucleic Acids Research N2 - FinO-domain proteins represent an emerging family of RNA-binding proteins (RBPs) with diverse roles in bacterial post-transcriptional control and physiology. They exhibit an intriguing targeting spectrum, ranging from an assumed single RNA pair (FinP/traJ) for the plasmid-encoded FinO protein, to transcriptome-wide activity as documented for chromosomally encoded ProQ proteins. Thus, the shared FinO domain might bear an unusual plasticity enabling it to act either selectively or promiscuously on the same cellular RNA pool. One caveat to this model is that the full suite of in vivo targets of the assumedly highly selective FinO protein is unknown. Here, we have extensively profiled cellular transcripts associated with the virulence plasmid-encoded FinO in Salmonella enterica. While our analysis confirms the FinP sRNA of plasmid pSLT as the primary FinO target, we identify a second major ligand: the RepX sRNA of the unrelated antibiotic resistance plasmid pRSF1010. FinP and RepX are strikingly similar in length and structure, but not in primary sequence, and so may provide clues to understanding the high selectivity of FinO-RNA interactions. Moreover, we observe that the FinO RBP encoded on the Salmonella virulence plasmid controls the replication of a cohabitating antibiotic resistance plasmid, suggesting cross-regulation of plasmids on the RNA level. KW - antisense RNA KW - Escherichia coli KW - chromosomal genes KW - protein KW - chaperone KW - virulence KW - family KW - HFQ KW - specificity KW - inhibition Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-261072 VL - 49 IS - 9 ER - TY - JOUR A1 - Mottola, Austin A1 - Ramírez-Zavala, Bernardo A1 - Hünninger, Kerstin A1 - Kurzai, Oliver A1 - Morschhäuser, Joachim T1 - The zinc cluster transcription factor Czf1 regulates cell wall architecture and integrity in Candida albicans JF - Molecular Microbiology N2 - The fungal cell wall is essential for the maintenance of cellular integrity and mediates interactions of the cells with the environment. It is a highly flexible organelle whose composition and organization is modulated in response to changing growth conditions. In the pathogenic yeast Candida albicans, a network of signaling pathways regulates the structure of the cell wall, and mutants with defects in these pathways are hypersensitive to cell wall stress. By harnessing a library of genetically activated forms of all C. albicans zinc cluster transcription factors, we found that a hyperactive Czf1 rescued the hypersensitivity to cell wall stress of different protein kinase deletion mutants. The hyperactive Czf1 induced the expression of many genes with cell wall-related functions and caused visible changes in the cell wall structure. C. albicans czf1Δ mutants were hypersensitive to the antifungal drug caspofungin, which inhibits cell wall biosynthesis. The changes in cell wall architecture caused by hyperactivity or absence of Czf1 resulted in an increased recognition of C. albicans by human neutrophils. Our results show that Czf1, which is known as a regulator of filamentous growth and white-opaque switching, controls the expression of cell wall genes and modulates the architecture of the cell wall. KW - cell wall KW - zinc cluster transcription factor KW - Candida albicans KW - protein kinases Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259583 VL - 116 IS - 2 ER - TY - JOUR A1 - Svensson, Sarah L. A1 - Sharma, Cynthia M. T1 - Small RNAs that target G-rich sequences are generated by diverse biogenesis pathways in Epsilonproteobacteria JF - Molecular Microbiology N2 - Bacterial small RNAs (sRNAs) are widespread post-transcriptional regulators that control bacterial stress responses and virulence. Nevertheless, little is known about how they arise and evolve. Homologs can be difficult to identify beyond the strain level using sequence-based approaches, and similar functionalities can arise by convergent evolution. Here, we found that the virulence-associated CJnc190 sRNA of the foodborne pathogen Campylobacter jejuni resembles the RepG sRNA from the gastric pathogen Helicobacter pylori. However, while both sRNAs bind G-rich sites in their target mRNAs using a C/U-rich loop, they largely differ in their biogenesis. RepG is transcribed from a stand-alone gene and does not require processing, whereas CJnc190 is transcribed from two promoters as precursors that are processed by RNase III and also has a cis-encoded antagonist, CJnc180. By comparing CJnc190 homologs in diverse Campylobacter species, we show that RNase III-dependent processing of CJnc190 appears to be a conserved feature even outside of C. jejuni. We also demonstrate the CJnc180 antisense partner is expressed in C. coli, yet here might be derived from the 3’UTR (untranslated region) of an upstream flagella-related gene. Our analysis of G-tract targeting sRNAs in Epsilonproteobacteria demonstrates that similar sRNAs can have markedly different biogenesis pathways. KW - sRNA biogenesis KW - Campylobacter jejuni KW - Helicobacter pylori KW - pathogenesis KW - RNase III Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259602 VL - 117 ER - TY - JOUR A1 - Prezza, Gianluca A1 - Ryan, Daniel A1 - Mädler, Gohar A1 - Reichardt, Sarah A1 - Barquist, Lars A1 - Westermann, Alexander J. T1 - Comparative genomics provides structural and functional insights into Bacteroides RNA biology JF - Molecular Microbiology N2 - Bacteria employ noncoding RNA molecules for a wide range of biological processes, including scaffolding large molecular complexes, catalyzing chemical reactions, defending against phages, and controlling gene expression. Secondary structures, binding partners, and molecular mechanisms have been determined for numerous small noncoding RNAs (sRNAs) in model aerobic bacteria. However, technical hurdles have largely prevented analogous analyses in the anaerobic gut microbiota. While experimental techniques are being developed to investigate the sRNAs of gut commensals, computational tools and comparative genomics can provide immediate functional insight. Here, using Bacteroides thetaiotaomicron as a representative microbiota member, we illustrate how comparative genomics improves our understanding of RNA biology in an understudied gut bacterium. We investigate putative RNA-binding proteins and predict a Bacteroides cold-shock protein homolog to have an RNA-related function. We apply an in silico protocol incorporating both sequence and structural analysis to determine the consensus structures and conservation of nine Bacteroides noncoding RNA families. Using structure probing, we validate and refine these predictions and deposit them in the Rfam database. Through synteny analyses, we illustrate how genomic coconservation can serve as a predictor of sRNA function. Altogether, this work showcases the power of RNA informatics for investigating the RNA biology of anaerobic microbiota members. KW - BT_1884 KW - cold-shock protein KW - GibS KW - RNA-binding proteins KW - secondary structure KW - 6S RNA Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259594 VL - 117 IS - 1 ER - TY - JOUR A1 - Masota, Nelson E. A1 - Vogg, Gerd A1 - Ohlsen, Knut A1 - Holzgrabe, Ulrike T1 - Reproducibility challenges in the search for antibacterial compounds from nature JF - PLoS One N2 - Background Reproducibility of reported antibacterial activities of plant extracts has long remained questionable. Although plant-related factors should be well considered in serious pharmacognostic research, they are often not addressed in many research papers. Here we highlight the challenges in reproducing antibacterial activities of plant extracts. Methods Plants with reported antibacterial activities of interest were obtained from a literature review. Antibacterial activities against Escherichia coli and Klebsiella pneumoniae were tested using extracts’ solutions in 10% DMSO and acetone. Compositions of working solutions from both solvents were established using LC-MS analysis. Moreover, the availability of details likely to affect reproducibility was evaluated in articles which reported antibacterial activities of studied plants. Results Inhibition of bacterial growth at MIC of 256–1024 μg/mL was observed in only 15.4% of identical plant species. These values were 4–16-fold higher than those reported earlier. Further, 18.2% of related plant species had MICs of 128–256 μg/mL. Besides, 29.2% and 95.8% of the extracts were soluble to sparingly soluble in 10% DMSO and acetone, respectively. Extracts’ solutions in both solvents showed similar qualitative compositions, with differing quantities of corresponding phytochemicals. Details regarding seasons and growth state at collection were missing in 65% and 95% of evaluated articles, respectively. Likewise, solvents used to dissolve the extracts were lacking in 30% of the articles, whereas 40% of them used unidentified bacterial isolates. Conclusion Reproducibility of previously reported activities from plants’ extracts is a multi-factorial aspect. Thus, collective approaches are necessary in addressing the highlighted challenges. KW - acetones KW - antibacterials KW - leaves KW - phytochemicals KW - solubility KW - plants KW - liquid chromatography-mass spectrometry KW - ethanol Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-260239 VL - 16 IS - 7 ER - TY - JOUR A1 - Wallaschek, Nina A1 - Reuter, Saskia A1 - Silkenat, Sabrina A1 - Wolf, Katharina A1 - Niklas, Carolin A1 - Özge, Kayisoglu A1 - Aguilar, Carmen A1 - Wiegering, Armin A1 - Germer, Christoph-Thomas A1 - Kircher, Stefan A1 - Rosenwald, Andreas A1 - Shannon-Lowe, Claire A1 - Bartfeld, Sina T1 - Ephrin receptor A2, the epithelial receptor for Epstein-Barr virus entry, is not available for efficient infection in human gastric organoids JF - PLoS Pathogens N2 - Epstein-Barr virus (EBV) is best known for infection of B cells, in which it usually establishes an asymptomatic lifelong infection, but is also associated with the development of multiple B cell lymphomas. EBV also infects epithelial cells and is associated with all cases of undifferentiated nasopharyngeal carcinoma (NPC). EBV is etiologically linked with at least 8% of gastric cancer (EBVaGC) that comprises a genetically and epigenetically distinct subset of GC. Although we have a very good understanding of B cell entry and lymphomagenesis, the sequence of events leading to EBVaGC remains poorly understood. Recently, ephrin receptor A2 (EPHA2) was proposed as the epithelial cell receptor on human cancer cell lines. Although we confirm some of these results, we demonstrate that EBV does not infect healthy adult stem cell-derived gastric organoids. In matched pairs of normal and cancer-derived organoids from the same patient, EBV only reproducibly infected the cancer organoids. While there was no clear pattern of differential expression between normal and cancer organoids for EPHA2 at the RNA and protein level, the subcellular location of the protein differed markedly. Confocal microscopy showed EPHA2 localization at the cell-cell junctions in primary cells, but not in cancer cell lines. Furthermore, histologic analysis of patient tissue revealed the absence of EBV in healthy epithelium and presence of EBV in epithelial cells from inflamed tissue. These data suggest that the EPHA2 receptor is not accessible to EBV on healthy gastric epithelial cells with intact cell-cell contacts, but either this or another, yet to be identified receptor may become accessible following cellular changes induced by inflammation or transformation, rendering changes in the cellular architecture an essential prerequisite to EBV infection. KW - Organoids KW - ephitelial cells KW - gastrointestinal infections KW - cancers and neoplasms KW - Epstein-Barr virus KW - flow cytometry KW - epithelium Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259206 VL - 17 IS - 2 ER - TY - JOUR A1 - Correia Santos, Sara A1 - Bischler, Thorsten A1 - Westermann, Alexander J. A1 - Vogel, Jörg T1 - MAPS integrates regulation of actin-targeting effector SteC into the virulence control network of Salmonella small RNA PinT JF - Cell Reports N2 - A full understanding of the contribution of small RNAs (sRNAs) to bacterial virulence demands knowledge of their target suites under infection-relevant conditions. Here, we take an integrative approach to capturing targets of the Hfq-associated sRNA PinT, a known post-transcriptional timer of the two major virulence programs of Salmonella enterica. Using MS2 affinity purification and RNA sequencing (MAPS), we identify PinT ligands in bacteria under in vitro conditions mimicking specific stages of the infection cycle and in bacteria growing inside macrophages. This reveals PinT-mediated translational inhibition of the secreted effector kinase SteC, which had gone unnoticed in previous target searches. Using genetic, biochemical, and microscopic assays, we provide evidence for PinT-mediated repression of steC mRNA, eventually delaying actin rearrangements in infected host cells. Our findings support the role of PinT as a central post-transcriptional regulator in Salmonella virulence and illustrate the need for complementary methods to reveal the full target suites of sRNAs. KW - gene expression KW - nondocing RNA KW - chaperone HFQ KW - soluble-RNA KW - SEQ KW - interactome KW - repression KW - secretion KW - infection KW - biology Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259134 VL - 34 IS - 5 ER - TY - THES A1 - Reuter-Weissenberger, Philipp T1 - The role of a fungal-specific transcription regulator on vacuolar biology and host interaction in \(Candida\) \(albicans\) T1 - Die Rolle eines pilzspezifischen Transkriptionsfaktors für die Vakuole und Wirtsinteraktion von \(Candida\) \(albicans\) N2 - Microorganisms that colonize the human body face large fluctuations in their surroundings. Therefore, those microbes developed sophisticated mechanisms that allow them to adapt their cell biology and maintain cellular homeostasis. One organelle vital to preserve cell physiology is the vacuole. The vacuole exhibits a wide range of functions and is able to adjust itself in response to both external and internal stimuli. Moreover, it plays an important role in host interaction and virulence in fungi such as Candida albicans. Despite this connection, only a few regulatory proteins have been described to modulate vacuolar biology in fungal pathogens. Furthermore, whether such regulation alters fungus-host interplay remains largely unknown. This thesis focuses on the characterization of ZCF8, a fungus-specific transcription regulator in the human-associated yeast C. albicans. To this end, I combined genome-wide protein-DNA interaction assays and gene expression analysis that identified genes regulated by Zcf8p. Fluorescence microscopy uncovered that several top targets of Zcf8p localize to the fungal vacuole. Moreover, deletion and overexpression of ZCF8 resulted in alterations in vacuolar morphology and in luminal pH and rendered the fungus resistant or susceptible to a vacuole-disturbing drug. Finally, in vitro adherence assays showed that Zcf8p modulates the attachment of C. albicans to human epithelial cells in a vacuole-dependent manner. Given those findings, I posit that the previously uncharacterized transcription regulator Zcf8p modulates fungal attachment to epithelial cells in a manner that depends on the status of the fungal vacuole. Furthermore, the results highlight that vacuolar physiology is a substantial factor influencing the physical interaction between Candida cells and mammalian mucosal surfaces. N2 - Mikroorganismen, die den Menschen besiedeln, sind großen Schwankungen in ihrer Umgebung ausgesetzt. Daher haben sie ausgeklügelte Mechanismen entwickelt, die es ihnen ermöglichen, ihre Zellbiologie anzupassen und die zelluläre Homöostase aufrechtzuerhalten. Eine für die Aufrechterhaltung der Zellphysiologie wichtige Organelle ist die Vakuole. Sie verfügt über ein breites Spektrum an Funktionen und ist in der Lage, auf externe und interne Stimuli zu reagieren. Außerdem spielt dieses Organell eine wichtige Rolle bei der Pilz-Wirt-Interaktion und somit für die Pathogenität von Pilzen wie Candida albicans. Trotz dieses Zusammenhangs wurden bisher nur wenige regulatorische Proteine beschrieben, welche die Biologie der Vakuolen in pathogenen Pilzen modulieren. Zudem ist weitgehend unbekannt, ob eine solche Regulierung das Zusammenspiel von Pilz und Wirt verändert. Diese Arbeit konzentriert sich auf die Charakterisierung von ZCF8, einem pilzspezifischen Transkriptionsregulator in der pathogenen Hefe C. albicans. Zu diesem Zweck wurden Protein-DNA-Interaktionstests und Genexpressionsanalysen kombiniert, um Gene zu identifizieren, die direkt von Zcf8p reguliert werden. Fluoreszenzmikroskopie zeigte zudem, dass mehrere der wichtigsten Ziele von Zcf8p in der Pilzvakuole lokalisiert sind. Darüber hinaus führte die Deletion und Überexpression von ZCF8 zu Veränderungen der Morphologie und des luminalen pH-Werts der Vakuole, und veränderte die Sensitivität des Pilzes gegenüber Stoffen, welche Funktionen der Vakuole beeinträchtigen. Schließlich deuteten In-vitro-Adhärenztests daraufhin, dass Zcf8p die Anheftung von C. albicans an menschliche Epithelzellen auf eine Weise moduliert, die abhängig von der Vakuole ist. Angesichts dieser Ergebnisse kann davon ausgegangen werden, dass der bisher unbekannte Transkriptionsregulator ZCF8 die Interaktion zwischen Pilz- und Epithelzellen des Wirts kontrolliert, und das auf eine Weise, die von der Pilzvakuole abhängig ist. Des Weiteren, unterstreichen die Ergebnisse, dass die Physiologie der Vakuole ein wesentlicher Faktor ist, welcher die Interaktion zwischen C. albicans und dem Wirt beeinflusst. KW - Vakuole KW - Transkriptionsfaktor KW - Candida albicans KW - vacuole KW - host colonization KW - Candida albicans KW - transcription regulator Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259287 ER - TY - JOUR A1 - Marincola, Gabriella A1 - Liong, Olivia A1 - Schoen, Christoph A1 - Abouelfetouh, Alaa A1 - Hamdy, Aisha A1 - Wencker, Freya D. R. A1 - Marciniak, Tessa A1 - Becker, Karsten A1 - Köck, Robin A1 - Ziebuhr, Wilma T1 - Antimicrobial Resistance Profiles of Coagulase-Negative Staphylococci in Community-Based Healthy Individuals in Germany JF - Frontiers in Public Health N2 - Coagulase-negative staphylococci (CoNS) are common opportunistic pathogens, but also ubiquitous human and animal commensals. Infection-associated CoNS from healthcare environments are typically characterized by pronounced antimicrobial resistance (AMR) including both methicillin- and multidrug-resistant isolates. Less is known about AMR patterns of CoNS colonizing the general population. Here we report on AMR in commensal CoNS recovered from 117 non-hospitalized volunteers in a region of Germany with a high livestock density. Among the 69 individuals colonized with CoNS, 29 had reported contacts to either companion or farm animals. CoNS were selectively cultivated from nasal swabs, followed by species definition by 16S rDNA sequencing and routine antibiotic susceptibility testing. Isolates displaying phenotypic AMR were further tested by PCR for presence of selected AMR genes. A total of 127 CoNS were isolated and Staphylococcus epidermidis (75%) was the most common CoNS species identified. Nine isolates (7%) were methicillin-resistant (MR) and carried the mecA gene, with seven individuals (10%) being colonized with at least one MR-CoNS isolate. While resistance against gentamicin, phenicols and spectinomycin was rare, high resistance rates were found against tetracycline (39%), erythromycin (33%) and fusidic acid (24%). In the majority of isolates, phenotypic resistance could be associated with corresponding AMR gene detection. Multidrug-resistance (MDR) was observed in 23% (29/127) of the isolates, with 33% (23/69) of the individuals being colonized with MDR-CoNS. The combined data suggest that MR- and MDR-CoNS are present in the community, with previous animal contact not significantly influencing the risk of becoming colonized with such isolates. KW - coagulase-negative staphylococci KW - antimicrobial resistance KW - One Health KW - community settings KW - Germany Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-240881 SN - 2296-2565 VL - 9 ER - TY - JOUR A1 - Marincola, Gabriella A1 - Jaschkowitz, Greta A1 - Kieninger, Ann-Katrin A1 - Wencker, Freya D.R. A1 - Feßler, Andrea T. A1 - Schwarz, Stefan A1 - Ziebuhr, Wilma T1 - Plasmid-Chromosome Crosstalk in Staphylococcus aureus: A Horizontally Acquired Transcription Regulator Controls Polysaccharide Intercellular Adhesin-Mediated Biofilm Formation JF - Frontiers in Cellular and Infection Microbiology N2 - Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of clonal complex CC398 typically carry various antimicrobial resistance genes, many of them located on plasmids. In the bovine LA-MRSA isolate Rd11, we previously identified plasmid pAFS11 in which resistance genes are co-localized with a novel ica-like gene cluster, harboring genes required for polysaccharide intercellular adhesin (PIA)-mediated biofilm formation. The ica genes on pAFS11 were acquired in addition to a pre-existing ica locus on the S. aureus Rd11 chromosomal DNA. Both loci consist of an icaADBC operon and icaR, encoding a corresponding icaADBC repressor. Despite carrying two biofilm gene copies, strain Rd11 did not produce PIA and transformation of pAFS11 into another S. aureus strain even slightly diminished PIA-mediated biofilm formation. By focusing on the molecular background of the biofilm-negative phenotype of pAFS11-carrying S. aureus, we identified the pAFS11-borne ica locus copy as functionally fully active. However, transcription of both plasmid- and core genome-derived icaADBC operons were efficiently suppressed involving IcaR. Surprisingly, although being different on the amino acid sequence level, the two IcaR repressor proteins are mutually replaceable and are able to interact with the icaA promoter region of the other copy. We speculate that this regulatory crosstalk causes the biofilm-negative phenotype in S. aureus Rd11. The data shed light on an unexpected regulatory interplay between pre-existing and newly acquired DNA traits in S. aureus. This also raises interesting general questions regarding functional consequences of gene transfer events and their putative implications for the adaptation and evolution of bacterial pathogens. KW - biofilm regulation KW - PIA/ica KW - IcaR KW - horizontal gene transfer KW - plasmid-chromosome crosstalk KW - Staphylococcus aureus Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232903 SN - 2235-2988 VL - 11 ER - TY - THES A1 - Venturini, Elisa T1 - Small proteins in \(Salmonella\): an updated annotation and a global analysis to find new regulators of virulence T1 - Kleine Proteine in \(Salmonella\): Eine aktualisierte Annotation und eine globale Analyse, um neue Regulatoren der Virulenz zu finden N2 - Small proteins, often defined as shorter than 50 amino acids, have been implicated in fundamental cellular processes. Despite this, they have been largely understudied throughout all domains of life, since their size often makes their identification and characterization challenging. This work addressed the knowledge gap surrounding small proteins with a focus on the model bacterial pathogen Salmonella Typhimurium. In a first step, new small proteins were identified with a combination of computational and experimental approaches. Infection-relevant datasets were then investigated with the updated Salmonella annotation to prioritize promising candidates involved in virulence. To implement the annotation of new small proteins, predictions from the algorithm sPepFinder were merged with those derived from Ribo-seq. These were added to the Salmonella annotation and used to (re)analyse different datasets. Information regarding expression during infection (dual RNA-seq) and requirement for virulence (TraDIS) was collected for each given coding sequence. In parallel, Grad-seq data were mined to identify small proteins engaged in intermolecular interactions. The combination of dual RNA-seq and TraDIS lead to the identification of small proteins with features of virulence factors, namely high intracellular induction and a virulence phenotype upon transposon insertion. As a proof of principle of the power of this approach in highlighting high confidence candidates, two small proteins were characterized in the context of Salmonella infection. MgrB, a known regulator of the PhoPQ two-component system, was shown to be essential for the infection of epithelial cells and macrophages, possibly via its stabilizing effect on flagella or by interacting with other sensor kinases of twocomponent systems. YjiS, so far uncharacterized in Salmonella, had an opposite role in infection, with its deletion rendering Salmonella hypervirulent. The mechanism underlying this, though still obscure, likely relies on the interaction with inner-membrane proteins. Overall, this work provides a global description of Salmonella small proteins in the context of infection with a combinatorial approach that expedites the identification of interesting candidates. Different high-throughput datasets available for a broad range of organisms can be analysed in a similar manner with a focus on small proteins. This will lead to the identification of key factors in the regulation of various processes, thus for example providing targets for the treatment of bacterial infections or, in the case of commensal bacteria, for the modulation of the microbiota composition. N2 - Kleine Proteine, oft definiert als kürzer als 50 Aminosäuren, sind in fundamentale zelluläre Prozesse involviert. Trotzdem sind sie in allen Domänen des Lebens noch weitgehend unerforscht, da ihre Größe ihre Identifizierung und Charakterisierung oft schwierig macht. Diese Arbeit adressiert die Wissenslücke um kleine Proteine mit einem Fokus auf das bakterielle Modellpathogen Salmonella Typhimurium. In einem ersten Schritt wurden neue kleine Proteine mit einer Kombination aus bioinformatischen und experimentellen Ansätzen identifiziert. Anschließend wurden infektionsrelevante Datensätze mit der aktualisierten Salmonella-Annotation untersucht, um vielversprechende Kandidaten zu priorisieren, die an der Virulenz beteiligt sind. Um die Annotation neuer kleiner Proteine zu implementieren, wurden die Vorhersagen aus dem Algorithmus sPepFinder mit denen aus Ribo-seq kombiniert. Diese wurden der Salmonella-Annotation hinzugefügt und zur (Re-)Analyse verschiedener Datensätze verwendet. Für jede gegebene kodierende Sequenz wurden Informationen zur Expression während der Infektion (duale RNA-seq) und zum Beitrag zur Virulenz (TraDIS) gesammelt. Parallel dazu wurden Grad-seq-Daten ausgewertet, um kleine Proteine zu identifizieren, die an intermolekularen Interaktionen beteiligt sind. Die Kombination von dualer RNA-seq und TraDIS führte zur Identifizierung von kleinen Proteinen mit Merkmalen von Virulenzfaktoren, nämlich einer hohen intrazellulären Induktion und einem Virulenz-Phänotyp nach Transposon- Insertion. Als Beweis für die Leistungsfähigkeit dieses Ansatzes Identifikation von vielversprechenden Kandidaten wurden zwei kleine Proteine im Kontext einer Salmonella-Infektion charakterisiert. MgrB, ein bekannter Regulator des PhoPQ-Zweikomponentensystems, erwies sich als ein für die Infektion von Epithelzellen und Makrophagen essentielles Protein, möglicherweise über seine stabilisierende Wirkung von Flagellen oder durch Interaktion mit Sensorkinasen von Zweikomponentensystemen. YjiS, das in Salmonella bisher nicht charakterisiert wurde, hatte eine entgegengesetzte Rolle bei der Infektion, wobei seine Deletion Salmonella hypervirulent macht. Der Mechanismus, der dem zugrunde liegt, ist zwar noch unklar, beruht aber wahrscheinlich auf der Interaktion mit inneneren Membranproteinen. Insgesamt liefert diese Arbeit eine globale Beschreibung der kleinen Salmonella- Proteine im Kontext der Infektion mit einem kombinatorischen Ansatz, der die Identifizierung interessanter Kandidaten beschleunigt. Verschiedene Hochdurchsatz- Datensätze, die für ein breites Spektrum von Organismen verfügbar sind, können auf ähnliche Weise mit einem Fokus auf kleine Proteine analysiert werden. Dies wird zur Identifizierung von Schlüsselfaktoren in der Regulation verschiedener Prozesse führen und damit z. B. Targets für die Behandlung bakterieller Infektionen oder, im Falle kommensaler Bakterien, für die Modulation der Mikrobiota- Zusammensetzung liefern. KW - Salmonella Typhimurium KW - Kleine Proteine KW - small proteins KW - dual RNA-seq KW - TraDIS KW - MgrB Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-247029 ER - TY - JOUR A1 - Esken, Jens A1 - Goris, Tobias A1 - Gadkari, Jennifer A1 - Bischler, Thorsten A1 - Förstner, Konrad U. A1 - Sharma, Cynthia M. A1 - Diekert, Gabriele A1 - Schubert, Torsten T1 - Tetrachloroethene respiration in Sulfurospirillum species is regulated by a two‐component system as unraveled by comparative genomics, transcriptomics, and regulator binding studies JF - MicrobiologyOpen N2 - Energy conservation via organohalide respiration (OHR) in dehalogenating Sulfurospirillum species is an inducible process. However, the gene products involved in tetrachloroethene (PCE) sensing and signal transduction have not been unambiguously identified. Here, genome sequencing of Sulfurospirillum strains defective in PCE respiration and comparative genomics, which included the PCE‐respiring representatives of the genus, uncovered the genetic inactivation of a two‐component system (TCS) in the OHR gene region of the natural mutants. The assumption that the TCS gene products serve as a PCE sensor that initiates gene transcription was supported by the constitutive low‐level expression of the TCS operon in fumarate‐adapted cells of Sulfurospirillum multivorans. Via RNA sequencing, eight transcriptional units were identified in the OHR gene region, which includes the TCS operon, the PCE reductive dehalogenase operon, the gene cluster for norcobamide biosynthesis, and putative accessory genes with unknown functions. The OmpR‐family response regulator (RR) encoded in the TCS operon was functionally characterized by promoter‐binding assays. The RR bound a cis‐regulatory element that contained a consensus sequence of a direct repeat (CTATW) separated by 17 bp. Its location either overlapping the −35 box or 50 bp further upstream indicated different regulatory mechanisms. Sequence variations in the regulator binding sites identified in the OHR gene region were in accordance with differences in the transcript levels of the respective gene clusters forming the PCE regulon. The results indicate the presence of a fine‐tuned regulatory network controlling PCE metabolism in dehalogenating Sulfurospirillum species, a group of metabolically versatile organohalide‐respiring bacteria. KW - genomics KW - organohalide respiration KW - RNA sequencing KW - tetrachloroethene KW - transcriptomics KW - two‐component system Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-225754 VL - 9 IS - 12 ER - TY - THES A1 - Popp, Christina T1 - Evolution of antifungal drug resistance of the human-pathogenic fungus \(Candida\) \(albicans\) T1 - Evolution der Antimykotikaresistenz im humanpathogenen Pilz \(Candida\) \(albicans\) N2 - Infections with the opportunistic yeast Candida albicans are frequently treated with the first-line drug fluconazole, which inhibits ergosterol biosynthesis. An alarming problem in clinics is the development of resistances against this azole, especially during long-term treatment of patients. Well-known resistance mechanisms include mutations in the zinc cluster transcription factors (ZnTFs) Mrr1 and Tac1, which cause an overexpression of efflux pump genes, and Upc2, which results in an overexpression of the drug target. C. albicans strains with such gain-of-function mutations (GOF) have an increased drug resistance conferring a selective advantage in the presence of the drug. It was previously shown that this advantage comes with a fitness defect in the absence of the drug. This was observed in different conditions and is presumably caused by a deregulated gene expression. One aim of the present study was to examine whether C. albicans can overcome the costs of drug resistance by further evolution. Therefore, the relative fitness of clinical isolates with one or a combination of different resistance mutations in Mrr1, Tac1 and/or Upc2 was analyzed in competition with the matched fluconazole-susceptible partner. Most fluconazole-resistant isolates had a decreased fitness in competition with their susceptible partner in vitro in rich medium. In contrast, three fluconazole-resistant strains with Mrr1 resistance mutations did not show a fitness defect in competition with their susceptible partner. In addition, the fitness of four selected clinical isolate pairs was examined in vivo in mouse models of gastrointestinal colonization (GI) and disseminated infection (IV). In the GI model all four fluconazole-resistant strains were outcompeted by their respective susceptible partner. In contrast, in the IV model only one out of four fluconazole-resistant isolates did show a slight fitness defect in competition with its susceptible partner during infection of the kidneys. It can be stated, that in the present work the in vitro fitness did not reflect the in vivo fitness and that the overall fitness was dependent on the tested conditions. In conclusion, C. albicans cannot easily overcome the costs of drug resistance caused by a deregulated gene expression. In addition to GOFs in Mrr1, Tac1 and Upc2, resistance mutations in the drug target Erg11 are a further key fluconazole resistance mechanism of C. albicans. Clinical isolates often harbor several resistance mechanisms, as the fluconazole resistance level is further increased in strains with a combination of different resistance mutations. In this regard, the question arises of how strains with multiple resistance mechanisms evolve. One possibility is that strains acquire mutations successively. In the present study it was examined whether highly drug-resistant C. albicans strains with multiple resistance mechanisms can evolve by parasexual recombination as another possibility. In a clonal population, cells with individually acquired resistance mutations could combine these advantageous traits by mating. Thereupon selection could act on the mating progeny resulting in even better adapted derivatives. Therefore, strains heterozygous for a resistance mutation and the mating type locus (MTL) were grown in the presence of fluconazole. Derivatives were isolated, which had become homozygous for the resistance mutation and at the same time for the MTL. This loss of heterozygosity was accompanied by increased drug resistance. In general, strains which are homozygous for one of both MTL configurations (MTLa and MTLα) can switch to the opaque phenotype, which is the mating-competent form of the yeast, and mate with cells of the opposite MTL. In the following, MTLa and MTLα homozygous strains in the opaque phenotype were mated in all possible combinations. The resulting mating products with combined genetic material from both parents did not show an increased drug resistance. Selected products of each mating cross were passaged with stepwise increasing concentrations of fluconazole. The isolated progeny showed high levels of drug resistance and loss of wild-type alleles of resistance-associated genes. In conclusion, selective pressure caused by fluconazole exposure selects for resistance mutations and at the same time induces genomic rearrangements, resulting in mating competence. Therefore, in a clonal population, cells with individually acquired resistance mutations can mate with each other and generate mating products with combined genetic backgrounds. Selection can act on these mating products and highly drug-resistant und thus highly adapted derivatives can evolve as a result. In summary, the present study contributes to the current understanding of the evolution of antifungal drug resistance by elucidating the effect of resistance mutations on the fitness of the strains in the absence of the drug selection pressure and investigates how highly drug-resistant strains could evolve within a mammalian host. N2 - Infektionen mit dem opportunistischen Hefepilz Candida albicans werden häufig mit dem First-Line-Medikament Fluconazol behandelt, welches die Ergosterol-Biosynthese hemmt. Ein besorgniserregendes Problem in der Klinik, insbesondere bei der Langzeitbehandlung von Patienten, ist die Entwicklung von Resistenzen gegen dieses Azol. Zu den bekannten Resistenzmechanismen gehören Resistenzmutationen in den Zink-Cluster-Transkriptionsfaktoren (ZnTFs) Mrr1 und Tac1, die eine Überexpression von Effluxpumpen-Genen bewirken und Resistenzmutationen in Upc2, die zu einer Überexpression des Wirkstofftargets führen. C. albicans Stämme mit solchen Gain-of-Function-Mutationen (GOF) weisen eine erhöhte Medikamentenresistenz auf, was einen selektiven Vorteil in Gegenwart des Medikaments bedeutet. Es wurde zuvor gezeigt, dass dieser Vorteil mit einem Fitnessdefekt in Abwesenheit des Medikaments einhergeht. Dies wurde in verschiedenen Bedingungen nachgewiesen und wird vermutlich durch eine deregulierte Genexpression verursacht. Ein Ziel der vorliegenden Studie war es zu untersuchen, ob C. albicans die Kosten der Medikamentenresistenz durch Evolution kompensieren kann. Daher wurde die relative Fitness von klinischen Isolaten mit einer oder einer Kombination verschiedener Resistenzmutationen in Mrr1, Tac1 und/oder Upc2 im Wettbewerb mit dem zugehörigen Fluconazol-sensitiven Partner analysiert. Die meisten Fluconazol-resistenten Isolate hatten eine verminderte Fitness im Wettbewerb mit ihrem sensitiven Partner in vitro in vollwertigem Medium. Dennoch zeigten drei Fluconazol-resistente Stämme mit Mrr1-Resistenzmutationen keinen Fitnessdefekt im Wettbewerb mit ihrem jeweiligen Partner. Zusätzlich wurde die Fitness von vier ausgewählten klinischen Isolat-Paaren in vivo in Mausmodellen für gastrointestinale Kolonisation (GI) und disseminierte Infektion (IV) untersucht. Im GI-Modell wurden alle vier Fluconazol-resistenten Stämme von ihren sensitiven Partnern überwachsen. Im Gegensatz dazu zeigte im IV-Modell nur einer der vier Fluconazol-resistenten Isolate einen leichten Fitnessdefekt im Wettbewerb mit dem jeweiligen Fluconazol-sensitiven Partner während der Infektion der Nieren. Es kann festgestellt werden, dass in der vorliegenden Arbeit die in vitro-Fitness nicht die in vivo-Fitness widerspiegelt und dass die Gesamtfitness von den getesteten Bedingungen abhängig ist. Zusammenfassend lässt sich sagen, dass C. albicans die Kosten der Medikamentenresistenz, die durch eine deregulierte Genexpression verursacht werden, nur schwer überwinden kann. Neben GOFs in Mrr1, Tac1 und Upc2 sind Resistenzmutationen im Wirkstofftarget Erg11 ein wichtiger Resistenzmechanismus von C. albicans. Klinische Isolate weißen oft mehrere Resistenzmechanismen auf, da die Kombination verschiedener Resistenzmutationen die Fluconazol-Resistenz potenziert. In diesem Zusammenhang stellt sich die Frage, wie sich Stämme mit mehreren Resistenzmechanismen entwickeln. Eine Möglichkeit ist, dass Stämme Mutationen sequenziell erwerben. In der vorliegenden Studie wurde untersucht, ob als weitere Möglichkeit hochresistente C. albicans Stämme mit multiplen Resistenzmechanismen durch parasexuelle Rekombination evolvieren können. In einer klonalen Population könnten Zellen mit individuell erworbenen Resistenzmutationen diese vorteilhaften Eigenschaften durch Paarung kombinieren. Daraufhin könnte Selektionsdruck auf die Matingprodukte wirken und so die Entstehung von besser angepassten Derivaten begünstigen. Daher wurden Resistenzmutation und Mating Type Locus (MTL) heterozygote Stämme in Gegenwart von Fluconazol kultiviert. So konnten Derivate isoliert werden, die homozygot für die Resistenzmutation und gleichzeitig für den MTL geworden waren. Dieser Verlust der Heterozygotie ging mit einer erhöhten Medikamentenresistenz einher. Generell können Stämme, die homozygot für eine der beiden MTL-Konfigurationen (MTLa und MTLα) sind, in den opaque Phänotyp wechseln, der die paarungskompetente Form der Hefe darstellt, und sich mit Zellen des gegensätzlichen MTL paaren. Im Folgenden wurden MTLa und MTLα homozygote Stämme im opaque Phänotyp in allen möglichen Kombinationen verpaart. Die resultierenden Matingprodukte mit kombiniertem genetischem Material beider Elternteile wiesen keine erhöhte Medikamentenresistenz auf. Ausgewählte Paarungsprodukte jeder Kreuzung wurden mit stufenweise ansteigenden Konzentrationen von Fluconazol passagiert. Die isolierten Nachkommen zeigten ein hohes Maß an Medikamentenresistenz und den Verlust von Wildtyp-Allelen der resistenzassoziierten Gene. Zusammenfassend lässt sich sagen, dass der selektive Druck, der durch die Fluconazol-Exposition verursacht wird, für Resistenzmutationen selektiert und gleichzeitig genomische Umlagerungen induziert, die eine Paarung ermöglichen. Daher können sich in einer klonalen Population Zellen mit individuell erworbenen Resistenzmutationen miteinander paaren und Matingprodukte mit kombiniertem genetischem Hintergrund generieren. Auf diese Matingprodukte kann die Selektion wirken, woraufhin sich hochresistente und damit stark an ihre Umwelt angepasste Derivate entwickeln können. Zusammenfassend trägt die vorliegende Studie zum aktuellen Verständnis der Evolution der Antimykotika-Resistenz bei, indem sie den Effekt von Resistenzmutationen auf die Fitness der Stämme in Abwesenheit des Medikamenten-Selektionsdrucks untersucht und aufklärt, wie sich hochgradig resistente Stämme in einem Säugetierwirt entwickeln könnten. KW - Evolution KW - Resistenz KW - Fitness KW - Candida albicans KW - Fluconazol KW - Resistance KW - Fluconazole KW - Drug resistance KW - Human-pathogenic KW - Yeast Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-243515 ER - TY - JOUR A1 - Umstätter, Florian A1 - Domhan, Cornelius A1 - Hertlein, Tobias A1 - Ohlsen, Knut A1 - Mühlberg, Eric A1 - Kleist, Christian A1 - Zimmermann, Stefan A1 - Beijer, Barbro A1 - Klika, Karel D. A1 - Haberkorn, Uwe A1 - Mier, Walter A1 - Uhl, Philipp T1 - Vancomycin Resistance Is Overcome by Conjugation of Polycationic Peptides JF - Angewandte Chemie International Edition N2 - Multidrug‐resistant bacteria represent one of the biggest challenges facing modern medicine. The increasing prevalence of glycopeptide resistance compromises the efficacy of vancomycin, for a long time considered as the last resort for the treatment of resistant bacteria. To reestablish its activity, polycationic peptides were conjugated to vancomycin. By site‐specific conjugation, derivatives that bear the peptide moiety at four different sites of the antibiotic were synthesized. The most potent compounds exhibited an approximately 1000‐fold increased antimicrobial activity and were able to overcome the most important types of vancomycin resistance. Additional blocking experiments using d‐Ala‐d‐Ala revealed a mode of action beyond inhibition of cell‐wall formation. The antimicrobial potential of the lead candidate FU002 for bacterial infection treatments could be demonstrated in an in vivo study. Molecular imaging and biodistribution studies revealed that conjugation engenders superior pharmacokinetics. KW - antibiotics KW - bacterial resistance KW - glycopeptide antibiotics KW - peptide conjugates KW - vancomycin Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215550 VL - 59 IS - 23 SP - 8823 EP - 8827 ER - TY - JOUR A1 - Barthels, Fabian A1 - Marincola, Gabriella A1 - Marciniak, Tessa A1 - Konhäuser, Matthias A1 - Hammerschmidt, Stefan A1 - Bierlmeier, Jan A1 - Distler, Ute A1 - Wich, Peter R. A1 - Tenzer, Stefan A1 - Schwarzer, Dirk A1 - Ziebuhr, Wilma A1 - Schirmeister, Tanja T1 - Asymmetric Disulfanylbenzamides as Irreversible and Selective Inhibitors of Staphylococcus aureus Sortase A JF - ChemMedChem N2 - Staphylococcus aureus is one of the most frequent causes of nosocomial and community‐acquired infections, with drug‐resistant strains being responsible for tens of thousands of deaths per year. S. aureus sortase A inhibitors are designed to interfere with virulence determinants. We have identified disulfanylbenzamides as a new class of potent inhibitors against sortase A that act by covalent modification of the active‐site cysteine. A broad series of derivatives were synthesized to derive structure‐activity relationships (SAR). In vitro and in silico methods allowed the experimentally observed binding affinities and selectivities to be rationalized. The most active compounds were found to have single‐digit micromolar Ki values and caused up to a 66 % reduction of S. aureus fibrinogen attachment at an effective inhibitor concentration of 10 μM. This new molecule class exhibited minimal cytotoxicity, low bacterial growth inhibition and impaired sortase‐mediated adherence of S. aureus cells. KW - antibiotics KW - biofilm KW - drug design KW - sortase A Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-214581 VL - 15 IS - 10 SP - 839 EP - 850 ER - TY - JOUR A1 - Bartfeld, Sina T1 - Realizing the potential of organoids — an interview with Hans Clevers JF - Journal of Molecular Medicine N2 - No abstract available. KW - organoids KW - interview Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235804 SN - Journal of Molecular Medicine VL - 99 ER - TY - JOUR A1 - Schielmann, Marta A1 - Szweda, Piotr A1 - Gucwa, Katarzyna A1 - Kawczyński, Marcin A1 - Milewska, Maria J. A1 - Martynow, Dorota A1 - Morschhäuser, Joachim A1 - Milewski, Sławomir T1 - Transport deficiency is the molecular basis of \(Candida\) \(albicans\) resistance to antifungal oligopeptides JF - Frontiers in Microbiology N2 - Oligopeptides incorporating \(N3\)-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP), an inhibitor of glucosamine-6-phosphate synthase, exhibited growth inhibitory activity against \(Candida\) \(albicans\), with minimal inhibitory concentration values in the 0.05–50 μg mL\(^{-1}\) range. Uptake by the peptide permeases was found to be the main factor limiting an anticandidal activity of these compounds. Di- and tripeptide containing FMDP (F2 and F3) were transported by Ptr2p/Ptr22p peptide transporters (PTR) and FMDP-containing hexa-, hepta-, and undecapeptide (F6, F7, and F11) were taken up by the oligopeptide transporters (OPT) oligopeptide permeases, preferably by Opt2p/Opt3p. A phenotypic, apparent resistance of \(C. albicans\) to FMDP-oligopeptides transported by OPT permeases was triggered by the environmental factors, whereas resistance to those taken up by the PTR system had a genetic basis. Anticandidal activity of longer FMDP-oligopeptides was strongly diminished in minimal media containing easily assimilated ammonium sulfate or L-glutamine as the nitrogen source, both known to downregulate expression of the OPT genes. All FMDP-oligopeptides tested were more active at lower pH and this effect was slightly more remarkable for peptides F6, F7, and F11, compared to F2 and F3. Formation of isolated colonies was observed inside the growth inhibitory zones induced by F2 and F3 but not inside those induced by F6, F7, and F11. The vast majority (98%) of those colonies did not originate from truly resistant cells. The true resistance of 2% of isolates was due to the impaired transport of di- and to a lower extent, tripeptides. The resistant cells did not exhibit a lower expression of \(PTR2\), \(PTR22\), or \(OPT1–3\) genes, but mutations in the \(PTR2\) gene resulting in T422H, A320S, D119V, and A320S substitutions in the amino acid sequence of Ptr2p were found. KW - microbiology KW - Candida albicans KW - oligopeptides KW - resistance mechanism KW - permease KW - antifungals Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173245 VL - 8 ER - TY - THES A1 - Mottola, Austin T1 - Molecular characterization of the SNF1 signaling pathway in \(Candida\) \(albicans\) T1 - Molekulare Charakterisierung des SNF1-Signalweges von \(Candida\) \(albicans\) N2 - The fungus Candida albicans is a typical member of the human microbiota, where it usually behaves as a commensal. It can also become pathogenic; often causing minor superficial infections in healthy people, but also potentially fatal invasive systemic infections in immunocompromised people. Unfortunately, there is only a fairly limited set of antifungal drugs, and evolution of drug resistance threatens their efficacy. Greater understanding of the mechanisms that C. albicans uses to survive in and infect the host can uncover candidate targets for novel antifungals. Protein kinases are central to a vast array of signalling pathways which govern practically all aspects of life, and furthermore are relatively straightforward to design drugs against. As such, investigation and characterization of protein kinases in C. albicans as well as their target proteins and the pathways they govern are important targets for research. AMP-activated kinases are well conserved proteins which respond to energy stress; they are represented in yeasts by the heterotrimeric SNF1 complex, which responds primarily to the absence of glucose. In this work, the SNF1 pathway was investigated with two primary goals: identify novel targets of this protein kinase and elucidate why SNF1 is essential. Two approaches were used to identify novel targets of SNF1. In one, suppressor mutants were evolved from a strain in which SNF1 activity is reduced, which exhibits defects in carbon source utilization and cell wall integrity. This revealed a suppressor mutation within SNF1 itself, coding for the catalytic subunit of the complex – SNF1Δ311-316. The second approach screened a library of artificially activated zinc cluster transcription factors, identifying Czf1 as one such transcription factor which, upon artificial activation, restored resistance to cell wall stress in a mutant of the SNF1 pathway. Finally, a, inducible gene deletion system revealed that SNF1 is not an essential gene. N2 - Der Pilz Candida albicans ist ein typisches Mitglied der menschlichen Mikrobiota, wo er sich normalerweise als Kommensale verhält. Als fakultativ pathogener Erreger kann er jedoch auch leichte, überfachliche Infektionen bei gesunden Menschen verursachen, sowie potenziell tödliche, invasive systemische Infektionen bei immungeschwächten Menschen. Leider gibt es nur eine recht begrenzte Anzahl von Antimykotika, und die Entwicklung von Resistenzen bedroht deren Wirksamkeit. Ein besseres Verständnis der Mechanismen, die C. albicans nutzt, um im Wirt zu überleben und ihn zu infizieren, kann mögliche Angriffspunkte für neue Antimykotika aufdecken. Proteinkinasen sind von zentraler Bedeutung für eine Vielzahl von Signalwegen, die praktisch alle Aspekte des Lebens steuern und gegen die sich zudem relativ einfach Medikamente entwickeln lassen. Daher ist die Untersuchung und Charakterisierung von Proteinkinasen in C. albicans sowie ihrer Zielproteine und der von ihnen gesteuerten Signalwege ein wichtiges Ziel für die Forschung. AMP-aktivierte Kinasen sind hoch konservierte Proteine, die auf Energiestress reagieren; sie sind in Hefen durch den heterotrimeren SNF1-Komplex vertreten, der vor allem auf das Fehlen von Glukose reagiert. In dieser Arbeit wurde der SNF1-Signalweg mit zwei primären Zielen untersucht: die Identifizierung neuer Zielproteine dieser Proteinkinase und die Klärung der Frage, warum SNF1 essentiell ist. Für die Identifikation neuer Zielproteine von SNF1 wurden zwei Ansätze verwendet. Zum einen wurde ein Stamm mit reduzierter SNF1-Aktivität, für die Entwicklung von Suppressor-Mutanten verwendet, die einen Defekte bei der Verwertung von Kohlenstoffquellen und eine eingeschränkte Zellwandintegrität aufweisen. Dabei wurde eine Suppressormutation in SNF1 selbst entdeckt, die für die katalytische Untereinheit des Komplexes – SNF1Δ311-316 - kodiert. Für den zweite Ansatz wurde eine Bibliothek von künstlich aktivierten Zink-Cluster-Transkriptionsfaktoren untersucht. Dies führte zur Identifikation von Czf1 als einen solchen Transkriptionsfaktor, der nach künstlicher Aktivierung die Resistenz gegen Zellwandstress in einer Mutante des SNF1- Signalweges wiederherstellte. Schließlich zeigte ein induzierbares Gendeletionssystem, dass SNF1 kein essentielles Gen ist. KW - candida albicans KW - yeast KW - fungus KW - candida KW - kinase KW - cell wall Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-238098 ER - TY - JOUR A1 - Hanzelmann, Dennis A1 - Joo, Hwang-Soo A1 - Franz-Wachtel, Mirita A1 - Hertlein, Tobias A1 - Stevanovic, Stefan A1 - Macek, Boris A1 - Wolz, Christiane A1 - Götz, Friedrich A1 - Otto, Michael A1 - Kretschmer, Dorothee A1 - Peschel, Andreas T1 - Toll-like receptor 2 activation depends on lipopeptide shedding by bacterial surfactants JF - Nature Communications N2 - Sepsis caused by Gram-positive bacterial pathogens is a major fatal disease but its molecular basis remains elusive. Toll-like receptor 2 (TLR2) has been implicated in the orchestration of inflammation and sepsis but its role appears to vary for different pathogen species and clones. Accordingly, Staphylococcus aureus clinical isolates differ substantially in their capacity to activate TLR2. Here we show that strong TLR2 stimulation depends on high-level production of phenol-soluble modulin (PSM) peptides in response to the global virulence activator Agr. PSMs are required for mobilizing lipoproteins, the TLR2 agonists, from the staphylococcal cytoplasmic membrane. Notably, the course of sepsis caused by PSM-deficient S. aureus is similar in wild-type and TLR2-deficient mice, but TLR2 is required for protection of mice against PSM-producing S. aureus. Thus, a crucial role of TLR2 depends on agonist release by bacterial surfactants. Modulation of this process may lead to new therapeutic strategies against Gram-positive infections. KW - Pathogens KW - Toll-like receptors Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165975 VL - 7 ER - TY - JOUR A1 - Mottola, Austin A1 - Schwanfelder, Sonja A1 - Morschhäuser, Joachim T1 - Generation of Viable Candida albicans Mutants Lacking the "Essential" Protein Kinase Snf1 by Inducible Gene Deletion JF - mSphere N2 - The protein kinase Snf1, a member of the highly conserved AMP-activated protein kinase family, is a central regulator of metabolic adaptation. In the pathogenic yeast Candida albicans, Snf1 is considered to be essential, as previous attempts by different research groups to generate homozygous snf1 Delta mutants were unsuccessful. We aimed to elucidate why Snf1 is required for viability in C. albicans by generating snf1 Delta null mutants through forced, inducible gene deletion and observing the terminal phenotype before cell death. Unexpectedly, we found that snf1 Delta mutants were viable and could grow, albeit very slowly, on rich media containing the preferred carbon source glucose. Growth was improved when the cells were incubated at 37 degrees C instead of 30 degrees C, and this phenotype enabled us to isolate homozygous snf1 Delta mutants also by conventional, sequential deletion of both SNF1 alleles in a wild-type C. albicans strain. All snf1 Delta mutants could grow slowly on glucose but were unable to utilize alternative carbon sources. Our results show that, under optimal conditions, C. albicans can live and grow without Snf1. Furthermore, they demonstrate that inducible gene deletion is a powerful method for assessing gene essentiality in C. albicans. IMPORTANCE Essential genes are those that are indispensable for the viability and growth of an organism. Previous studies indicated that the protein kinase Snf1, a central regulator of metabolic adaptation, is essential in the pathogenic yeast Candida albicans, because no homozygous snf1 deletion mutants of C. albicans wild-type strains could be obtained by standard approaches. In order to investigate the lethal consequences of SNF1 deletion, we generated conditional mutants in which SNF1 could be deleted by forced, inducible excision from the genome. Unexpectedly, we found that snf1 null mutants were viable and could grow slowly under optimal conditions. The growth phenotypes of the snf1 Delta mutants explain why such mutants were not recovered in previous attempts. Our study demonstrates that inducible gene deletion is a powerful method for assessing gene essentiality in C. albicans. KW - Candida albicans KW - Snf1 KW - conditional mutants KW - essential genes KW - protein kinases Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230524 VL - 5 IS - 4 ER - TY - JOUR A1 - Bauriedl, Saskia A1 - Gerovac, Milan A1 - Heidrich, Nadja A1 - Bischler, Thorsten A1 - Barquist, Lars A1 - Vogel, Jörg A1 - Schoen, Christoph T1 - The minimal meningococcal ProQ protein has an intrinsic capacity for structure-based global RNA recognition JF - Nature Communications N2 - FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition. FinO-domain proteins are bacterial RNA-binding proteins with a wide range of target specificities. Here, the authors employ UV CLIP-seq and show that minimal ProQ protein of Neisseria meningitidis binds to various small non-coding RNAs and mRNAs involved in virulence. KW - Neisseria meningitidis KW - natural transformation KW - dual function KW - FinO family KW - HFQ KW - chaperone KW - transcriptome KW - regulator KW - sequence KW - in vivo Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230040 VL - 11 ER - TY - JOUR A1 - Vogel, Jörg T1 - An RNA biology perspective on species‐specific programmable RNA antibiotics JF - Molecular Microbiology N2 - Our body is colonized by a vast array of bacteria the sum of which forms our microbiota. The gut alone harbors >1,000 bacterial species. An understanding of their individual or synergistic contributions to human health and disease demands means to interfere with their functions on the species level. Most of the currently available antibiotics are broad‐spectrum, thus too unspecific for a selective depletion of a single species of interest from the microbiota. Programmable RNA antibiotics in the form of short antisense oligonucleotides (ASOs) promise to achieve precision manipulation of bacterial communities. These ASOs are coupled to small peptides that carry them inside the bacteria to silence mRNAs of essential genes, for example, to target antibiotic‐resistant pathogens as an alternative to standard antibiotics. There is already proof‐of‐principle with diverse bacteria, but many open questions remain with respect to true species specificity, potential off‐targeting, choice of peptides for delivery, bacterial resistance mechanisms and the host response. While there is unlikely a one‐fits‐all solution for all microbiome species, I will discuss how recent progress in bacterial RNA biology may help to accelerate the development of programmable RNA antibiotics for microbiome editing and other applications. KW - antibiotic KW - microbiome KW - RNA-seq KW - small RNA Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-214869 VL - 113 IS - 3 SP - 550 EP - 559 ER - TY - JOUR A1 - Mayr, Eva-Maria A1 - Ramírez-Zavala, Bernardo A1 - Krüger, Ines A1 - Morschhäuser, Joachim T1 - A Zinc Cluster Transcription Factor Contributes to the Intrinsic Fluconazole Resistance of Candida auris JF - mSphere N2 - ABSTRACT The recently emerged pathogenic yeast Candida auris is a major concern for human health, because it is easily transmissible, difficult to eradicate from hospitals, and highly drug resistant. Most C. auris isolates are resistant to the widely used antifungal drug fluconazole due to mutations in the target enzyme Erg11 and high activity of efflux pumps, such as Cdr1. In the well-studied, distantly related yeast Candida albicans, overexpression of drug efflux pumps also is a major mechanism of acquired fluconazole resistance and caused by gain-of-function mutations in the zinc cluster transcription factors Mrr1 and Tac1. In this study, we investigated a possible involvement of related transcription factors in efflux pump expression and fluconazole resistance of C. auris. The C. auris genome contains three genes encoding Mrr1 homologs and two genes encoding Tac1 homologs, and we generated deletion mutants lacking these genes in two fluconazole-resistant strains from clade III and clade IV. Deletion of TAC1b decreased the resistance to fluconazole and voriconazole in both strain backgrounds, demonstrating that the encoded transcription factor contributes to azole resistance in C. auris strains from different clades. CDR1 expression was not or only minimally affected in the mutants, indicating that Tac1b can confer increased azole resistance by a CDR1-independent mechanism. IMPORTANCE Candida auris is a recently emerged pathogenic yeast that within a few years after its initial description has spread all over the globe. C. auris is a major concern for human health, because it can cause life-threatening systemic infections, is easily transmissible, and is difficult to eradicate from hospital environments. Furthermore, C. auris is highly drug resistant, especially against the widely used antifungal drug fluconazole. Mutations in the drug target and high activity of efflux pumps are associated with azole resistance, but it is not known how drug resistance genes are regulated in C. auris. We have investigated the potential role of several candidate transcriptional regulators in the intrinsic fluconazole resistance of C. auris and identified a transcription factor that contributes to the high resistance to fluconazole and voriconazole of two C. auris strains from different genetic clades, thereby providing insight into the molecular basis of drug resistance of this medically important yeast." KW - Candida auris KW - fluconazole resistance KW - transcription factor Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229937 VL - 5 IS - 2 ER - TY - JOUR A1 - Alzheimer, Mona A1 - Svensson, Sarah L. A1 - König, Fabian A1 - Schweinlin, Matthias A1 - Metzger, Marco A1 - Walles, Heike A1 - Sharma, Cynthia M. T1 - A three-dimensional intestinal tissue model reveals factors and small regulatory RNAs important for colonization with Campylobacter jejuni JF - PLoS Pathogens N2 - The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens. KW - in vitro KW - stem cells KW - invasion KW - host KW - adhesion KW - epithelial cells KW - translocation KW - virulence KW - responses KW - microenvironment Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229454 VL - 16 IS - 2 ER - TY - JOUR A1 - Schulte, Leon N. A1 - Schweinlin, Matthias A1 - Westermann, Alexander J. A1 - Janga, Harshavardhan A1 - Santos, Sara C. A1 - Appenzeller, Silke A1 - Walles, Heike A1 - Vogel, Jörg A1 - Metzger, Marco T1 - An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Salmonella Infection JF - mBio N2 - A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens. IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host. KW - Salmonella KW - gene expression KW - infectious disease Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229428 VL - 11, 2020 IS - 1 ER - TY - JOUR A1 - Dingemans, Josef A1 - Monsieurs, Pieter A1 - Yu, Sung-Huan A1 - Crabbé, Aurélie A1 - Förstner, Konrad U. A1 - Malfroot, Anne A1 - Cornelis, Pierre A1 - Van Houdt, Rob T1 - Effect of Shear Stress on Pseudomonas aeruginosa Isolated from the Cystic Fibrosis Lung JF - mBio N2 - Chronic colonization of the lungs by Pseudomonas aeruginosa is one of the major causes of morbidity and mortality in cystic fibrosis (CF) patients. To gain insights into the characteristic biofilm phenotype of P. aeruginosa in the CF lungs, mimicking the CF lung environment is critical. We previously showed that growth of the non-CF-adapted P. aeruginosa PAO1 strain in a rotating wall vessel, a device that simulates the low fluid shear (LS) conditions present in the CF lung, leads to the formation of in-suspension, self-aggregating biofilms. In the present study, we determined the phenotypic and transcriptomic changes associated with the growth of a highly adapted, transmissible P. aeruginosa CF strain in artificial sputum medium under LS conditions. Robust self-aggregating biofilms were observed only under LS conditions. Growth under LS conditions resulted in the upregulation of genes involved in stress response, alginate biosynthesis, denitrification, glycine betaine biosynthesis, glycerol metabolism, and cell shape maintenance, while genes involved in phenazine biosynthesis, type VI secretion, and multidrug efflux were downregulated. In addition, a number of small RNAs appeared to be involved in the response to shear stress. Finally, quorum sensing was found to be slightly but significantly affected by shear stress, resulting in higher production of autoinducer molecules during growth under high fluid shear (HS) conditions. In summary, our study revealed a way to modulate the behavior of a highly adapted P. aeruginosa CF strain by means of introducing shear stress, driving it from a biofilm lifestyle to a more planktonic lifestyle. KW - biology Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165821 VL - 7 IS - 4 ER - TY - JOUR A1 - Ashraf, Kerolos A1 - Yasrebi, Kaveh A1 - Hertlein, Tobias A1 - Ohlsen, Knut A1 - Lalk, Michael A1 - Hilgeroth, Andreas T1 - Novel effective small-molecule antibacterials against \(Enterococcus\) strains JF - Molecules N2 - \(Enterococcus\) species cause increasing numbers of infections in hospitals. They contribute to the increasing mortality rates, mostly in patients with comorbidities, who suffer from severe diseases. \(Enterococcus\) resistances against most antibiotics have been described, including novel antibiotics. Therefore, there is an ongoing demand for novel types of antibiotics that may overcome bacterial resistances. We discovered a novel class of antibiotics resulting from a simple one-pot reaction of indole and \(o\)-phthaldialdehyde. Differently substituted indolyl benzocarbazoles were yielded. Both the indole substitution and the positioning at the molecular scaffold influence the antibacterial activity towards the various strains of \(Enterococcus\) species with the highest relevance to nosocomial infections. Structure-activity relationships are discussed, and the first lead compounds were identified as also being effective in the case of a vancomycin resistance. KW - medicine KW - antibacterial activity KW - synthesis KW - derivatives KW - structure-activity KW - lead structure Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172628 VL - 22 IS - 12 ER - TY - JOUR A1 - Hassan, Musa A. A1 - Vasquez, Juan J. A1 - Guo-Liang, Chew A1 - Meissner, Markus A1 - Siegel, T. Nicolai T1 - Comparative ribosome profiling uncovers a dominant role for translational control in \(Toxoplasma\) \(gondii\) JF - BMC Genomics N2 - Background The lytic cycle of the protozoan parasite \(Toxoplasma\) \(gondii\), which involves a brief sojourn in the extracellular space, is characterized by defined transcriptional profiles. For an obligate intracellular parasite that is shielded from the cytosolic host immune factors by a parasitophorous vacuole, the brief entry into the extracellular space is likely to exert enormous stress. Due to its role in cellular stress response, we hypothesize that translational control plays an important role in regulating gene expression in \(Toxoplasma\) during the lytic cycle. Unlike transcriptional profiles, insights into genome-wide translational profiles of \(Toxoplasma\) \(gondii\) are lacking. Methods We have performed genome-wide ribosome profiling, coupled with high throughput RNA sequencing, in intracellular and extracellular \(Toxoplasma\) \(gondii\) parasites to investigate translational control during the lytic cycle. Results Although differences in transcript abundance were mostly mirrored at the translational level, we observed significant differences in the abundance of ribosome footprints between the two parasite stages. Furthermore, our data suggest that mRNA translation in the parasite is potentially regulated by mRNA secondary structure and upstream open reading frames. Conclusion We show that most of the \(Toxoplasma\) genes that are dysregulated during the lytic cycle are translationally regulated. KW - Biology KW - Ribosome profiling KW - RNA-sequencing KW - Translation efficiency KW - Toxoplasma gondii KW - Apicomplexan Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172376 VL - 18 ER - TY - JOUR A1 - Dugar, Gaurav A1 - Svensson, Sarah L. A1 - Bischler, Thorsten A1 - Waldchen, Sina A1 - Reinhardt, Richard A1 - Sauer, Markus A1 - Sharma, Cynthia M. T1 - The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni JF - Nature Communications N2 - The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA. KW - bacterial genetics KW - cell signalling KW - translation KW - Campylobacter jejuni Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173201 VL - 7 ER - TY - JOUR A1 - Selle, Martina A1 - Hertlein, Tobias A1 - Oesterreich, Babett A1 - Klemm, Theresa A1 - Kloppot, Peggy A1 - Müller, Elke A1 - Ehricht, Ralf A1 - Stentzel, Sebastian A1 - Bröker, Barbara M. A1 - Engelmann, Susanne A1 - Ohlsen, Knut T1 - Global antibody response to Staphylococcus aureus live-cell vaccination JF - Scientific Reports N2 - The pathogen Staphylococcus aureus causes a broad range of severe diseases and is feared for its ability to rapidly develop resistance to antibiotic substances. The increasing number of highly resistant S. aureus infections has accelerated the search for alternative treatment options to close the widening gap in anti-S. aureus therapy. This study analyses the humoral immune response to vaccination of Balb/c mice with sublethal doses of live S. aureus. The elicited antibody pattern in the sera of intravenously and intramuscularly vaccinated mice was determined using of a recently developed protein array. We observed a specific antibody response against a broad set of S. aureus antigens which was stronger following i.v. than i.m. vaccination. Intravenous but not intramuscular vaccination protected mice against an intramuscular challenge infection with a high bacterial dose. Vaccine protection was correlated with the strength of the anti-S. aureus antibody response. This study identified novel vaccine candidates by using protein microarrays as an effective tool and showed that successful vaccination against S. aureus relies on the optimal route of administration. KW - pathogens KW - bacterial infection KW - cell vaccines KW - Staphylococcus aureus Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-181245 VL - 6 ER - TY - JOUR A1 - Michaux, Charlotte A1 - Hansen, Elisabeth E. A1 - Jenniches, Laura A1 - Gerovac, Milan A1 - Barquist, Lars A1 - Vogel, Jörg T1 - Single-Nucleotide RNA Maps for the Two Major Nosocomial Pathogens Enterococcus faecalis and Enterococcus faecium JF - Frontiers in Cellular and Infection Microbiology N2 - Enterococcus faecalis and faecium are two major representative clinical strains of the Enterococcus genus and are sadly notorious to be part of the top agents responsible for nosocomial infections. Despite their critical implication in worldwide public healthcare, essential and available resources such as deep transcriptome annotations remain poor, which also limits our understanding of post-transcriptional control small regulatory RNA (sRNA) functions in these bacteria. Here, using the dRNA-seq technique in combination with ANNOgesic analysis, we successfully mapped and annotated transcription start sites (TSS) of both E. faecalis V583 and E. faecium AUS0004 at single nucleotide resolution. Analyzing bacteria in late exponential phase, we capture ~40% (E. faecalis) and 43% (E. faecium) of the annotated protein-coding genes, determine 5′ and 3′ UTR (untranslated region) length, and detect instances of leaderless mRNAs. The transcriptome maps revealed sRNA candidates in both bacteria, some found in previous studies and new ones. Expression of candidate sRNAs is being confirmed under biologically relevant environmental conditions. This comprehensive global TSS mapping atlas provides a valuable resource for RNA biology and gene expression analysis in the Enterococci. It can be accessed online at www.helmholtz-hiri.de/en/datasets/enterococcus through an instance of the genomic viewer JBrowse. KW - transcription start sites KW - RNA-seq KW - sRNA atlas KW - Gram-positive bacteria KW - post-transcriptional regulation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-217947 SN - 2235-2988 VL - 10 ER - TY - JOUR A1 - Ene, Iuliana V. A1 - Lohse, Matthew B. A1 - Vladu, Adrian V. A1 - Morschhäuser, Joachim A1 - Johnson, Alexander D. A1 - Bennett, Richard J. T1 - Phenotypic Profiling Reveals that Candida albicans Opaque Cells Represent a Metabolically Specialized Cell State Compared to Default White Cells JF - mBio N2 - The white-opaque switch is a bistable, epigenetic transition affecting multiple traits in Candida albicans including mating, immunogenicity, and niche specificity. To compare how the two cell states respond to external cues, we examined the fitness, phenotypic switching, and filamentation properties of white cells and opaque cells under 1,440 different conditions at 25°C and 37°C. We demonstrate that white and opaque cells display striking differences in their integration of metabolic and thermal cues, so that the two states exhibit optimal fitness under distinct conditions. White cells were fitter than opaque cells under a wide range of environmental conditions, including growth at various pHs and in the presence of chemical stresses or antifungal drugs. This difference was exacerbated at 37°C, consistent with white cells being the default state of C. albicans in the mammalian host. In contrast, opaque cells showed greater fitness than white cells under select nutritional conditions, including growth on diverse peptides at 25°C. We further demonstrate that filamentation is significantly rewired between the two states, with white and opaque cells undergoing filamentous growth in response to distinct external cues. Genetic analysis was used to identify signaling pathways impacting the white-opaque transition both in vitro and in a murine model of commensal colonization, and three sugar sensing pathways are revealed as regulators of the switch. Together, these findings establish that white and opaque cells are programmed for differential integration of metabolic and thermal cues and that opaque cells represent a more metabolically specialized cell state than the default white state. KW - biology Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165818 VL - 7 IS - 6 ER - TY - JOUR A1 - Westermann, Alexander J. A1 - Barquist, Lars A1 - Vogel, Jörg T1 - Resolving host-pathogen interactions by dual RNA-seq JF - PLoS Pathogens N2 - The transcriptome is a powerful proxy for the physiological state of a cell, healthy or diseased. As a result, transcriptome analysis has become a key tool in understanding the molecular changes that accompany bacterial infections of eukaryotic cells. Until recently, such transcriptomic studies have been technically limited to analyzing mRNA expression changes in either the bacterial pathogen or the infected eukaryotic host cell. However, the increasing sensitivity of high-throughput RNA sequencing now enables “dual RNA-seq” studies, simultaneously capturing all classes of coding and noncoding transcripts in both the pathogen and the host. In the five years since the concept of dual RNA-seq was introduced, the technique has been applied to a range of infection models. This has not only led to a better understanding of the physiological changes in pathogen and host during the course of an infection but has also revealed hidden molecular phenotypes of virulence-associated small noncoding RNAs that were not visible in standard infection assays. Here, we use the knowledge gained from these recent studies to suggest experimental and computational guidelines for the design of future dual RNA-seq studies. We conclude this review by discussing prospective applications of the technique. KW - Medicine KW - RNA sequencing KW - Salmonellosis KW - Transcriptome analysis KW - Gene expression KW - Bacterial pathogens KW - Salmonella KW - Host cells KW - Lysis (medicine) Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171921 VL - 13 IS - 2 ER - TY - JOUR A1 - Balasubramanian, Srikkanth A1 - Othman, Eman M. A1 - Kampik, Daniel A1 - Stopper, Helga A1 - Hentschel, Ute A1 - Ziebuhr, Wilma A1 - Oelschlaeger, Tobias A. A1 - Abdelmohsen, Usama R. T1 - Marine sponge-derived Streptomyces sp SBT343 extract inhibits staphylococcal biofilm formation JF - Frontiers in Microbiology N2 - Staphylococcus epidermidis and Staphylococcus aureus are opportunistic pathogens that cause nosocomial and chronic biofilm-associated infections. Indwelling medical devices and contact lenses are ideal ecological niches for formation of staphylococcal biofilms. Bacteria within biofilms are known to display reduced susceptibilities to antimicrobials and are protected from the host immune system. High rates of acquired antibiotic resistances in staphylococci and other biofilm-forming bacteria further hamper treatment options and highlight the need for new anti-biofilm strategies. Here, we aimed to evaluate the potential of marine sponge-derived actinomycetes in inhibiting biofilm formation of several strains of S. epidermidis, S. aureus, and Pseudomonas aeruginosa. Results from in vitro biofilm-formation assays, as well as scanning electron and confocal microscopy, revealed that an organic extract derived from the marine sponge-associated bacterium Streptomyces sp. SBT343 significantly inhibited staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces, without affecting bacterial growth. The extract also displayed similar antagonistic effects towards the biofilm formation of other S. epidermidis and S. aureus strains tested but had no inhibitory effects towards Pseudomonas biofilms. Interestingly the extract, at lower effective concentrations, did not exhibit cytotoxic effects on mouse fibroblast, macrophage and human corneal epithelial cell lines. Chemical analysis by High Resolution Fourier Transform Mass Spectrometry (HRMS) of the Streptomyces sp. SBT343 extract proportion revealed its chemical richness and complexity. Preliminary physico-chemical characterization of the extract highlighted the heat-stable and non-proteinaceous nature of the active component(s). The combined data suggest that the Streptomyces sp. SBT343 extract selectively inhibits staphylococcal biofilm formation without interfering with bacterial cell viability. Due to absence of cell toxicity, the extract might represent a good starting material to develop a future remedy to block staphylococcal biofilm formation on contact lenses and thereby to prevent intractable contact lens-mediated ocular infections. KW - medicine KW - marine sponges KW - actinomycetes KW - Streptomyces KW - staphilococci KW - biofilms KW - contact lens Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171844 VL - 8 ER - TY - JOUR A1 - Mietrach, Nicole A1 - Schlosser, Andreas A1 - Geibel, Sebastian T1 - An extracellular domain of the EsaA membrane component of the type VIIb secretion system: expression, purification and crystallization JF - Acta Crystallographica Section F N2 - The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48 kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α‐helices with a melting point of 34.5°C. Size‐exclusion chromatography combined with multi‐angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedral crystals were grown in 0.2 M ammonium citrate tribasic pH 7.0, 16% PEG 3350 using the hanging‐drop vapor‐diffusion method. Diffraction data were analyzed to 4.0 Å resolution, showing that the crystals belonged to the enantiomorphic tetragonal space groups P41212 or P43212, with unit‐cell parameters a = 197.5, b = 197.5, c = 368.3 Å, α = β = γ = 90°. KW - ESAT‐6‐like secretion system KW - ESS KW - type VII secretion system KW - EsaA KW - extracellular domain KW - Staphylococcus aureus USA300 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213681 VL - 75 IS - 12 ER - TY - JOUR A1 - Barquist, Lars A1 - Mayho, Matthew A1 - Cummins, Carla A1 - Cain, Amy K. A1 - Boinett, Christine J. A1 - Page, Andrew J. A1 - Langridge, Gemma C. A1 - Quail, Michael A. A1 - Keane, Jacqueline A. A1 - Parkhill, Julian T1 - The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries JF - Bioinformatics N2 - Transposon insertion sequencing is a high-throughput technique for assaying large libraries of otherwise isogenic transposon mutants providing insight into gene essentiality, gene function and genetic interactions. We previously developed the Transposon Directed Insertion Sequencing (TraDIS) protocol for this purpose, which utilizes shearing of genomic DNA followed by specific PCR amplification of transposon-containing fragments and Illumina sequencing. Here we describe an optimized high-yield library preparation and sequencing protocol for TraDIS experiments and a novel software pipeline for analysis of the resulting data. The Bio-Tradis analysis pipeline is implemented as an extensible Perl library which can either be used as is, or as a basis for the development of more advanced analysis tools. This article can serve as a general reference for the application of the TraDIS methodology. KW - mechanisms KW - Transposon insertion sequencing KW - sequencing protocol KW - TraDIS Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-189667 VL - 32 IS - 7 ER - TY - JOUR A1 - Blättner, Sebastian A1 - Das, Sudip A1 - Paprotka, Kerstin A1 - Eilers, Ursula A1 - Krischke, Markus A1 - Kretschmer, Dorothee A1 - Remmele, Christian W. A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Schuelein-Voelk, Christina A1 - Hertlein, Tobias A1 - Mueller, Martin J. A1 - Huettel, Bruno A1 - Reinhardt, Richard A1 - Ohlsen, Knut A1 - Rudel, Thomas A1 - Fraunholz, Martin J. T1 - Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes JF - PLoS Pathogens N2 - Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection. KW - cell death KW - cytotoxicity KW - Staphylococcus aureus KW - host cells KW - neutrophils KW - macrophages KW - transposable elements KW - epithelial cells Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-180380 VL - 12 IS - 9 ER - TY - JOUR A1 - Fröhlich, Kathrin S. A1 - Haneke, Katharina A1 - Papenfort, Kai A1 - Vogel, Jörg T1 - The target spectrum of SdsR small RNA in Salmonella JF - Nucleic Acids Research N2 - Model enteric bacteria such as Escherichia coli and Salmonella enterica express hundreds of small non-coding RNAs (sRNAs), targets for most of which are yet unknown. Some sRNAs are remarkably well conserved, indicating that they serve cellular functions that go beyond the necessities of a single species. One of these ‘core sRNAs’ of largely unknown function is the abundant ∼100-nucleotide SdsR sRNA which is transcribed by the general stress σ-factor, σ\(^{S}\) and accumulates in stationary phase. In Salmonella, SdsR was known to inhibit the synthesis of the species-specific porin, OmpD. However, sdsR genes are present in almost all enterobacterial genomes, suggesting that additional, conserved targets of this sRNA must exist. Here, we have combined SdsR pulse-expression with whole genome transcriptomics to discover 20 previously unknown candidate targets of SdsR which include mRNAs coding for physiologically important regulators such as the carbon utilization regulator, CRP, the nucleoid-associated chaperone, StpA and the antibiotic resistance transporter, TolC. Processing of SdsR by RNase E results in two cellular SdsR variants with distinct target spectra. While the overall physiological role of this orphan core sRNA remains to be fully understood, the new SdsR targets present valuable leads to determine sRNA functions in resting bacteria. KW - sRNA KW - Salmonella enterica KW - SdsR Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175365 VL - 44 IS - 21 ER - TY - THES A1 - Hör, Jens T1 - Discovery of RNA/protein complexes by Grad-seq T1 - Ermittlung von RNA/Protein-Komplexen mittels Grad-seq N2 - Complex formation between macromolecules constitutes the foundation of most cellular processes. Most known complexes are made up of two or more proteins interacting in order to build a functional entity and therefore enabling activities which the single proteins could otherwise not fulfill. With the increasing knowledge about noncoding RNAs (ncRNAs) it has become evident that, similar to proteins, many of them also need to form a complex to be functional. This functionalization is usually executed by specific or global RNA-binding proteins (RBPs) that are specialized binders of a certain class of ncRNAs. For instance, the enterobacterial global RBPs Hfq and ProQ together bind >80 % of the known small regulatory RNAs (sRNAs), a class of ncRNAs involved in post-transcriptional regulation of gene expression. However, identification of RNA-protein interactions so far was performed individually by employing low-throughput biochemical methods and thereby hindered the discovery of such interactions, especially in less studied organisms such as Gram-positive bacteria. Using gradient profiling by sequencing (Grad-seq), the present thesis aimed to establish high-throughput, global RNA/protein complexome resources for Escherichia coli and Streptococcus pneumoniae in order to provide a new way to investigate RNA-protein as well as protein-protein interactions in these two important model organisms. In E. coli, Grad-seq revealed the sedimentation profiles of 4,095 (∼85 % of total) transcripts and 2,145 (∼49 % of total) proteins and with that reproduced its major ribonucleoprotein particles. Detailed analysis of the in-gradient distribution of the RNA and protein content uncovered two functionally unknown molecules—the ncRNA RyeG and the small protein YggL—to be ribosomeassociated. Characterization of RyeG revealed it to encode for a 48 aa long, toxic protein that drastically increases lag times when overexpressed. YggL was shown to be bound by the 50S subunit of the 70S ribosome, possibly indicating involvement of YggL in ribosome biogenesis or translation of specific mRNAs. S. pneumoniae Grad-seq detected 2,240 (∼88 % of total) transcripts and 1,301 (∼62 % of total) proteins, whose gradient migration patterns were successfully reconstructed, and thereby represents the first RNA/protein complexome resource of a Gram-positive organism. The dataset readily verified many conserved major complexes for the first time in S. pneumoniae and led to the discovery of a specific interaction between the 3’!5’ exonuclease Cbf1 and the competence-regulating ciadependent sRNAs (csRNAs). Unexpectedly, trimming of the csRNAs by Cbf1 stabilized the former, thereby promoting their inhibitory function. cbf1 was further shown to be part of the late competence genes and as such to act as a negative regulator of competence. N2 - Makromoleküle, die Komplexe bilden, sind die Grundlage der meisten zellulären Prozesse. Die meisten bekannten Komplexe bestehen aus zwei oder mehr Proteinen, die interagieren, um eine funktionelle Einheit zu bilden. Diese Interaktionen ermöglichen Funktionen, die die einzelnen Proteine nicht erfüllen könnten. Wachsende wissenschaftliche Erkenntnisse über nichtkodierende RNAs (ncRNAs) haben gezeigt, dass, analog zu Proteinen, auch viele ncRNAs Komplexe bilden müssen, um ihre Funktionen ausüben zu können. Diese Funktionalisierung wird normalerweise von spezifischen oder globalen RNA-bindenden Proteinen (RBPs), die auf eine bestimmte Klasse an ncRNAs spezialisiert sind, durchgeführt. So binden beispielsweise die in Enterobakterien verbreiteten globalen RBPs Hfq und ProQ zusammen >80 % der bekannten kleinen regulatorischen RNAs (sRNAs)—eine Klasse der ncRNAs, die in die posttranskriptionelle Genexpressionsregulation involviert ist. RNA-Protein-Interaktionen wurden bisher anhand einzelner Moleküle und mithilfe von biochemischen Methoden mit niedrigem Durchsatz identifiziert, was die Entdeckung solcher Interaktionen erschwert hat. Dies gilt insbesondere für Organismen, die seltener Gegenstand der Forschung sind, wie beispielsweise grampositive Bakterien. Das Ziel dieser Doktorarbeit war es, mittels gradient profiling by sequencing (Grad-seq) globale Hochdurchsatzkomplexomdatensätze der RNA-ProteinInteraktionen in Escherichia coli und Streptococcus pneumoniae zu generieren. Diese Datensätze ermöglichen es auf eine neue Art und Weise RNA-Protein- und ProteinProtein-Interaktionen in diesen wichtigen Modellorganismen zu untersuchen. Die E. coli Grad-seq-Daten beinhalten die Sedimentationsprofile von 4095 Transkripten (∼85 % des Transkriptoms) und 2145 Proteinen (∼49 % des Proteoms), mit denen die wichtigsten Ribonukleoproteine reproduziert werden konnten. Die detaillierte Analyse der Verteilung von RNAs und Proteinen im Gradienten zeigte, dass zwei Moleküle, deren Funktionen bisher unbekannt waren—die ncRNA RyeG und das kleine Protein YggL—ribosomenassoziiert sind. Durch weitere Charakterisierung konnte gezeigt werden, dass RyeG für ein toxisches Protein mit einer Länge von 48 Aminosäuren kodiert, das bei Überexpression die Latenzphase drastisch verlängert. Für YggL konnte eine Interaktion mit der 50S Untereinheit von 70S Ribosomen nachgewiesen werden, was auf eine potenzielle Funktion in der Biogenese von Ribosomen oder bei der Translation bestimmter mRNAs hindeutet. Die S. pneumoniae Grad-seq Daten beinhalten 2240 Transkripte (∼88 % des Transkriptoms) und 1301 Proteine (∼62 % des Proteoms), deren Migrationsprofile im Gradienten erfolgreich rekonstruiert werden konnten. Dieser RNA/ProteinKomplexomdatensatz eines grampositiven Organismus ermöglichte erstmalig die Verifizierung der wichtigsten konservierten Komplexe von S. pneumoniae. Weiterhin konnte eine spezifische Interaktion der 3’!5’-Exonuklease Cbf1 mit den ciadependent sRNAs (csRNAs), die an der Regulation von Kompetenz beteiligt sind, nachgewiesen werden. Überraschenderweise stabilisiert das von Cbf1 durchgeführte Kürzen der csRNAs die selbigen, was deren inhibitorische Funktion unterstützt. Darüber hinaus konnte gezeigt werden, dass cbf1 eines der späten Kompetenzgene ist und als solches als negativer Regulator der Kompetenz agiert. KW - Multiproteinkomplex KW - RNS-Bindungsproteine KW - RNS KW - Escherichia coli KW - Streptococcus pneumoniae KW - Complexome KW - RNA-binding protein KW - RNA KW - Escherichia coli KW - Streptococcus pneumoniae KW - Grad-seq KW - Bacteria Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-211811 ER - TY - JOUR A1 - Bar-Yosef, Hagit A1 - Gildor, Tsvia A1 - Ramírez-Zavala, Bernardo A1 - Schmauch, Christian A1 - Weissman, Ziva A1 - Pinsky, Mariel A1 - Naddaf, Rawi A1 - Morschhäuser, Joachim A1 - Arkowitz, Robert A. A1 - Kornitzer, Daniel T1 - A global analysis of kinase function in Candida albicans hyphal morphogenesis reveals a role for the endocytosis regulator Akl1 JF - Frontiers in Cellular and Infection Microbiology N2 - The human pathogenic fungus Candida albicans can switch between yeast and hyphal morphologies as a function of environmental conditions and cellular physiology. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. In order to identify possible inhibitory pathways of the yeast-to-hyphae transition, we interrogated a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TETon promoter. Proportionately more phosphatases than kinases were identified that inhibited hyphal morphogenesis, consistent with the known role of protein phosphorylation in hyphal induction. Among the kinases, we identified AKL1 as a gene that significantly suppressed hyphal morphogenesis in serum. Akl1 specifically affected hyphal elongation rather than initiation: overexpression of AKL1 repressed hyphal growth, and deletion of AKL1 resulted in acceleration of the rate of hyphal elongation. Akl1 suppressed fluid-phase endocytosis, probably via Pan1, a putative clathrin-mediated endocytosis scaffolding protein. In the absence of Akl1, the Pan1 patches were delocalized from the sub-apical region, and fluid-phase endocytosis was intensified. These results underscore the requirement of an active endocytic pathway for hyphal morphogenesis. Furthermore, these results suggest that under standard conditions, endocytosis is rate-limiting for hyphal elongation. KW - hyphae KW - endocytosis KW - Pan1 KW - functional genomics Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197204 SN - 2235-2988 VL - 8 ER - TY - JOUR A1 - Papenfort, Kai A1 - Vogel, Jörg T1 - Small RNA functions in carbon metabolism and virulence of enteric pathogens JF - Frontiers in Cellular and Infection Microbiology N2 - Enteric pathogens often cycle between virulent and saprophytic lifestyles. To endure these frequent changes in nutrient availability and composition bacteria possess an arsenal of regulatory and metabolic genes allowing rapid adaptation and high flexibility. While numerous proteins have been characterized with regard to metabolic control in pathogenic bacteria, small non-coding RNAs have emerged as additional regulators of metabolism. Recent advances in sequencing technology have vastly increased the number of candidate regulatory RNAs and several of them have been found to act at the interface of bacterial metabolism and virulence factor expression. Importantly, studying these riboregulators has not only provided insight into their metabolic control functions but also revealed new mechanisms of post-transcriptional gene control. This review will focus on the recent advances in this area of host-microbe interaction and discuss how regulatory small RNAs may help coordinate metabolism and virulence of enteric pathogens. KW - sRNA KW - carbon metabolism KW - Hfq KW - CsrA KW - virulence Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197520 SN - 2235-2988 VL - 4 IS - 91 ER - TY - JOUR A1 - Wheeler, Nicole E. A1 - Barquist, Lars A1 - Kingsley, Robert A. A1 - Gardner, Paul P. T1 - A profile-based method for identifying functional divergence of orthologous genes in bacterial genomes JF - Bioinformatics N2 - Motivation: Next generation sequencing technologies have provided us with a wealth of information on genetic variation, but predi cting the functional significance of this variation is a difficult task. While many comparative genomics studies have focused on gene flux and large scale changes, relatively little attention has been paid to quantifying the effects of single nucleotide polymorphisms and indels on protein function, particularly in bacterial genomics. Results: We present a hidden Markov model based approach we call delta-bitscore (DBS) for identifying orthologous proteins that have diverged at the amino acid sequence level in a way that is likely to impact biological function. We benchmark this approach with several widely used datasets and apply it to a proof-of-concept study of orthologous proteomes in an investigation of host adaptation in Salmonella enterica. We highlight the value of the method in identifying functional divergence of genes, and suggest that this tool may be a better approach than the commonly used dN/dS metric for identifying functionally significant genetic changes occurring in recently diverged organisms. KW - Host adaptation KW - Salmonella-enteritidis KW - Sequence identity KW - Rapid evolution KW - Variants KW - Cystic-fibriosis KW - Strains KW - Pathogenicity KW - Typhimurium KW - Yersinia Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-186502 VL - 32 IS - 23 ER - TY - JOUR A1 - Mottola, Austin A1 - Morschhäuser, Joachim T1 - An intragenic recombination event generates a Snf4-independent form of the essential protein kinase SNF1 in Candida albicans JF - mSphere N2 - The heterotrimeric protein kinase SNF1 plays a key role in the metabolic adaptation of the pathogenic yeast Candida albicans. It consists of the essential catalytic α-subunit Snf1, the γ-subunit Snf4, and one of the two β-subunits Kis1 and Kis2. Snf4 is required to release the N-terminal catalytic domain of Snf1 from autoinhibition by the C-terminal regulatory domain, and snf4Δ mutants cannot grow on carbon sources other than glucose. In a screen for suppressor mutations that restore growth of a snf4Δ mutant on alternative carbon sources, we isolated a mutant in which six amino acids between the N-terminal kinase domain and the C-terminal regulatory domain of Snf1 were deleted. The deletion was caused by an intragenic recombination event between two 8-bp direct repeats flanking six intervening codons. In contrast to truncated forms of Snf1 that contain only the kinase domain, the Snf4-independent Snf1\(^{Δ311 − 316}\) was fully functional and could replace wild-type Snf1 for normal growth, because it retained the ability to interact with the Kis1 and Kis2 β-subunits via its C-terminal domain. Indeed, the Snf4-independent Snf1\(^{Δ311 − 316}\) still required the β-subunits of the SNF1 complex to perform its functions and did not rescue the growth defects of kis1Δ mutants. Our results demonstrate that a preprogrammed in-frame deletion event within the SNF1 coding region can generate a mutated form of this essential kinase which abolishes autoinhibition and thereby overcomes growth deficiencies caused by a defect in the γ-subunit Snf4. KW - AMP-activated kinases KW - Candida albicans KW - genetic recombination KW - metabolic adaptation KW - suppressor mutation Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202170 VL - 4 IS - 3 ER - TY - JOUR A1 - Popp, Christina A1 - Ramírez-Zavala, Bernardo A1 - Schwanfelder, Sonja A1 - Krüger, Ines A1 - Morschhäuser, Joachim T1 - Evolution of fluconazole-resistant Candida albicans strains by drug-induced mating competence and parasexual recombination JF - mBio N2 - The clonal population structure of Candida albicans suggests that (para)sexual recombination does not play an important role in the lifestyle of this opportunistic fungal pathogen, an assumption that is strengthened by the fact that most C. albicans strains are heterozygous at the mating type locus (MTL) and therefore mating-incompetent. On the other hand, mating might occur within clonal populations and allow the combination of advantageous traits that were acquired by individual cells to adapt to adverse conditions. We have investigated if parasexual recombination may be involved in the evolution of highly drug-resistant strains exhibiting multiple resistance mechanisms against fluconazole, an antifungal drug that is commonly used to treat infections by C. albicans. Growth of strains that were heterozygous for MTL and different fluconazole resistance mutations in the presence of the drug resulted in the emergence of derivatives that had become homozygous for the mutated allele and the mating type locus and exhibited increased drug resistance. When MTLa/a and MTLα/α cells of these strains were mixed in all possible combinations, we could isolate mating products containing the genetic material from both parents. The initial mating products did not exhibit higher drug resistance than their parental strains, but further propagation under selective pressure resulted in the loss of the wild-type alleles and increased fluconazole resistance. Therefore, fluconazole treatment not only selects for resistance mutations but also promotes genomic alterations that confer mating competence, which allows cells in an originally clonal population to exchange individually acquired resistance mechanisms and generate highly drug-resistant progeny. KW - Candida albicans KW - drug resistance evolution KW - mating KW - parasexual recombination Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200901 VL - 10 IS - 1 ER - TY - JOUR A1 - Jiang, Yuxiang A1 - Oron, Tal Ronnen A1 - Clark, Wyatt T. A1 - Bankapur, Asma R. A1 - D'Andrea, Daniel A1 - Lepore, Rosalba A1 - Funk, Christopher S. A1 - Kahanda, Indika A1 - Verspoor, Karin M. A1 - Ben-Hur, Asa A1 - Koo, Da Chen Emily A1 - Penfold-Brown, Duncan A1 - Shasha, Dennis A1 - Youngs, Noah A1 - Bonneau, Richard A1 - Lin, Alexandra A1 - Sahraeian, Sayed M. E. A1 - Martelli, Pier Luigi A1 - Profiti, Giuseppe A1 - Casadio, Rita A1 - Cao, Renzhi A1 - Zhong, Zhaolong A1 - Cheng, Jianlin A1 - Altenhoff, Adrian A1 - Skunca, Nives A1 - Dessimoz, Christophe A1 - Dogan, Tunca A1 - Hakala, Kai A1 - Kaewphan, Suwisa A1 - Mehryary, Farrokh A1 - Salakoski, Tapio A1 - Ginter, Filip A1 - Fang, Hai A1 - Smithers, Ben A1 - Oates, Matt A1 - Gough, Julian A1 - Törönen, Petri A1 - Koskinen, Patrik A1 - Holm, Liisa A1 - Chen, Ching-Tai A1 - Hsu, Wen-Lian A1 - Bryson, Kevin A1 - Cozzetto, Domenico A1 - Minneci, Federico A1 - Jones, David T. A1 - Chapman, Samuel A1 - BKC, Dukka A1 - Khan, Ishita K. A1 - Kihara, Daisuke A1 - Ofer, Dan A1 - Rappoport, Nadav A1 - Stern, Amos A1 - Cibrian-Uhalte, Elena A1 - Denny, Paul A1 - Foulger, Rebecca E. A1 - Hieta, Reija A1 - Legge, Duncan A1 - Lovering, Ruth C. A1 - Magrane, Michele A1 - Melidoni, Anna N. A1 - Mutowo-Meullenet, Prudence A1 - Pichler, Klemens A1 - Shypitsyna, Aleksandra A1 - Li, Biao A1 - Zakeri, Pooya A1 - ElShal, Sarah A1 - Tranchevent, Léon-Charles A1 - Das, Sayoni A1 - Dawson, Natalie L. A1 - Lee, David A1 - Lees, Jonathan G. A1 - Sillitoe, Ian A1 - Bhat, Prajwal A1 - Nepusz, Tamás A1 - Romero, Alfonso E. A1 - Sasidharan, Rajkumar A1 - Yang, Haixuan A1 - Paccanaro, Alberto A1 - Gillis, Jesse A1 - Sedeño-Cortés, Adriana E. A1 - Pavlidis, Paul A1 - Feng, Shou A1 - Cejuela, Juan M. A1 - Goldberg, Tatyana A1 - Hamp, Tobias A1 - Richter, Lothar A1 - Salamov, Asaf A1 - Gabaldon, Toni A1 - Marcet-Houben, Marina A1 - Supek, Fran A1 - Gong, Qingtian A1 - Ning, Wei A1 - Zhou, Yuanpeng A1 - Tian, Weidong A1 - Falda, Marco A1 - Fontana, Paolo A1 - Lavezzo, Enrico A1 - Toppo, Stefano A1 - Ferrari, Carlo A1 - Giollo, Manuel A1 - Piovesan, Damiano A1 - Tosatto, Silvio C. E. A1 - del Pozo, Angela A1 - Fernández, José M. A1 - Maietta, Paolo A1 - Valencia, Alfonso A1 - Tress, Michael L. A1 - Benso, Alfredo A1 - Di Carlo, Stefano A1 - Politano, Gianfranco A1 - Savino, Alessandro A1 - Rehman, Hafeez Ur A1 - Re, Matteo A1 - Mesiti, Marco A1 - Valentini, Giorgio A1 - Bargsten, Joachim W. A1 - van Dijk, Aalt D. J. A1 - Gemovic, Branislava A1 - Glisic, Sanja A1 - Perovic, Vladmir A1 - Veljkovic, Veljko A1 - Almeida-e-Silva, Danillo C. A1 - Vencio, Ricardo Z. N. A1 - Sharan, Malvika A1 - Vogel, Jörg A1 - Kansakar, Lakesh A1 - Zhang, Shanshan A1 - Vucetic, Slobodan A1 - Wang, Zheng A1 - Sternberg, Michael J. E. A1 - Wass, Mark N. A1 - Huntley, Rachael P. A1 - Martin, Maria J. A1 - O'Donovan, Claire A1 - Robinson, Peter N. A1 - Moreau, Yves A1 - Tramontano, Anna A1 - Babbitt, Patricia C. A1 - Brenner, Steven E. A1 - Linial, Michal A1 - Orengo, Christine A. A1 - Rost, Burkhard A1 - Greene, Casey S. A1 - Mooney, Sean D. A1 - Friedberg, Iddo A1 - Radivojac, Predrag A1 - Veljkovic, Nevena T1 - An expanded evaluation of protein function prediction methods shows an improvement in accuracy JF - Genome Biology N2 - Background A major bottleneck in our understanding of the molecular underpinnings of life is the assignment of function to proteins. While molecular experiments provide the most reliable annotation of proteins, their relatively low throughput and restricted purview have led to an increasing role for computational function prediction. However, assessing methods for protein function prediction and tracking progress in the field remain challenging. Results We conducted the second critical assessment of functional annotation (CAFA), a timed challenge to assess computational methods that automatically assign protein function. We evaluated 126 methods from 56 research groups for their ability to predict biological functions using Gene Ontology and gene-disease associations using Human Phenotype Ontology on a set of 3681 proteins from 18 species. CAFA2 featured expanded analysis compared with CAFA1, with regards to data set size, variety, and assessment metrics. To review progress in the field, the analysis compared the best methods from CAFA1 to those of CAFA2. Conclusions The top-performing methods in CAFA2 outperformed those from CAFA1. This increased accuracy can be attributed to a combination of the growing number of experimental annotations and improved methods for function prediction. The assessment also revealed that the definition of top-performing algorithms is ontology specific, that different performance metrics can be used to probe the nature of accurate predictions, and the relative diversity of predictions in the biological process and human phenotype ontologies. While there was methodological improvement between CAFA1 and CAFA2, the interpretation of results and usefulness of individual methods remain context-dependent. KW - Protein function prediction KW - Disease gene prioritization Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166293 VL - 17 IS - 184 ER - TY - JOUR A1 - Gomes, Sara F. Martins A1 - Westermann, Alexander J. A1 - Sauerwein, Till A1 - Hertlein, Tobias A1 - Förstner, Konrad U. A1 - Ohlsen, Knut A1 - Metzger, Marco A1 - Shusta, Eric V. A1 - Kim, Brandon J. A1 - Appelt-Menzel, Antje A1 - Schubert-Unkmeir, Alexandra T1 - Induced pluripotent stem cell-derived brain endothelial cells as a cellular model to study Neisseria meningitidis infection JF - Frontiers in Microbiology N2 - Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs. KW - Neisseria meningitidis KW - meningococcus KW - bacteria KW - stem cells KW - blood-cerebrospinal fluid barrier KW - blood-brain barrier KW - brain endothelial cells Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201562 VL - 10 IS - 1181 ER - TY - JOUR A1 - Weidner, Magdalena T. A1 - Lardenoije, Roy A1 - Eijssen, Lars A1 - Mogavero, Floriana A1 - De Groodt, Lilian P. M. T. A1 - Popp, Sandy A1 - Palme, Rupert A1 - Förstner, Konrad U. A1 - Strekalova, Tatyana A1 - Steinbusch, Harry W. M. A1 - Schmitt-Böhrer, Angelika G. A1 - Glennon, Jeffrey C. A1 - Waider, Jonas A1 - van den Hove, Daniel L. A. A1 - Lesch, Klaus-Peter T1 - Identification of cholecystokinin by genome-wide profiling as potential mediator of serotonin-dependent behavioral effects of maternal separation in the amygdala JF - Frontiers in Neuroscience N2 - Converging evidence suggests a role of serotonin (5-hydroxytryptamine, 5-HT) and tryptophan hydroxylase 2 (TPH2), the rate-limiting enzyme of 5-HT synthesis in the brain, in modulating long-term, neurobiological effects of early-life adversity. Here, we aimed at further elucidating the molecular mechanisms underlying this interaction, and its consequences for socio-emotional behaviors, with a focus on anxiety and social interaction. In this study, adult, male Tph2 null mutant (Tph2\(^{-/-}\)) and heterozygous (Tph2\(^{+/-}\)) mice, and their wildtype littermates (Tph2\(^{+/+}\)) were exposed to neonatal, maternal separation (MS) and screened for behavioral changes, followed by genome-wide RNA expression and DNA methylation profiling. In Tph2\(^{-/-}\) mice, brain 5-HT deficiency profoundly affected socio-emotional behaviors, i.e., decreased avoidance of the aversive open arms in the elevated plus-maze (EPM) as well as decreased prosocial and increased rule breaking behavior in the resident-intruder test when compared to their wildtype littermates. Tph2\(^{+/-}\) mice showed an ambiguous profile with context-dependent, behavioral responses. In the EPM they showed similar avoidance of the open arm but decreased prosocial and increased rule breaking behavior in the resident-intruder test when compared to their wildtype littermates. Notably, MS effects on behavior were subtle and depended on the Tph2 genotype, in particular increasing the observed avoidance of EPM open arms in wildtype and Tph2\(^{+/-}\) mice when compared to their Tph2\(^{-/-}\) littermates. On the genomic level, the interaction of Tph2 genotype with MS differentially affected the expression of numerous genes, of which a subset showed an overlap with DNA methylation profiles at corresponding loci. Remarkably, changes in methylation nearby and expression of the gene encoding cholecystokinin, which were inversely correlated to each other, were associated with variations in anxiety-related phenotypes. In conclusion, next to various behavioral alterations, we identified gene expression and DNA methylation profiles to be associated with TPH2 inactivation and its interaction with MS, suggesting a gene-by-environment interaction-dependent, modulatory function of brain 5-HT availability. KW - serotonin KW - maternal separation KW - mouse KW - emotional behavior KW - DNA methylation KW - RNA expression Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201340 VL - 13 ER - TY - JOUR A1 - Čuklina, Jelena A1 - Hahn, Julia A1 - Imakaev, Maxim A1 - Omasits, Ulrich A1 - Förstner, Konrad U. A1 - Ljubimov, Nikolay A1 - Goebel, Melanie A1 - Pessi, Gabriella A1 - Fischer, Hans-Martin A1 - Ahrens, Christian H. A1 - Gelfand, Mikhail S. A1 - Evguenieva-Hackenberg, Elena T1 - Genome-wide transcription start site mapping of Bradyrhizobium japonicum grown free-living or in symbiosis - a rich resource to identify new transcripts, proteins and to study gene regulation JF - BMC Genomics N2 - Background Differential RNA-sequencing (dRNA-seq) is indispensable for determination of primary transcriptomes. However, using dRNA-seq data to map transcriptional start sites (TSSs) and promoters genome-wide is a bioinformatics challenge. We performed dRNA-seq of Bradyrhizobium japonicum USDA 110, the nitrogen-fixing symbiont of soybean, and developed algorithms to map TSSs and promoters. Results A specialized machine learning procedure for TSS recognition allowed us to map 15,923 TSSs: 14,360 in free-living bacteria, 4329 in symbiosis with soybean and 2766 in both conditions. Further, we provide proteomic evidence for 4090 proteins, among them 107 proteins corresponding to new genes and 178 proteins with N-termini different from the existing annotation (72 and 109 of them with TSS support, respectively). Guided by proteomics evidence, previously identified TSSs and TSSs experimentally validated here, we assign a score threshold to flag 14 % of the mapped TSSs as a class of lower confidence. However, this class of lower confidence contains valid TSSs of low-abundant transcripts. Moreover, we developed a de novo algorithm to identify promoter motifs upstream of mapped TSSs, which is publicly available, and found motifs mainly used in symbiosis (similar to RpoN-dependent promoters) or under both conditions (similar to RpoD-dependent promoters). Mapped TSSs and putative promoters, proteomic evidence and updated gene annotation were combined into an annotation file. Conclusions The genome-wide TSS and promoter maps along with the extended genome annotation of B. japonicum represent a valuable resource for future systems biology studies and for detailed analyses of individual non-coding transcripts and ORFs. Our data will also provide new insights into bacterial gene regulation during the agriculturally important symbiosis between rhizobia and legumes. KW - Bradyrhizobium KW - RNA-seq KW - Promoter prediction KW - Genome re-annotation KW - Internal transcription start site KW - Nodule KW - Transcription start site KW - Proteogenomics KW - Antisense RNA Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164565 VL - 17 ER - TY - JOUR A1 - Babski, Julia A1 - Haas, Karina A. A1 - Näther-Schindler, Daniela A1 - Pfeiffer, Friedhelm A1 - Förstner, Konrad U. A1 - Hammelmann, Matthias A1 - Hilker, Rolf A1 - Becker, Anke A1 - Sharma, Cynthia M. A1 - Marchfelder, Anita A1 - Soppa, Jörg T1 - Genome-wide identification of transcriptional start sites in the haloarchaeon Haloferax volcanii based on differential RNA-Seq (dRNA-Seq) JF - BMC Genomics N2 - Background Differential RNA-Seq (dRNA-Seq) is a recently developed method of performing primary transcriptome analyses that allows for the genome-wide mapping of transcriptional start sites (TSSs) and the identification of novel transcripts. Although the transcriptomes of diverse bacterial species have been characterized by dRNA-Seq, the transcriptome analysis of archaeal species is still rather limited. Therefore, we used dRNA-Seq to characterize the primary transcriptome of the model archaeon Haloferax volcanii. Results Three independent cultures of Hfx. volcanii grown under optimal conditions to the mid-exponential growth phase were used to determine the primary transcriptome and map the 5′-ends of the transcripts. In total, 4749 potential TSSs were detected. A position weight matrix (PWM) was derived for the promoter predictions, and the results showed that 64 % of the TSSs were preceded by stringent or relaxed basal promoters. Of the identified TSSs, 1851 belonged to protein-coding genes. Thus, fewer than half (46 %) of the 4040 protein-coding genes were expressed under optimal growth conditions. Seventy-two percent of all protein-coding transcripts were leaderless, which emphasized that this pathway is the major pathway for translation initiation in haloarchaea. A total of 2898 of the TSSs belonged to potential non-coding RNAs, which accounted for an unexpectedly high fraction (61 %) of all transcripts. Most of the non-coding TSSs had not been previously described (2792) and represented novel sequences (59 % of all TSSs). A large fraction of the potential novel non-coding transcripts were cis-antisense RNAs (1244 aTSSs). A strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs was found, which suggested that the negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts corresponded to internal transcripts overlapping with mRNAs (1153 iTSSs) and intergenic small RNA (sRNA) candidates (395 TSSs). Conclusion This study provides a comprehensive map of the primary transcriptome of Hfx. volcanii grown under optimal conditions. Fewer than half of all protein-coding genes have been transcribed under these conditions. Unexpectedly, more than half of the detected TSSs belonged to several classes of non-coding RNAs. Thus, RNA-based regulation appears to play a more important role in haloarchaea than previously anticipated. KW - Archaea KW - dRNA-Seq KW - Promoter KW - Non-coding RNAs KW - sRNA KW - Haloferax volcanii KW - Transcriptome KW - Leaderless transcript KW - Antisense RNA Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164553 VL - 17 IS - 629 ER - TY - JOUR A1 - Heidrich, Nadja A1 - Bauriedl, Saskia A1 - Barquist, Lars A1 - Li, Lei A1 - Schoen, Christoph A1 - Vogel, Jörg T1 - The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq JF - Nucleic Acids Research N2 - Neisseria meningitidis is a human commensal that can also cause life-threatening meningitis and septicemia. Despite growing evidence for RNA-based regulation in meningococci, their transcriptome structure and output of regulatory small RNAs (sRNAs) are incompletely understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptome of N. meningitidis strain 8013. Annotation of 1625 transcriptional start sites defines transcription units for most protein-coding genes but also reveals a paucity of classical σ70-type promoters, suggesting the existence of activators that compensate for the lack of −35 consensus sequences in N. meningitidis. The transcriptome maps also reveal 65 candidate sRNAs, a third of which were validated by northern blot analysis. Immunoprecipitation with the RNA chaperone Hfq drafts an unexpectedly large post-transcriptional regulatory network in this organism, comprising 23 sRNAs and hundreds of potential mRNA targets. Based on this data, using a newly developed gfp reporter system we validate an Hfq-dependent mRNA repression of the putative colonization factor PrpB by the two trans-acting sRNAs RcoF1/2. Our genome-wide RNA compendium will allow for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx. KW - RNA KW - Neisseria meningitidis KW - dRNA-seq KW - transcriptome KW - RNA chaperone Hfq Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170828 VL - 45 IS - 10 ER - TY - JOUR A1 - García-Betancur, Juan-Carlos A1 - Goñi-Moreno, Angel A1 - Horger, Thomas A1 - Schott, Melanie A1 - Sharan, Malvika A1 - Eikmeier, Julian A1 - Wohlmuth, Barbara A1 - Zernecke, Alma A1 - Ohlsen, Knut A1 - Kuttler, Christina A1 - Lopez, Daniel T1 - Cell differentiation defines acute and chronic infection cell types in Staphylococcus aureus JF - eLife N2 - A central question to biology is how pathogenic bacteria initiate acute or chronic infections. Here we describe a genetic program for cell-fate decision in the opportunistic human pathogen Staphylococcus aureus, which generates the phenotypic bifurcation of the cells into two genetically identical but different cell types during the course of an infection. Whereas one cell type promotes the formation of biofilms that contribute to chronic infections, the second type is planktonic and produces the toxins that contribute to acute bacteremia. We identified a bimodal switch in the agr quorum sensing system that antagonistically regulates the differentiation of these two physiologically distinct cell types. We found that extracellular signals affect the behavior of the agr bimodal switch and modify the size of the specialized subpopulations in specific colonization niches. For instance, magnesium-enriched colonization niches causes magnesium binding to S. aureusteichoic acids and increases bacterial cell wall rigidity. This signal triggers a genetic program that ultimately downregulates the agr bimodal switch. Colonization niches with different magnesium concentrations influence the bimodal system activity, which defines a distinct ratio between these subpopulations; this in turn leads to distinct infection outcomes in vitro and in an in vivo murine infection model. Cell differentiation generates physiological heterogeneity in clonal bacterial infections and helps to determine the distinct infection types. KW - Staphylococcus aureus KW - infection KW - cell differentiation KW - pathogenic bacteria Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170346 VL - 6 IS - e28023 ER - TY - JOUR A1 - Mielich-Süss, Benjamin A1 - Wagner, Rabea M. A1 - Mietrach, Nicole A1 - Hertlein, Tobias A1 - Marincola, Gabriella A1 - Ohlsen, Knut A1 - Geibel, Sebastian A1 - Lopez, Daniel T1 - Flotillin scaffold activity contributes to type VII secretion system assembly in Staphylococcus aureus JF - PLoS Pathogens N2 - Scaffold proteins are ubiquitous chaperones that promote efficient interactions between partners of multi-enzymatic protein complexes; although they are well studied in eukaryotes, their role in prokaryotic systems is poorly understood. Bacterial membranes have functional membrane microdomains (FMM), a structure homologous to eukaryotic lipid rafts. Similar to their eukaryotic counterparts, bacterial FMM harbor a scaffold protein termed flotillin that is thought to promote interactions between proteins spatially confined to the FMM. Here we used biochemical approaches to define the scaffold activity of the flotillin homolog FloA of the human pathogen Staphylococcus aureus, using assembly of interacting protein partners of the type VII secretion system (T7SS) as a case study. Staphylococcus aureus cells that lacked FloA showed reduced T7SS function, and thus reduced secretion of T7SS-related effectors, probably due to the supporting scaffold activity of flotillin. We found that the presence of flotillin mediates intermolecular interactions of T7SS proteins. We tested several small molecules that interfere with flotillin scaffold activity, which perturbed T7SS activity in vitro and in vivo. Our results suggest that flotillin assists in the assembly of S. aureus membrane components that participate in infection and influences the infective potential of this pathogen. KW - flotillin KW - scaffold protein KW - Staphylococcus aureus KW - type VII secretion system Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170035 VL - 13 IS - 11 ER - TY - JOUR A1 - Müller, Anna A. A1 - Dolowschiak, Tamas A1 - Sellin, Mikael E. A1 - Felmy, Boas A1 - Verbree, Carolin A1 - Gadient, Sandra A1 - Westermann, Alexander J. A1 - Vogel, Jörg A1 - LeibundGut-Landmann, Salome A1 - Hardt, Wolf-Dietrich T1 - An NK Cell Perforin Response Elicited via IL-18 Controls Mucosal Inflammation Kinetics during Salmonella Gut Infection JF - PLoS Pathogens N2 - Salmonella Typhimurium (S.Tm) is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S.Tm infections in knockout mice, cytokine inhibition and –injection experiments revealed that caspase-1 (not -11) dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf\(^{-/-}\) ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S.Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens. KW - NK cells KW - Salmonella Typhimurium KW - mucosal inflammation KW - diarrhea Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167429 VL - 12 IS - 6 ER - TY - JOUR A1 - Hershko-Shalev, Tal A1 - Odenheimer-Bergman, Ahuva A1 - Elgrably-Weiss, Maya A1 - Ben-Zvi, Tamar A1 - Govindarajan, Sutharsan A1 - Seri, Hemda A1 - Papenfort, Kai A1 - Vogel, Jörg A1 - Altuvia, Shoshy T1 - Gifsy-1 Prophage IsrK with Dual Function as Small and Messenger RNA Modulates Vital Bacterial Machineries JF - PLoS Genetics N2 - While an increasing number of conserved small regulatory RNAs (sRNAs) are known to function in general bacterial physiology, the roles and modes of action of sRNAs from horizontally acquired genomic regions remain little understood. The IsrK sRNA of Gifsy-1 prophage of Salmonella belongs to the latter class. This regulatory RNA exists in two isoforms. The first forms, when a portion of transcripts originating from isrK promoter reads-through the IsrK transcription-terminator producing a translationally inactive mRNA target. Acting in trans, the second isoform, short IsrK RNA, binds the inactive transcript rendering it translationally active. By switching on translation of the first isoform, short IsrK indirectly activates the production of AntQ, an antiterminator protein located upstream of isrK. Expression of antQ globally interferes with transcription termination resulting in bacterial growth arrest and ultimately cell death. Escherichia coli and Salmonella cells expressing AntQ display condensed chromatin morphology and localization of UvrD to the nucleoid. The toxic phenotype of AntQ can be rescued by co-expression of the transcription termination factor, Rho, or RNase H, which protects genomic DNA from breaks by resolving R-loops. We propose that AntQ causes conflicts between transcription and replication machineries and thus promotes DNA damage. The isrK locus represents a unique example of an island-encoded sRNA that exerts a highly complex regulatory mechanism to tune the expression of a toxic protein. KW - prophage KW - Gifsy-1 KW - sRNA KW - IsrK Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166717 VL - 12 IS - 4 ER - TY - JOUR A1 - Read, Hannah M. A1 - Mills, Grant A1 - Johnson, Sarah A1 - Tsai, Peter A1 - Dalton, James A1 - Barquist, Lars A1 - Print, Cristin G. A1 - Patrick, Wayne M. A1 - Wiles, Siouxsie T1 - The in vitro and in vivo effects of constitutive light expression on a bioluminescent strain of the mouse enteropathogen Citrobacter rodentium JF - PeerJ N2 - Bioluminescent reporter genes, such as those from fireflies and bacteria, let researchers use light production as a non-invasive and non-destructive surrogate measure of microbial numbers in a wide variety of environments. As bioluminescence needs microbial metabolites, tagging microorganisms with luciferases means only live metabolically active cells are detected. Despite the wide use of bioluminescent reporter genes, very little is known about the impact of continuous (also called constitutive) light expression on tagged bacteria. We have previously made a bioluminescent strain of Citrobacter rodentium, a bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infect humans. In this study, we compared the growth of the bioluminescent C. rodentium strain ICC180 with its non-bioluminescent parent (strain ICC169) in a wide variety of environments. To understand more about the metabolic burden of expressing light, we also compared the growth profiles of the two strains under approximately 2,000 different conditions. We found that constitutive light expression in ICC180 was near-neutral in almost every non-toxic environment tested. However, we also found that the non-bioluminescent parent strain has a competitive advantage over ICC180 during infection of adult mice, although this was not enough for ICC180 to be completely outcompeted. In conclusion, our data suggest that constitutive light expression is not metabolically costly to C. rodentium and supports the view that bioluminescent versions of microbes can be used as a substitute for their non-bioluminescent parents to study bacterial behaviour in a wide variety of environments. KW - bioluminescence KW - lux KW - luciferase KW - biophotonic imaging KW - bioluminescence imaging KW - enteric pathogens KW - animal model KW - reporter genes KW - phenotypic microarray KW - biolog Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166576 VL - 4 IS - e2130 ER - TY - JOUR A1 - Schneider, György A1 - Dobrindt, Ulrich A1 - Middendorf, Barbara A1 - Hochhut, Bianca A1 - Szijártó, Valeria A1 - Emódy, Levente A1 - Hacker, Jörg T1 - Mobilisation and remobilisation of a large archetypal pathogenicity island of uropathogenic \(Escherichia\) \(coli\) \(in\) \(vitro\) support the role of conjugation for horizontal transfer of genomic islands JF - BMC Microbiology N2 - Background: A substantial amount of data has been accumulated supporting the important role of genomic islands (GEIs) - including pathogenicity islands (PAIs) - in bacterial genome plasticity and the evolution of bacterial pathogens. Their instability and the high level sequence similarity of different (partial) islands suggest an exchange of PAIs between strains of the same or even different bacterial species by horizontal gene transfer (HGT). Transfer events of archetypal large genomic islands of enterobacteria which often lack genes required for mobilisation or transfer have been rarely investigated so far. Results: To study mobilisation of such large genomic regions in prototypic uropathogenic E. coli (UPEC) strain 536, PAI II(536) was supplemented with the mob(RP4) region, an origin of replication (oriV(R6K)), an origin of transfer (oriT(RP4)) and a chloramphenicol resistance selection marker. In the presence of helper plasmid RP4, conjugative transfer of the 107-kb PAI II(536) construct occured from strain 536 into an E. coli K-12 recipient. In transconjugants, PAI II(536) existed either as a cytoplasmic circular intermediate (CI) or integrated site-specifically into the recipient's chromosome at the leuX tRNA gene. This locus is the chromosomal integration site of PAI II(536) in UPEC strain 536. From the E. coli K-12 recipient, the chromosomal PAI II(536) construct as well as the CIs could be successfully remobilised and inserted into leuX in a PAI II(536) deletion mutant of E. coli 536. Conclusions: Our results corroborate that mobilisation and conjugal transfer may contribute to evolution of bacterial pathogens through horizontal transfer of large chromosomal regions such as PAIs. Stabilisation of these mobile genetic elements in the bacterial chromosome result from selective loss of mobilisation and transfer functions of genomic islands. KW - Recombination directionality factor KW - Staphylococcus-aureus KW - Yersinia-pseudotuberculosis KW - Pseudomonas-aeruginosa KW - Bacterial conjugation KW - Suicide vector KW - Gene-transfer KW - Excision KW - Family KW - Evolution Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-140975 VL - 11 ER - TY - JOUR A1 - Westermann, Alexander J. A1 - Venturini, Elisa A1 - Sellin, Mikael E. A1 - Förstner, Konrad U. A1 - Hardt, Wolf-Dietrich A1 - Vogel, Jörg T1 - The major RNA-binding protein ProQ impacts virulence gene expression in Salmonella enterica serovar Typhimurium JF - mBio N2 - FinO domain proteins such as ProQ of the model pathogen Salmonella enterica have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as Salmonella infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host. Comparison with the other major RNA chaperone in Salmonella, Hfq, reinforces the notion that these two global RNA-binding proteins work in parallel to ensure full virulence. Of newly discovered infection-associated ProQ-bound small noncoding RNAs (sRNAs), we show that the 3′UTR-derived sRNA STnc540 is capable of repressing an infection-induced magnesium transporter mRNA in a ProQ-dependent manner. Together, this comprehensive study uncovers the relevance of ProQ for Salmonella pathogenesis and highlights the importance of RNA-binding proteins in regulating bacterial virulence programs. IMPORTANCE The protein ProQ has recently been discovered as the centerpiece of a previously overlooked “third domain” of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukaryotic cells. Here, we have determined how ProQ shapes Salmonella virulence and how the activities of this RNA-binding protein compare with those of Hfq, another central protein in RNA-based gene regulation in this and other bacteria. To this end, we apply global transcriptomics of pathogen and host cells during infection. In doing so, we reveal ProQ-dependent transcript changes in key virulence and host immune pathways. Moreover, we differentiate the roles of ProQ from those of Hfq during infection, for both coding and noncoding transcripts, and provide an important resource for those interested in ProQ-dependent small RNAs in enteric bacteria. KW - Hfq KW - noncoding RNA KW - ProQ KW - RNA-seq KW - bacterial pathogen KW - posttranscriptional control Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177722 VL - 10 IS - 1 ER - TY - JOUR A1 - Jarick, Marcel A1 - Bertsche, Ute A1 - Stahl, Mark A1 - Schultz, Daniel A1 - Methling, Karen A1 - Lalk, Michael A1 - Stigloher, Christian A1 - Steger, Mirco A1 - Schlosser, Andreas A1 - Ohlsen, Knut T1 - The serine/threonine kinase Stk and the phosphatase Stp regulate cell wall synthesis in Staphylococcus aureus JF - Scientific Reports N2 - The cell wall synthesis pathway producing peptidoglycan is a highly coordinated and tightly regulated process. Although the major components of bacterial cell walls have been known for decades, the complex regulatory network controlling peptidoglycan synthesis and many details of the cell division machinery are not well understood. The eukaryotic-like serine/threonine kinase Stk and the cognate phosphatase Stp play an important role in cell wall biosynthesis and drug resistance in S. aureus. We show that stp deletion has a pronounced impact on cell wall synthesis. Deletion of stp leads to a thicker cell wall and decreases susceptibility to lysostaphin. Stationary phase Δstp cells accumulate peptidoglycan precursors and incorporate higher amounts of incomplete muropeptides with non-glycine, monoglycine and monoalanine interpeptide bridges into the cell wall. In line with this cell wall phenotype, we demonstrate that the lipid II:glycine glycyltransferase FemX can be phosphorylated by the Ser/Thr kinase Stk in vitro. Mass spectrometric analyses identify Thr32, Thr36 and Ser415 as phosphoacceptors. The cognate phosphatase Stp dephosphorylates these phosphorylation sites. Moreover, Stk interacts with FemA and FemB, but is unable to phosphorylate them. Our data indicate that Stk and Stp modulate cell wall synthesis and cell division at several levels. KW - bacterial transcription KW - pathogens KW - cell wall synthesis Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177333 VL - 8 IS - 13693 ER - TY - JOUR A1 - Förstner, Konrad U A1 - Reuscher, Carina M A1 - Haberzettl, Kerstin A1 - Weber, Lennart A1 - Klug, Gabriele T1 - RNase E cleavage shapes the transcriptome of Rhodobacter sphaeroides and strongly impacts phototrophic growth JF - Life Science Alliance N2 - Bacteria adapt to changing environmental conditions by rapid changes in their transcriptome. This is achieved not only by adjusting rates of transcription but also by processing and degradation of RNAs. We applied TIER-Seq (transiently inactivating an endoribonuclease followed by RNA-Seq) for the transcriptome-wide identification of RNase E cleavage sites and of 5′ RNA ends, which are enriched when RNase E activity is reduced in Rhodobacter sphaeroides. These results reveal the importance of RNase E for the maturation and turnover of mRNAs, rRNAs, and sRNAs in this guanine-cytosine-rich α-proteobacterium, some of the latter have well-described functions in the oxidative stress response. In agreement with this, a role of RNase E in the oxidative stress response is demonstrated. A remarkably strong phenotype of a mutant with reduced RNase E activity was observed regarding the formation of photosynthetic complexes and phototrophic growth, whereas there was no effect on chemotrophic growth. KW - Rhodobacter sphaeroides KW - phototrophic growth KW - RNase E Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177139 VL - 1 IS - 4 ER - TY - JOUR A1 - Yu, Sung-Huan A1 - Vogel, Jörg A1 - Förstner, Konrad U. T1 - ANNOgesic: a Swiss army knife for the RNA-seq based annotation of bacterial/archaeal genomes JF - GigaScience N2 - To understand the gene regulation of an organism of interest, a comprehensive genome annotation is essential. While some features, such as coding sequences, can be computationally predicted with high accuracy based purely on the genomic sequence, others, such as promoter elements or noncoding RNAs, are harder to detect. RNA sequencing (RNA-seq) has proven to be an efficient method to identify these genomic features and to improve genome annotations. However, processing and integrating RNA-seq data in order to generate high-resolution annotations is challenging, time consuming, and requires numerous steps. We have constructed a powerful and modular tool called ANNOgesic that provides the required analyses and simplifies RNA-seq-based bacterial and archaeal genome annotation. It can integrate data from conventional RNA-seq and differential RNA-seq and predicts and annotates numerous features, including small noncoding RNAs, with high precision. The software is available under an open source license (ISCL) at https://pypi.org/project/ANNOgesic/. KW - genome annotation KW - RNA-seq KW - transcriptomics Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-178942 VL - 7 ER - TY - JOUR A1 - Gentschev, Ivaylo A1 - Müller, Meike A1 - Adelfinger, Marion A1 - Weibel, Stephanie A1 - Grummt, Friedrich A1 - Zimmermann, Martina A1 - Bitzer, Michael A1 - Heisig, Martin A1 - Zhang, Qian A1 - Yu, Yong A. A1 - Chen, Nanhai G. A1 - Stritzker, Jochen A1 - Lauer, Ulrich M. A1 - Szalay, Aladar A. T1 - Efficient Colonization and Therapy of Human Hepatocellular Carcinoma (HCC) Using the Oncolytic Vaccinia Virus Strain GLV-1h68 JF - PLOS ONE N2 - Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man. KW - Breast-tumors KW - Nude-mice KW - In-vivo KW - Cancer KW - Inhibitor KW - Tissue KW - Agent KW - COX-2 Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135319 VL - 6 IS - 7 ER - TY - JOUR A1 - Schierack, Peter A1 - Kleta, Sylvia A1 - Tedin, Karsten A1 - Babila, Julius Tachu A1 - Oswald, Sibylle A1 - Oelschlaeger, Tobias A. A1 - Hiemann, Rico A1 - Paetzold, Susanne A1 - Wieler, Lothar H. T1 - E. coli Nissle 1917 Affects Salmonella Adhesion to Porcine Intestinal Epithelial Cells JF - PLoS ONE N2 - Background: The probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known. Methodology/Principal Findings: In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra-and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN. Conclusions: We propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion. KW - Nonpathogenic Escherichia-coli KW - Enterica serovar typhimurium KW - Strain nissle-1917 KW - In-vitro KW - Invasion genes KW - Diarrhea KW - Growth KW - Expression KW - Infection KW - PPGPP Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135298 VL - 6 IS - 2 ER - TY - JOUR A1 - Hertlein, Tobias A1 - Sturm, Volker A1 - Kircher, Stefan A1 - Basse-Lüsebrink, Thomas A1 - Haddad, Daniel A1 - Ohlsen, Knut A1 - Jakob, Peter T1 - Visualization of Abscess Formation in a Murine Thigh Infection Model of \(Staphylococcus\) \(aureus\) by (19)F-Magnetic Resonance Imaging (MRI) JF - PLoS ONE N2 - Background: During the last years, (19)F-MRI and perfluorocarbon nanoemulsion (PFC) emerged as a powerful contrast agent methodology to track cells and to visualize inflammation. We applied this new modality to visualize deep tissue abscesses during acute and chronic phase of inflammation caused by Staphylococcus aureus infection. Methodology and Principal Findings: In this study, a murine thigh infection model was used to induce abscess formation and PFC or CLIO (cross linked ironoxides) was administered during acute or chronic phase of inflammation. 24 h after inoculation, the contrast agent accumulation was imaged at the site of infection by MRI. Measurements revealed a strong accumulation of PFC at the abscess rim at acute and chronic phase of infection. The pattern was similar to CLIO accumulation at chronic phase and formed a hollow sphere around the edema area. Histology revealed strong influx of neutrophils at the site of infection and to a smaller extend macrophages during acute phase and strong influx of macrophages at chronic phase of inflammation. Conclusion and Significance: We introduce (19)F-MRI in combination with PFC nanoemulsions as a new platform to visualize abscess formation in a murine thigh infection model of S. aureus. The possibility to track immune cells in vivo by this modality offers new opportunities to investigate host immune response, the efficacy of antibacterial therapies and the influence of virulence factors for pathogenesis. KW - Soft-tissue infection KW - In-vivo KW - Iron-oxide KW - F-19 MRI KW - Inflammation KW - Particles KW - Tracking KW - Lesions KW - Images KW - Rats Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-142846 VL - 6 IS - 3 ER - TY - JOUR A1 - Cull, Benjamin A1 - Lima Prado Godinho, Joseane A1 - Fernandes Rodrigues, Juliany Cola A1 - Frank, Benjamin A1 - Schurigt, Uta A1 - Williams, Roderick AM A1 - Coombs, Graham H A1 - Mottram, Jeremy C T1 - Glycosome turnover in Leishmania major is mediated by autophagy JF - Autophagy N2 - Autophagy is a central process behind the cellular remodeling that occurs during differentiation of Leishmania, yet the cargo of the protozoan parasite's autophagosome is unknown. We have identified glycosomes, peroxisome-like organelles that uniquely compartmentalize glycolytic and other metabolic enzymes in Leishmania and other kinetoplastid parasitic protozoa, as autophagosome cargo. It has been proposed that the number of glycosomes and their content change during the Leishmania life cycle as a key adaptation to the different environments encountered. Quantification of RFP-SQL-labeled glycosomes showed that promastigotes of L. major possess ~20 glycosomes per cell, whereas amastigotes contain ~10. Glycosome numbers were significantly greater in promastigotes and amastigotes of autophagy-defective L. major Δatg5 mutants, implicating autophagy in glycosome homeostasis and providing a partial explanation for the previously observed growth and virulence defects of these mutants. Use of GFP-ATG8 to label autophagosomes showed glycosomes to be cargo in ~15% of them; glycosome-containing autophagosomes were trafficked to the lysosome for degradation. The number of autophagosomes increased 10-fold during differentiation, yet the percentage of glycosome-containing autophagosomes remained constant. This indicates that increased turnover of glycosomes was due to an overall increase in autophagy, rather than an upregulation of autophagosomes containing this cargo. Mitophagy of the single mitochondrion was not observed in L. major during normal growth or differentiation; however, mitochondrial remnants resulting from stress-induced fragmentation colocalized with autophagosomes and lysosomes, indicating that autophagy is used to recycle these damaged organelles. These data show that autophagy in Leishmania has a central role not only in maintaining cellular homeostasis and recycling damaged organelles but crucially in the adaptation to environmental change through the turnover of glycosomes. KW - ATG8 KW - Leishmania KW - TEM KW - glycosome KW - protozoan parasite KW - ATG KW - autophagy-related KW - GFP KW - green fluorescent protein KW - MVT KW - multivesicular tubule KW - RFP KW - red fluorescent protein KW - transmission electron microscopy KW - adaptation KW - autophagy KW - mC KW - mCherry KW - fluorescent protein Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-150277 VL - 10 IS - 12 ER - TY - JOUR A1 - Bergmiller, Tobias A1 - Pena-Miller, Rafael A1 - Boehm, Alexander A1 - Ackermann, Martin T1 - Single-cell time-lapse analysis of depletion of the universally conserved essential protein YgjD JF - BMC Microbiology N2 - Background: The essential Escherichia coli gene ygjD belongs to a universally conserved group of genes whose function has been the focus of a number of recent studies. Here, we put ygjD under control of an inducible promoter, and used time-lapse microscopy and single cell analysis to investigate the phenotypic consequences of the depletion of YgjD protein from growing cells. Results: We show that loss of YgjD leads to a marked decrease in cell size and termination of cell division. The transition towards smaller size occurs in a controlled manner: cell elongation and cell division remain coupled, but cell size at division decreases. We also find evidence that depletion of YgjD leads to the synthesis of the intracellular signaling molecule (p) ppGpp, inducing a cellular reaction resembling the stringent response. Concomitant deletion of the relA and spoT genes - leading to a strain that is uncapable of synthesizing (p) ppGpp abrogates the decrease in cell size, but does not prevent termination of cell division upon YgjD depletion. Conclusions: Depletion of YgjD protein from growing cells leads to a decrease in cell size that is contingent on (p) ppGpp, and to a termination of cell division. The combination of single-cell time-lapse microscopy and statistical analysis can give detailed insights into the phenotypic consequences of the loss of essential genes, and can thus serve as a new tool to study the function of essential genes. KW - Transfer-RNA modification KW - Escherichia-coli K-12 KW - Gene KW - Division KW - Expression KW - Inactivation KW - Maintenance KW - Growth KW - Level KW - Ftsz Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-142324 VL - 11 IS - 118 ER - TY - JOUR A1 - Chen, Nanhai G. A1 - Yu, Yong A. A1 - Zhang, Qian A1 - Szalay, Aladar A. T1 - Replication efficiency of oncolytic vaccinia virus in cell cultures prognosticates the virulence and antitumor efficacy in mice JF - Journal of Translational Medicine N2 - Background: We have shown that insertion of the three vaccinia virus (VACV) promoter-driven foreign gene expression cassettes encoding Renilla luciferase-Aequorea GFP fusion protein, beta-galactosidase, and beta-glucuronidase into the F14.5L, J2R, and A56R loci of the VACV LIVP genome, respectively, results in a highly attenuated mutant strain GLV 1h68. This strain shows tumor specific replication and is capable of eradicating tumors with little or no virulence in mice. This study aimed to distinguish the contribution of added VACV promoter-driven transcriptional units as inserts from the effects of insertional inactivation of three viral genes, and to determine the correlation between replication efficiency of oncolytic vaccinia virus in cell cultures and the virulence and antitumor efficacy in mice Methods: A series of recombinant VACV strains was generated by replacing one, two, or all three of the expression cassettes in GLV 1h68 with short non coding DNA sequences. The replication efficiency and tumor cell killing capacity of these newly generated VACV strains were compared with those of the parent virus GLV-1h68 in cell cultures. The virus replication efficiency in tumors and antitumor efficacy as well as the virulence were evaluated in nu/nu (nude) mice bearing human breast tumor xenografts. Results: we found that virus replication efficiency increased with removal of each of the expression cassettes. The increase in virus replication efficiency was proportionate to the strength of removed VACV promoters linked to foreign genes. The replication efficiency of the new VACV strains paralleled their cytotoxicity in cell cultures. The increased replication efficiency in tumor xenografts resulted in enhanced antitumor efficacy in nude mice. Similarly, the enhanced virus replication efficiency was indicative of increased virulence in nude mice. Conclusions: These data demonstrated that insertion of VACV promoter-driven transcriptional units into the viral genome for the purpose of insertional mutagenesis did modulate the efficiency of virus replication together with antitumor efficacy as well as virulence. Replication efficiency of oncolytic VACV in cell cultures can predict the virulence and therapeutic efficacy in nude mice. These findings may be essential for rational design of safe and potent VACV strains for vaccination and virotherapy of cancer in humans and animals. KW - Recombinant vaccinia KW - Nude-mice KW - Cancer KW - GLV-1H68 KW - Therapy KW - Agent KW - Regression KW - Carcinoma KW - Deletion KW - Protein KW - modulation of virus replication KW - GI-101A tumor xenografts KW - oncolytic virotherapy Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-142268 VL - 9 IS - 164 ER - TY - THES A1 - Lerch, Maike Franziska T1 - Characterisation of a novel non-coding RNA and its involvement in polysaccharide intercellular adhesin (PIA)-mediated biofilm formation of \(Staphylococcus\) \(epidermidis\) T1 - Charakterisierung einer neuen nicht-kodierenden RNA und deren Beteiligung an der PIA-vermittelten Biofilmbildung von \(Staphylococcus\) \(epidermidis\) N2 - Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, have been recognised as an important cause of health care-associated infections due to catheterisation, and livestock-associated infections. The colonisation of indwelling medical devices is achieved by the formation of biofilms, which are large cell-clusters surrounded by an extracellular matrix. This extracellular matrix consists mainly of PIA (polysaccharide intercellular adhesin), which is encoded by the icaADBC-operon. The importance of icaADBC in clinical strains provoking severe infections initiated numerous investigations of this operon and its regulation within the last two decades. The discovery of a long transcript being located next to icaADBC, downstream of the regulator gene icaR, led to the hypothesis of a possible involvement of this transcript in the regulation of biofilm formation (Eckart, 2006). Goal of this work was to characterise this transcript, named ncRNA IcaZ, in molecular detail and to uncover its functional role in S. epidermidis. The ~400 nt long IcaZ is specific for ica-positive S. epidermidis and is transcribed in early- and mid-exponential growth phase as primary transcript. The promotor sequence and the first nucleotides of icaZ overlap with the 3' UTR of the preceding icaR gene, whereas the terminator sequence is shared by tRNAThr-4, being located convergently to icaZ. Deletion of icaZ resulted in a macroscopic biofilm-negative phenotype with highly diminished PIA-biofilm. Biofilm composition was analysed in vitro by classical crystal violet assays and in vivo by confocal laser scanning microscopy under flow conditions to display biofilm formation in real-time. The mutant showed clear defects in initial adherence and decreased cell-cell adherence, and was therefore not able to form a proper biofilm under flow in contrast to the wildtype. Restoration of PIA upon providing icaZ complementation from plasmids revealed inconsistent results in the various mutant backgrounds. To uncover the functional role of IcaZ, transcriptomic and proteomic analysis was carried out, providing some hints on candidate targets, but the varying biofilm phenotypes of wildtype and icaZ mutants made it difficult to identify direct IcaZ mRNA targets. Pulse expression of icaZ was then used as direct fishing method and computational target predictions were executed with candidate mRNAs from aforesaid approaches. The combined data of these analyses suggested an involvement of icaR in IcaZ-mediated biofilm control. Therefore, RNA binding assays were established for IcaZ and icaR mRNA. A positive gel shift was maintained with icaR 3' UTR and with 5'/3' icaR mRNA fusion product, whereas no gel shift was obtained with icaA mRNA. From these assays, it was assumed that IcaZ regulates icaR mRNA expression in S. epidermidis. S. aureus instead lacks ncRNA IcaZ and its icaR mRNA was shown to undergo autoregulation under so far unknown circumstances by intra- or intermolecular binding of 5' UTR and 3' UTR (Ruiz de los Mozos et al., 2013). Here, the Shine-Dalgarno sequence is blocked through 5'/3' UTR base pairing and RNase III, an endoribonuclease, degrades icaR mRNA, leading to translational blockade. In this work, icaR mRNA autoregulation was therefore analysed experimentally in S. epidermidis and results showed that this specific autoregulation does not take place in this organism. An involvement of RNase III in the degradation process could not be verified here. GFP-reporter plasmids were generated to visualise the interaction, but have to be improved for further investigations. In conclusion, IcaZ was found to interact with icaR mRNA, thereby conceivably interfering with translation initiation of repressor IcaR, and thus to promote PIA synthesis and biofilm formation. In addition, the environmental factor ethanol was found to induce icaZ expression, while only weak or no effects were obtained with NaCl and glucose. Ethanol, actually is an ingredient of disinfectants in hospital settings and known as efficient effector for biofilm induction. As biofilm formation on medical devices is a critical factor hampering treatment of S. epidermidis infections in clinical care, the results of this thesis do not only contribute to better understanding of the complex network of biofilm regulation in staphylococci, but may also have practical relevance in the future. N2 - Koagulase-negative Staphylokokken besiedeln die menschliche und tierische Haut, sowie die Schleimhäute. Durch Läsionen oder das Einbringen von medizinischen Instrumenten wie Kathetern gelangen sie in tiefere Hautschichten oder die Blutbahn und können dort schwerwiegende Infektionen auslösen, vor Allem bei Risikopersonen. Besonders Staphylococcus epidermidis hat sich als Verursacher von nosokomialen Infektionen, aber auch als Pathogen in der Tierhaltung etabliert. Die Bakterien bilden bei der Besiedlung sogenannte Biofilme aus (d.h. eine Akkumulation der Keime, die von einer extrazellulären Matrix umgeben sind). Diese Matrix besteht neben Proteinen und eDNA hauptsächlich aus einem Polysaccharid, dem interzellulären Adhäsin PIA (engl.: polysaccharide intercellular adhesin). Dieses wird durch die Ica-Proteine synthetisiert, die im icaADBC-Operon (engl.: intercellular adhesin operon) kodiert sind. Das Operon hat große Bedeutung in klinischen Stämmen und wurde daher innerhalb der letzten beiden Jahrzehnte eingehend untersucht, auch im Hinblick auf seine Regulation. In der unmittelbaren Umgebung des icaADBC-Operons, stromabwärts des icaR Gens, das für den Repressor des ica-Operons (IcaR) kodiert, wurde ein großes Transkript identifiziert, von dem vermutet wird, dass es möglicherweise an der Regulation der Biofilmbildung beteiligt ist (Eckart, 2006). Ziel dieser Arbeit war es, dieses Transkript zu charakterisieren und seine Funktion in S. epidermidis aufzudecken. Die nicht-kodierende RNA, genannt IcaZ, hat eine Länge von ~400 nt und ist spezifisch für ica-positive S. epidermidis. Sie wird in der frühen bis mittleren exponentiellen Phase temperaturabhängig exprimiert. Stromaufwärts überlappt das icaZ-Gen und dessen Promotor mit der 3' UTR vom icaR-Gen. Stromabwärts wird das icaZ-Gen vom einem Transkriptionsterminator begrenzt, der auch für das tRNAThr-4-Gen benutzt wird, das auf dem gegenüberliegenden Strang in Richtung des icaZ-Gens lokalisiert ist. Die Deletion der RNA führte zu einem makroskopisch sichtbaren Biofilm-negativen Phänotyp mit deutlich verminderter PIA Bildung. Die Biofilmzusammensetzung wurde in vitro mittels eines klassischen Kristallviolett-Assays gemessen und die Biofilmbildung in vivo in Echtzeit mittels konfokaler Mikroskopie (CLSM) betrachtet. Dabei wurde mit einer peristaltischen Pumpe ein Mediumfluss appliziert. Die Mutante zeigte klare Defekte in der initialen Adhärenz und in der Zell-Zell Adhäsion. Sie bildete im Gegensatz zum Wildtyp keinen strukturierten Biofilm aus. Zur Komplementierung des Biofilms wurde die IcaZ von einem Plasmid exprimiert und die Biofilmzusammensetzung nach 18-20 Stunden Wachstum gemessen. Die Ergebnisse dieser Untersuchungen in den verschiedenen Mutanten waren nicht eindeutig. Um die Funktion von IcaZ aufzudecken, wurden Transkriptom- und Proteomvergleiche zwischen Wildtyp und Mutante gemacht. Diese lieferten einige Hinweise, aber da der metabolische Unterschied eines Biofilmbildners zu einem Nicht-Biofilmbildner zu groß war, wurde eine direktere Methode angewandt, die induzierte Expression (Pulsexpression). Zudem wurden potentielle Interaktionspartner der IcaZ mittels computer-basierter Bindungsvorhersagen analysiert. Die icaR mRNA kristallisierte sich dabei als Target heraus und die Interaktion zwischen IcaZ und icaR mRNA wurde mit Gelshift-Assays (EMSA) untersucht. Eine Bandenverschiebung wurde mit icaR 3' UTR und mit dem icaR-5'-3' UTR-Fusionsprodukt detektiert, wohingegen keine Interaktion zwischen IcaZ und icaA mRNA stattfand. Aufgrund dieser Assays wurde vermutet, dass IcaZ die Translation von icaR in S. epidermidis reguliert. In S. aureus fehlt die nicht-kodierende RNA IcaZ und für icaR mRNA wurde eine Autoregulation gezeigt, bei der die icaR 5' UTR mit der icaR 3' UTR intramolekular oder intermolekular durch Basenpaarung interagiert, wodurch die Shine-Dalgarno Sequenz blockiert wird und es aufgrund dessen zu einer Hemmung der Translation kommt. Die Umweltfaktoren, die dazu führen sind bisher unbekannt. Der Komplex wird durch eine Endoribonuklease, RNase III, abgebaut (Ruiz de los Mozos et al., 2013). In S. epidermidis wurde eine solche Interaktion theoretisch ausgeschlossen. Experimentelle Analysen dieser Arbeit haben gezeigt, dass diese Autoregulation in S. epidermidis nicht stattfinden kann und es wird angenommen, dass IcaZ diese Regulation übernimmt. Um die Interaktion zu visualisieren wurden GFP-Reporter Plasmide generiert, die aber für weitere Experimente noch zu verbessern sind. Zusammenfassend lässt sich sagen, dass IcaZ mit der icaR mRNA interagiert, was höchstwahrscheinlich zu einer Hemmung der Translation des Repressors IcaR führt und damit letztlich PIA-Synthese und Biofilmbildung positiv reguliert. Zusätzlich wurde gefunden, dass Ethanol die Expression der IcaZ-RNA induziert, während NaCl nur schwache Effekte zeigte und Glucose keinen Einfluss auf die Expression von icaZ hatte. Ethanol ist ein Bestandteil von Desinfektionsmitteln, die in Krankenhäusern verwendet werden und ist bekannt dafür Biofilmbildung auszulösen. Da die Bildung von Biofilmen auf medizinischen Geräten kritisch ist und diese die Behandlung von S. epidermidis Infektionen erschweren, tragen die Ergebnisse dieser Arbeit nicht nur zu einem besseren Verständnis des komplexen Netzwerks der Biofilmregulation bei, sondern haben möglicherweise auch praktischen Nutzen in der Zukunft. KW - Biofilm KW - Staphylococcus epidermidis KW - Non-coding RNA KW - Hospitalismus KW - icaADBC KW - Nosocomial Infections KW - Polysaccharide intercellular adhesin (PIA) KW - Biofilm formation KW - non-coding RNA KW - ncRNA KW - Nosokomiale Infektionen Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-155777 ER - TY - JOUR A1 - Boes, Alexander A1 - Spiegel, Holger A1 - Voepel, Nadja A1 - Edgue, Gueven A1 - Beiss, Veronique A1 - Kapelski, Stephanie A1 - Fendel, Rolf A1 - Scheuermayer, Matthias A1 - Pradel, Gabriele A1 - Bolscher, Judith M. A1 - Behet, Marije C. A1 - Dechering, Koen J. A1 - Hermsen, Cornelus C. A1 - Sauerwein, Robert W. A1 - Schillberg, Stefan A1 - Reimann, Andreas A1 - Fischer, Rainer T1 - Analysis of a multi-component multi-stage malaria vaccine candidate—tackling the cocktail challenge JF - PLoS ONE N2 - Combining key antigens from the different stages of the P. falciparum life cycle in the context of a multi-stage-specific cocktail offers a promising approach towards the development of a malaria vaccine ideally capable of preventing initial infection, the clinical manifestation as well as the transmission of the disease. To investigate the potential of such an approach we combined proteins and domains (11 in total) from the pre-erythrocytic, blood and sexual stages of P. falciparum into a cocktail of four different components recombinantly produced in plants. After immunization of rabbits we determined the domain-specific antibody titers as well as component-specific antibody concentrations and correlated them with stage specific in vitro efficacy. Using purified rabbit immune IgG we observed strong inhibition in functional in vitro assays addressing the pre-erythrocytic (up to 80%), blood (up to 90%) and sexual parasite stages (100%). Based on the component-specific antibody concentrations we calculated the IC50 values for the pre-erythrocytic stage (17–25 μg/ml), the blood stage (40–60 μg/ml) and the sexual stage (1.75 μg/ml). While the results underline the feasibility of a multi-stage vaccine cocktail, the analysis of component-specific efficacy indicates significant differences in IC50 requirements for stage-specific antibody concentrations providing valuable insights into this complex scenario and will thereby improve future approaches towards malaria vaccine cocktail development regarding the selection of suitable antigens and the ratios of components, to fine tune overall and stage-specific efficacy. KW - malaria KW - vaccines KW - antibodies KW - P. falciparum Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173092 VL - 10 IS - 7 ER - TY - JOUR A1 - Hill, Philip J. A1 - Stritzker, Jochen A1 - Scadeng, Miriam A1 - Geissinger, Ulrike A1 - Haddad, Daniel A1 - Basse-Lüsebrink, Thomas C. A1 - Gbureck, Uwe A1 - Jakob, Peter A1 - Szalay, Aladar A. T1 - Magnetic Resonance Imaging of Tumors Colonized with Bacterial Ferritin-Expressing \(Escherichia\) \(coli\) JF - PLoS ONE N2 - Background: Recent studies have shown that human ferritin can be used as a reporter of gene expression for magnetic resonance imaging (MRI). Bacteria also encode three classes of ferritin-type molecules with iron accumulation properties. Methods and Findings: Here, we investigated whether these bacterial ferritins can also be used as MRI reporter genes and which of the bacterial ferritins is the most suitable reporter. Bacterial ferritins were overexpressed in probiotic E. coli Nissle 1917. Cultures of these bacteria were analyzed and those generating highest MRI contrast were further investigated in tumor bearing mice. Among members of three classes of bacterial ferritin tested, bacterioferritin showed the most promise as a reporter gene. Although all three proteins accumulated similar amounts of iron when overexpressed individually, bacterioferritin showed the highest contrast change. By site-directed mutagenesis we also show that the heme iron, a unique part of the bacterioferritin molecule, is not critical for MRI contrast change. Tumor-specific induction of bacterioferritin-expression in colonized tumors resulted in contrast changes within the bacteria-colonized tumors. Conclusions: Our data suggest that colonization and gene expression by live vectors expressing bacterioferritin can be monitored by MRI due to contrast changes. KW - Blood-brain barrier KW - Gene-expression KW - Salmonella-typhimurium KW - Sugar-transport KW - Breast-tumors KW - MRI reporter KW - Iron-uptake KW - Proteins KW - Therapy KW - Mice Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-140920 VL - 6 IS - 10 ER - TY - JOUR A1 - Firdessa, Rebuma A1 - Good, Liam A1 - Amstalden, Maria Cecilia A1 - Chindera, Kantaraja A1 - Kamaruzzaman, Nor Fadhilah A1 - Schultheis, Martina A1 - Röger, Bianca A1 - Hecht, Nina A1 - Oelschlaeger, Tobias A. A1 - Meinel, Lorenz A1 - Lühmann, Tessa A1 - Moll, Heidrun T1 - Pathogen- and host-directed antileishmanial effects mediated by polyhexanide (PHMB) JF - PLoS Neglected Tropical Diseases N2 - Background Cutaneous leishmaniasis (CL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed. Methodology/Principal Findings Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide), a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major) via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB's DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN). PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB. Conclusions Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators. KW - resistance KW - activation KW - dendritic cells KW - Cutaneous leishmaniasis KW - topical treatment KW - biocide polyhexamethylene biguanide KW - experimental visceral leishmaniasis KW - drug-delivery systems KW - therapy KW - paromomycin Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148162 VL - 9 IS - 10 ER - TY - JOUR A1 - Belair, Cédric A1 - Baud, Jessica A1 - Chabas, Sandrine A1 - Sharma, Cynthia M A1 - Vogel, Jörg A1 - Staedel, Cathy A1 - Darfeuille, Fabien T1 - Helicobacter pylori interferes with an embryonic stem cell micro RNA cluster to block cell cycle progression JF - Silence : a Journal of RNA regulation N2 - Background MicroRNAs, post-transcriptional regulators of eukaryotic gene expression, are implicated in host defense against pathogens. Viruses and bacteria have evolved strategies that suppress microRNA functions, resulting in a sustainable infection. In this work we report that Helicobacter pylori, a human stomach-colonizing bacterium responsible for severe gastric inflammatory diseases and gastric cancers, downregulates an embryonic stem cell microRNA cluster in proliferating gastric epithelial cells to achieve cell cycle arrest. Results Using a deep sequencing approach in the AGS cell line, a widely used cell culture model to recapitulate early events of H. pylori infection of gastric mucosa, we reveal that hsa-miR-372 is the most abundant microRNA expressed in this cell line, where, together with hsa-miR-373, it promotes cell proliferation by silencing large tumor suppressor homolog 2 (LATS2) gene expression. Shortly after H. pylori infection, miR-372 and miR-373 synthesis is highly inhibited, leading to the post-transcriptional release of LATS2 expression and thus, to a cell cycle arrest at the G1/S transition. This downregulation of a specific cell-cycle-regulating microRNA is dependent on the translocation of the bacterial effector CagA into the host cells, a mechanism highly associated with the development of severe atrophic gastritis and intestinal-type gastric carcinoma. Conclusions These data constitute a novel example of host-pathogen interplay involving microRNAs, and unveil the couple LATS2/miR-372 and miR-373 as an unexpected mechanism in infection-induced cell cycle arrest in proliferating gastric cells, which may be relevant in inhibition of gastric epithelium renewal, a major host defense mechanism against bacterial infections. KW - MicroRNAs KW - cell cycle KW - Helicobacter pylori KW - gastric cancer Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-140438 VL - 2 IS - 7 ER - TY - JOUR A1 - Okoro, Chinyere K. A1 - Barquist, Lars A1 - Connor, Thomas R. A1 - Harris, Simon R. A1 - Clare, Simon A1 - Stevens, Mark P. A1 - Arends, Mark J. A1 - Hale, Christine A1 - Kane, Leanne A1 - Pickard, Derek J. A1 - Hill, Jennifer A1 - Harcourt, Katherine A1 - Parkhill, Julian A1 - Dougan, Gordon A1 - Kingsley, Robert A. T1 - Signatures of adaptation in human invasive Salmonella Typhimurium ST313 populations from sub-Saharan Africa JF - PLoS Neglected Tropical Diseases N2 - Two lineages of Salmonella enterica serovar Typhimurium (S. Typhimurium) of multi-locus sequence type ST313 have been linked with the emergence of invasive Salmonella disease across sub-Saharan Africa. The expansion of these lineages has a temporal association with the HIV pandemic and antibiotic usage. We analysed the whole genome sequence of 129 ST313 isolates representative of the two lineages and found evidence of lineage-specific genome degradation, with some similarities to that observed in S. Typhi. Individual ST313 S. Typhimurium isolates exhibit a distinct metabolic signature and modified enteropathogenesis in both a murine and cattle model of colitis, compared to S. Typhimurium outside of the ST313 lineages. These data define phenotypes that distinguish ST313 isolates from other S. Typhimurium and may represent adaptation to a distinct pathogenesis and lifestyle linked to an-immuno-compromised human population. KW - genome sequence KW - infection KW - pathogenicity KW - children KW - disease KW - adults KW - identification KW - Escherichia coli KW - virulence Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143779 VL - 9 IS - 3 ER - TY - JOUR A1 - Dembek, Marcin A1 - Barquist, Lars A1 - Boinett, Christine J. A1 - Cain, Amy K. A1 - Mayho, Matthew A1 - Lawley, Trevor D. A1 - Fairweather, Neil F. A1 - Fagan, Robert P. T1 - High-throughput analysis of gene essentiality and sporulation in Clostridium difficile JF - mBio N2 - Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen. KW - Bacillus subtilis KW - expression KW - spores KW - toxin KW - transcription KW - germination KW - transposition KW - metabolism KW - infection KW - in vitro Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143745 VL - 6 IS - 2 ER - TY - JOUR A1 - Berg, Stefan A1 - Schelling, Esther A1 - Hailu, Elena A1 - Firdessa, Rebuma A1 - Gumi, Balako A1 - Erenso, Girume A1 - Gadisa, Endalamaw A1 - Mengistu, Araya A1 - Habtamu, Meseret A1 - Hussein, Jemal A1 - Kiros, Teklu A1 - Bekele, Shiferaw A1 - Mekonnen, Wondale A1 - Derese, Yohannes A1 - Zinsstag, Jakob A1 - Ameni, Gobena A1 - Gagneux, Sebastien A1 - Robertson, Brian D A1 - Tschopp, Rea A1 - Hewinson, Glyn A1 - Yamuah, Lawrence A1 - Gordon, Stephen V A1 - Aseffa, Abraham T1 - Investigation of the high rates of extrapulmonary tuberculosis in Ethiopia reveals no single driving factor and minimal evidence for zoonotic transmission of Mycobacterium bovis infection JF - BMC Infectious Diseases N2 - Background: Ethiopia, a high tuberculosis (TB) burden country, reports one of the highest incidence rates of extra-pulmonary TB dominated by cervical lymphadenitis (TBLN). Infection with Mycobacterium bovis has previously been excluded as the main reason for the high rate of extra-pulmonary TB in Ethiopia. Methods: Here we examined demographic and clinical characteristics of 953 pulmonary (PTB) and 1198 TBLN patients visiting 11 health facilities in distinct geographic areas of Ethiopia. Clinical characteristics were also correlated with genotypes of the causative agent, Mycobacterium tuberculosis. Results: No major patient or bacterial strain factor could be identified as being responsible for the high rate of TBLN, and there was no association with HIV infection. However, analysis of the demographic data of involved patients showed that having regular and direct contact with live animals was more associated with TBLN than with PTB, although no M. bovis was isolated from patients with TBLN. Among PTB patients, those infected with Lineage 4 reported "contact with other TB patient" more often than patients infected with Lineage 3 did (OR = 1.6, CI 95% 1.0-2.7; p = 0.064). High fever, in contrast to low and moderate fever, was significantly associated with Lineage 4 (OR = 2.3; p = 0.024). On the other hand, TBLN cases infected with Lineage 4 tended to get milder symptoms overall for the constitutional symptoms than those infected with Lineage 3. Conclusions: The study suggests a complex role for multiple interacting factors in the epidemiology of extra-pulmonary TB in Ethiopia, including factors that can only be derived from population-based studies, which may prove to be significant for TB control in Ethiopia. KW - zoonotic KW - Mycobacterium KW - Ethiopia KW - tuberculosis KW - Bovis KW - pulmonary KW - extrapulmonary KW - lymphadenitis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143935 VL - 15 IS - 112 ER - TY - JOUR A1 - Böhm, Lena A1 - Torsin, Sanda A1 - Tint, Su Hlaing A1 - Eckstein, Marie Therese A1 - Ludwig, Tobias A1 - Pérez, J. Christian T1 - The yeast form of the fungus Candida albicans promotes persistence in the gut of gnotobiotic mice JF - PLoS Pathogens N2 - Many microorganisms that cause systemic, life-threatening infections in humans reside as harmless commensals in our digestive tract. Yet little is known about the biology of these microbes in the gut. Here, we visualize the interface between the human commensal and pathogenic fungus Candida albicans and the intestine of mice, a surrogate host. Because the indigenous mouse microbiota restricts C. albicans settlement, we compared the patterns of colonization in the gut of germ free and antibiotic-treated conventionally raised mice. In contrast to the heterogeneous morphologies found in the latter, we establish that in germ free animals the fungus almost uniformly adopts the yeast cell form, a proxy of its commensal state. By screening a collection of C. albicans transcription regulator deletion mutants in gnotobiotic mice, we identify several genes previously unknown to contribute to in vivo fitness. We investigate three of these regulators—ZCF8, ZFU2 and TRY4—and show that indeed they favor the yeast form over other morphologies. Consistent with this finding, we demonstrate that genetically inducing non-yeast cell morphologies is detrimental to the fitness of C. albicans in the gut. Furthermore, the identified regulators promote adherence of the fungus to a surface covered with mucin and to mucus-producing intestinal epithelial cells. In agreement with this result, histology sections indicate that C. albicans dwells in the murine gut in close proximity to the mucus layer. Thus, our findings reveal a set of regulators that endows C. albicans with the ability to endure in the intestine through multiple mechanisms. KW - Candida albicans KW - deletion mutagenesis KW - gastrointestinal tract KW - fungi KW - regulator genes KW - gene regulation KW - mouse models KW - fungal genetics Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159120 VL - 13 IS - 10 ER - TY - JOUR A1 - Hampe, Irene A. I. A1 - Friedman, Justin A1 - Edgerton, Mira A1 - Morschhäuser, Joachim T1 - An acquired mechanism of antifungal drug resistance simultaneously enables Candida albicans to escape from intrinsic host defenses JF - PLoS Pathogens N2 - The opportunistic fungal pathogen Candida albicans frequently produces genetically altered variants to adapt to environmental changes and new host niches in the course of its life-long association with the human host. Gain-of-function mutations in zinc cluster transcription factors, which result in the constitutive upregulation of their target genes, are a common cause of acquired resistance to the widely used antifungal drug fluconazole, especially during long-term therapy of oropharyngeal candidiasis. In this study, we investigated if C. albicans also can develop resistance to the antimicrobial peptide histatin 5, which is secreted in the saliva of humans to protect the oral mucosa from pathogenic microbes. As histatin 5 has been shown to be transported out of C. albicans cells by the Flu1 efflux pump, we screened a library of C. albicans strains that contain artificially activated forms of all zinc cluster transcription factors of this fungus for increased FLU1 expression. We found that a hyperactive Mrr1, which confers fluconazole resistance by upregulating the multidrug efflux pump MDR1 and other genes, also causes FLU1 overexpression. Similarly to the artificially activated Mrr1, naturally occurring gain-of-function mutations in this transcription factor also caused FLU1 upregulation and increased histatin 5 resistance. Surprisingly, however, Mrr1-mediated histatin 5 resistance was mainly caused by the upregulation of MDR1 instead of FLU1, revealing a previously unrecognized function of the Mdr1 efflux pump. Fluconazole-resistant clinical C. albicans isolates with different Mrr1 gain-of-function mutations were less efficiently killed by histatin 5, and this phenotype was reverted when MRR1 was deleted. Therefore, antimycotic therapy can promote the evolution of strains that, as a consequence of drug resistance mutations, simultaneously have acquired increased resistance against an innate host defense mechanism and are thereby better adapted to certain host niches. KW - antimicrobial resistance KW - transcriptional control KW - Candida albicans KW - transcription factors KW - mutation KW - hyperexpression techniques KW - antifungals KW - point mutation Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158883 VL - 13 IS - 9 ER - TY - JOUR A1 - Tawk, Caroline A1 - Sharan, Malvika A1 - Eulalio, Ana A1 - Vogel, Jörg T1 - A systematic analysis of the RNA-targeting potential of secreted bacterial effector proteins JF - Scientific Reports N2 - Many pathogenic bacteria utilize specialized secretion systems to deliver proteins called effectors into eukaryotic cells for manipulation of host pathways. The vast majority of known effector targets are host proteins, whereas a potential targeting of host nucleic acids remains little explored. There is only one family of effectors known to target DNA directly, and effectors binding host RNA are unknown. Here, we take a two-pronged approach to search for RNA-binding effectors, combining biocomputational prediction of RNA-binding domains (RBDs) in a newly assembled comprehensive dataset of bacterial secreted proteins, and experimental screening for RNA binding in mammalian cells. Only a small subset of effectors were predicted to carry an RBD, indicating that if RNA targeting was common, it would likely involve new types of RBDs. Our experimental evaluation of effectors with predicted RBDs further argues for a general paucity of RNA binding activities amongst bacterial effectors. We obtained evidence that PipB2 and Lpg2844, effector proteins of Salmonella and Legionella species, respectively, may harbor novel biochemical activities. Our study presenting the first systematic evaluation of the RNA-targeting potential of bacterial effectors offers a basis for discussion of whether or not host RNA is a prominent target of secreted bacterial proteins. KW - pathogens KW - bacterial secretion Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158815 VL - 7 ER - TY - JOUR A1 - Rakette, Sonja A1 - Donat, Stefanie A1 - Ohlsen, Knut A1 - Stehle, Thilo T1 - Structural Analysis of Staphylococcus aureus Serine/Threonine Kinase PknB JF - PLoS One N2 - Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 angstrom resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation. KW - SER/THR kinase KW - domain KW - subunit KW - dependent protein-kinase KW - mycobacterium-tuberculosis KW - activation mechanism KW - crystal structure KW - antibiotic resistance KW - catalytic KW - methicillin KW - inhibitor Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135369 VL - 7 IS - 6 ER - TY - THES A1 - Hampe, Irene Aurelia Ida T1 - Analysis of the mechanism and the regulation of histatin 5 resistance in \(Candida\) \(albicans\) T1 - Analyse des Mechanismus und der Regulierung von Histatin 5 Resistenz in \(Candida\) \(albicans\) N2 - Antimycotics such as fluconazole are frequently used to treat C. albicans infections of the oral mucosa. Prolonged treatment of the fungal infection with fluconazole pose a risk to resistance development. C. albicans can adapt to these stressful environmental changes by regulation of gene expression or by producing genetically altered variants that arise in the population. Adapted variants frequently carry activating mutations in zinc cluster transcription factors, which cause the upregulation of their target genes, including genes encoding efflux pumps that confer drug resistance. MDR1, regulated by the zinc cluster transcription factor Mrr1, as well as CDR1 and CDR2, regulated by the zinc cluster transcription factor Tac1, are well-known examples of genes encoding efflux pumps that extrude the antimycotic fluconazole from the fungal cell and thus contribute to the survival of the fungus. In this study, it was investigated if C. albicans can develop resistance to the antimicrobial peptide histatin 5, which serves as the first line of defence in the oral cavity of the human host. Recently, it was shown that C. albicans transports histatin 5 outside of the Candia cell via the efflux pump Flu1. As efflux pumps are often regulated by zinc cluster transcription factors, the Flu1 efflux pump could also be regulated by a zinc cluster transcription factor which could in a hyperactive form upregulate the expression of the efflux pump, resulting in increased export of histatin 5 and consequently in histatin 5 resistance. In order to find a zinc cluster transcription factor that upregulates FLU1 expression, a comprehensive library of C. albicans strains containing artificially activated forms of zinc cluster transcription factors was screened for suitable candidates. The screening was conducted on medium containing mycophenolic acid because mycophenolic acid is also a substrate of Flu1 and a strain expressing a hyperactive zinc cluster transcription factor that upregulates FLU1 expression should exhibit an easily recognisable mycophenolic acid-resistant phenotype. Further, FACS analysis, quantitative real-time RT-PCR analysis, broth microdilution assays as well as histatin 5 assays were conducted to analyse the mechanism and the regulation of histatin 5 resistance. Several zinc cluster transcription factors caused mycophenolic acid resistance and upregulated FLU1 expression. Of those, only hyperactive Mrr1 was able to confer increased histatin 5 resistance. Finding Mrr1 to confer histatin 5 resistance was highly interesting as fluconazole-resistant strains with naturally occurring Mrr1 gain of function mutations exist, which were isolated from HIV-infected patients with oral candidiasis. These Mrr1 gain of function mutations as well as artificially activated Mrr1 cause fluconazole resistance by upregulation of the efflux pump MDR1 and other target genes. In the course of the study, it was found that expression of different naturally occurring MRR1 gain-of-function mutations in the SC5314 wild type background caused increased FLU1 expression and increased histatin 5 resistance. The same was true for fluconazole-resistant clinical isolates with Mrr1 gain of function mutations, which also caused the overexpression of FLU1. Those cells were less efficiently killed by histatin 5 dependent on Mrr1. Surprisingly, FLU1 contributed only little to histatin 5 resistance, rather, overexpression of MDR1 mainly contributed to the Mrr1-mediated histatin 5 resistance, but also additional Mrr1-target genes were involved. These target genes are yet to be uncovered. Moreover, if a link between the yet unknown Mrr1-target genes contributing to fluconazole resistance and increased histatin 5 resistance can be drawn remains to be discovered upon finding of the responsible target genes. Collectively, this study contributes to the understanding of the impact of prolonged antifungal exposure on the interaction between host and fungus. Drug therapy can give rise to resistance evolution resulting in strains that have not only developed resistance to fluconazole but also to an innate host mechanism, which allows adaption to the host niche even in the absence of the drug. N2 - Antimykotika wie Fluconazol werden häufig zur Behandlung von C. albicans Infektionen der Mundschleimhaut verwendet. Dabei stellt eine langzeitige Behandlung der Pilzinfektion mit Fluconazol ein Risiko zur Resistenzentwicklung dar. C. albicans kann sich an solche Umweltveränderungen anpassen, indem es die Genexpression reguliert oder genetisch veränderte Varianten produziert, welche in der Population entstehen. Adaptierte Varianten tragen häufig aktivierende Mutationen in Zink-Cluster-Transkriptionsfaktoren, welche die Hochregulierung der Expression von Genen verursachen, darunter solche, die für Multidrug-Effluxpumpen kodieren und dadurch Antimykotikaresistenz verleihen können. MDR1, reguliert durch den Zink-Cluster-Transkriptionsfaktor Mrr1, sowie CDR1 und CDR2, reguliert durch den Zink-Cluster-Transkriptionsfaktor Tac1, sind bekannte Beispiele für Effluxpumpen, die das Antimykotikum Fluconazol aus der Pilzzelle extrudieren und somit zum Überleben der Pilzzelle beitragen. In dieser Arbeit wurde untersucht, ob C. albicans eine Resistenz gegen das antimikrobielle Peptid Histatin 5 entwickeln kann, das in der Mundhöhle des menschlichen Wirtes als erste Verteidigungsbarriere gegen den Pilz dient. Kürzlich wurde gezeigt, dass C. albicans Histatin 5 über die Effluxpumpe Flu1 aus der Candia-Zelle heraustransportiert (Li et al., 2013). Da Effluxpumpen häufig durch Zink-Cluster-Transkriptionsfaktoren reguliert werden, könnte auch die Flu1-Effluxpumpe durch solch einen Transkriptionsfaktor reguliert werden, der in einer hyperaktiven Form die Expression der Effluxpumpe hochregulieren könnte, was wiederrum zu einem erhöhten Export von Histatin 5 und folglich zur Histatin 5 Resistenz führen könnte. Um einen Zink-Cluster-Transkriptionsfaktor zu finden, der die FLU1-Expression hochreguliert, wurde mit Hilfe einer Bibliothek von C. albicans-Stämmen, die künstlich aktivierte Formen von Zink-Cluster-Transkriptionsfaktoren enthält, nach geeigneten Kandidaten gesucht. Das Screening wurde auf Mycophenolsäure-haltigem Medium durchgeführt, da Mycophenolsäure ebenfalls ein Substrat von Flu1 ist. Folglich sollte ein Stamm mit hyperaktivem Zink-Cluster-Transkriptionsfaktor, welcher die FLU1-Expression hochreguliert, einen leicht erkennbaren Mycophenolsäure-resistenten Phänotyp aufweisen. Weiterhin wurden FACS-Analysen, quantitative real-time RT-PCR-Analysen, Broth microdilution-Assays sowie Histatin 5-Assays durchgeführt, um den Mechanismus und die Regulierung der Histatin-5-Resistenz zu analysieren. Mehrere Zink-Cluster-Transkriptionsfaktoren verursachten Mycophenolsäure-Resistenz und erhöhten die FLU1-Expression. Von diesen war nur hyperaktives Mrr1 in der Lage, eine erhöhte Histatin-5-Resistenz zu verleihen. Das Auffinden von Mrr1 als Regulator der Histatin 5-Resistenz war hochinteressant, da fluconazolresistente Stämme mit natürlich vorkommenden MRR1 gain-of-function Mutationen existieren, die aus HIV-infizierten Patienten mit oropharyngealer Candidiasis isoliert wurden. Diese gain-of-function Mutationen sowie künstlich aktivierendes Mrr1 verursachen Fluconazol-Resistenz durch Hochregulation der Effluxpumpe MDR1 und anderer Zielgene. Im Verlauf der Studie wurde herausgefunden, dass verschiedene natürlich vorkommende MRR1 gain-of-function Mutationen im SC5314 Wildtyp Hintergrund eine erhöhte FLU1-Expression und eine erhöhte Histatin-5-Resistenz verursachten. Das Gleiche galt für Fluconazol-resistente klinische Isolate mit Mrr1 gain-of-function Mutationen, welche die Überexpression von FLU1 verursachten. Zellen dieser Isolate wurden, abhängig von Mrr1, weniger wirksam durch Histatin 5 abgetötet. Überraschenderweise trug FLU1 nur wenig zur Histatin-5-Resistenz bei, vielmehr trug die Überexpression von MDR1 hauptsächlich zur Mrr1-vermittelten Histatin-5-Resistenz bei, aber auch weitere Mrr1-Zielgene waren daran beteiligt. Diese Mrr1-Zielgene gilt es nun noch zu entdecken. Ob ein Zusammenhang zwischen diesen noch unbekannten Mrr1-Zielgenen hergestellt werden kann, die zur Fluconazolresistenz sowie zu einer erhöhten Histatin-5-Resistenz beitragen, wird erst nach dem Auffinden der verantwortlichen Zielgene geprüft werden können. Zusammenfassend trägt diese Studie zum Verständnis der Auswirkungen einer anhaltenden antimykotischen Exposition auf die Interaktion zwischen Wirt und Pilz bei. Eine medikamentöse Therapie kann zu einer Resistenzentwicklung führen, aus der Stämme hervorgehen, welche nicht nur eine Resistenz gegen Fluconazol entwickelt haben, sondern gleichzeitig eine Resistenz gegen einen angeborenen Wirtsabwehrmechanismus, der eine Adaption an die Wirtsnische auch in Abwesenheit des Antimykotikums ermöglicht. KW - Histatin 5 KW - Candida albicans KW - Efflux pump KW - MDR1 KW - MRR1 KW - Mrr1 KW - MDR1 KW - Fluconazole KW - Efflux pump Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159634 ER - TY - JOUR A1 - Sunkavalli, Ushasree A1 - Aguilar, Carmen A1 - Silva, Ricardo Jorge A1 - Sharan, Malvika A1 - Cruz, Ana Rita A1 - Tawk, Caroline A1 - Maudet, Claire A1 - Mano, Miguel A1 - Eulalio, Ana T1 - Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia JF - PLoS Pathogens N2 - MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells. KW - hos tcells KW - Salmonellosis KW - Shigellosis KW - microRNAs KW - Shigella KW - small interfering RNAs KW - HeLa cells KW - Cell binding Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158204 VL - 13 IS - 4 ER - TY - JOUR A1 - Sharan, Malvika A1 - Förstner, Konrad U. A1 - Eulalio, Ana A1 - Vogel, Jörg T1 - APRICOT: an integrated computational pipeline for the sequence-based identification and characterization of RNA-binding proteins JF - Nucleic Acids Research N2 - RNA-binding proteins (RBPs) have been established as core components of several post-transcriptional gene regulation mechanisms. Experimental techniques such as cross-linking and co-immunoprecipitation have enabled the identification of RBPs, RNA-binding domains (RBDs) and their regulatory roles in the eukaryotic species such as human and yeast in large-scale. In contrast, our knowledge of the number and potential diversity of RBPs in bacteria is poorer due to the technical challenges associated with the existing global screening approaches. We introduce APRICOT, a computational pipeline for the sequence-based identification and characterization of proteins using RBDs known from experimental studies. The pipeline identifies functional motifs in protein sequences using position-specific scoring matrices and Hidden Markov Models of the functional domains and statistically scores them based on a series of sequence-based features. Subsequently, APRICOT identifies putative RBPs and characterizes them by several biological properties. Here we demonstrate the application and adaptability of the pipeline on large-scale protein sets, including the bacterial proteome of Escherichia coli. APRICOT showed better performance on various datasets compared to other existing tools for the sequence-based prediction of RBPs by achieving an average sensitivity and specificity of 0.90 and 0.91 respectively. The command-line tool and its documentation are available at https://pypi.python.org/pypi/bio-apricot. KW - RNA-binding proteins KW - identification KW - characterization Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157963 VL - 45 IS - 11 ER - TY - JOUR A1 - Jäger, Dominik A1 - Pernitzsch, Sandy R. A1 - Richter, Andreas S. A1 - Backofen, Rolf A1 - Sharma, Cynthia M. A1 - Schmitz, Ruth A. T1 - An archaeal sRNA targeting cis- and trans-encoded mRNAs via two distinct domains JF - Nucleic Acids Research N2 - We report on the characterization and target analysis of the small (s) RNA\(_{162}\) in the methanoarchaeon Methanosarcina mazei. Using a combination of genetic approaches, transcriptome analysis and computational predictions, the bicistronic MM2441-MM2440 mRNA encoding the transcription factor MM2441 and a protein of unknown function was identified as a potential target of this sRNA, which due to processing accumulates as three stabile 5' fragments in late exponential growth. Mobility shift assays using various mutants verified that the non-structured single-stranded linker region of sRNA\(_{162}\) (SLR) base-pairs with the MM2440-MM2441 mRNA internally, thereby masking the predicted ribosome binding site of MM2441. This most likely leads to translational repression of the second cistron resulting in dis-coordinated operon expression. Analysis of mutant RNAs in vivo confirmed that the SLR of sRNA\(_{162}\) is crucial for target interactions. Furthermore, our results indicate that sRNA\(_{162}\)-controlled MM2441 is involved in regulating the metabolic switch between the carbon sources methanol and methylamine. Moreover, biochemical studies demonstrated that the 50 end of sRNA\(_{162}\) targets the 5'-untranslated region of the cis-encoded MM2442 mRNA. Overall, this first study of archaeal sRNA/mRNA-target interactions unraveled that sRNA\(_{162}\) acts as an antisense (as) RNA on cis- and trans-encoded mRNAs via two distinct domains, indicating that cis-encoded asRNAs can have larger target regulons than previously anticipated. KW - strain KW - escherichia coli KW - methanosarcina mazei GO1 KW - methanol methyltransferase isozymes KW - small nucleolar RNAs KW - acetivorans C2A KW - antisense RNAs KW - GO1 KW - transcriptional regulator KW - translational initiation KW - pyrococcus furiosus Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134972 VL - 40 IS - 21 ER - TY - THES A1 - Oesterreich, Babett T1 - Preclinical development of an immunotherapy against antibiotic-resistant Staphylococcus aureus T1 - Präklinische Entwicklung einer Immuntherapie zur Behandlung Antibiotika-resistenter Staphylococcus aureus N2 - The Gram-positive bacterium Staphylococcus aureus is the leading cause of nosocomial infections. In particular, diseases caused by methicillin-resistant S. aureus (MRSA) are associated with higher morbidity, mortality and medical costs due to showing resistance to several classes of established antibiotics and their ability to develop resistance mechanisms against new antibiotics rapidly. Therefore, strategies based on immunotherapy approaches have the potential to close the gap for an efficient treatment of MRSA. In this thesis, a humanized antibody specific for the immunodominant staphylococcal antigen A (IsaA) was generated and thoroughly characterized as potential candidate for an antibody based therapy. A murine monoclonal antibody was selected for humanization based on its binding characteristics and the ability of efficient staphylococcal killing in mouse infection models. The murine antibody was humanized by CDR grafting and mouse and humanized scFv as well as scFv-Fc fragments were constructed for comparative binding studies to analyse the successful humanization. After these studies, the full antibody with the complete Fc region was constructed as isotype IgG1, IgG2 and IgG4, respectively to assess effector functions, including antibody-dependent killing of S. aureus. The biological activity of the humanized antibody designated hUK-66 was analysed in vitro with purified human PMNs and whole blood samples taken from healthy donors and patients at high risk of S. aureus infections, such as those with diabetes, end-stage renal disease, or artery occlusive disease (AOD). Results of the in vitro studies show, that hUK-66 was effective in antibody-dependent killing of S. aureus in blood from both healthy controls and patients vulnerable to S. aureus infections. Moreover, the biological activity of hUK-66 and hUK-66 combined with a humanized anti-alpha-toxin antibody (hUK-tox) was investigated in vivo using a mouse pneumonia model. The in vivo results revealed the therapeutic efficacy of hUK-66 and the antibody combination of hUK-66 and hUK-tox to prevent staphylococcal induced pneumonia in a prophylactic set up. Based on the experimental data, hUK-66 represents a promising candidate for an antibody-based therapy against antibiotic resistant MRSA. N2 - Staphylococcus aureus ist ein bedeutender nosokomialer Erreger, der eine Vielzahl von Infektionen im Menschen verursacht. Besonders Krankheiten, die durch Methicillin resistente S. aureus (MRSA) verursacht werden, sind mit einer erhöhten Morbidität, einer höheren Sterblichkeitsrate und hohen medizinischen Kosten verbunden. Seine besondere medizinische Bedeutung erlangte S. aureus durch die Ausbildung von Resistenzen gegen eine Vielzahl von Antibiotika und seiner Fähigkeit auch gegen neu entwickelte Antibiotika schnell Resistenzmechanismen auszubilden. Aus diesem Grund, ist die Entwicklung von neuen Therapieansätzen von besonderer Bedeutung, um die entstandene Lücke für eine effektive MRSA-Therapie zu schließen. In dieser Arbeit wurde ein humanisierter monoklonaler Antikörper entwickelt und charakterisiert, der spezifisch an das „immunodominant staphylococcal antigen A“ (IsaA) bindet. Dieser Antiköper wurde auf Grund seiner Eigenschaft, in einem Mausmodell effektiv S. aureus abzutöten, als vielversprechender Kandidat für eine Antikörper-Therapie ausgewählt. Der murine Vorläuferantikörper wurde mittels „CDR grafting“ humanisiert und durch die Generierung von humanisierten und murinen scFv und scFv-Fc Fragmenten, die in vergleichenden Bindungsstudien getestet wurden, konnte der Erfolg der Humanisierung beurteilt werden. Im Anschluss wurde der vollständige Antikörper mit vollständig funktionaler Fc-Region in den Isotypen IgG1, IgG2 und IgG4 hergestellt. Die Funktionalität des humanisierten Antikörpers wurde in vitro mittels aufgereinigter PMNs und Blutproben von gesunden Spendern und Patienten bestimmt, die ein hohes Risiko für S. aureus Infektionen besitzen wie Diabetiker, Dialyse-Patienten und Patienten mit arterieller Verschlusskrankheit. Die Ergebnisse der in vitro-Studien zeigen, dass der anti-IsaA-Antikörper hUK-66 nicht nur S. aureus effektiv in Blutproben von gesunden Spendern abtötet, sondern auch in Blutproben von Patienten mit erhöhter Anfälligkeit für S. aureus Infektionen. Darüber hinaus wurde die biologische Aktivität des humanisierten Antikörpers gegen IsaA als Monotherapie und in Kombination mit einem humanisierten anti-alpha-Toxin-Antikörper (hUK-tox) in vivo in einem Maus Pneumonie Modell untersucht. Hierbei konnte gezeigt werden, dass die prophylaktische Verabreichung von hUK-66 sowie die Kombination von hUK-66 und hUK-tox, die Bildung einer Staphylokokken-induzierten Pneumonie mit Todesfolge signifikant senkt. KW - Staphylococcus KW - Immunotherapy Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123237 ER - TY - THES A1 - Leimbach, Andreas T1 - Genomics of pathogenic and commensal \(Escherichia\) \(coli\) T1 - Genomik pathogener und kommensaler \(Escherichia\) \(coli\) N2 - High-throughput sequencing (HTS) has revolutionized bacterial genomics. Its unparalleled sensitivity has opened the door to analyzing bacterial evolution and population genomics, dispersion of mobile genetic elements (MGEs), and within-host adaptation of pathogens, such as Escherichia coli. One of the defining characteristics of intestinal pathogenic E. coli (IPEC) pathotypes is a specific repertoire of virulence factors (VFs). Many of these IPEC VFs are used as typing markers in public health laboratories to monitor outbreaks and guide treatment options. Instead, extraintestinal pathogenic E. coli (ExPEC) isolates are genotypically diverse and harbor a varied set of VFs -- the majority of which also function as fitness factors (FFs) for gastrointestinal colonization. The aim of this thesis was the genomic characterization of pathogenic and commensal E. coli with respect to their virulence- and antibiotic resistance-associated gene content as well as phylogenetic background. In order to conduct the comparative analyses, I created a database of E. coli VFs, ecoli_VF_collection, with a focus on ExPEC virulence-associated proteins (Leimbach, 2016b). Furthermore, I wrote a suite of scripts and pipelines, bac-genomics-scripts, that are useful for bacterial genomics (Leimbach, 2016a). This compilation includes tools for assembly and annotation as well as comparative genomics analyses, like multi-locus sequence typing (MLST), assignment of Clusters of Orthologous Groups (COG) categories, searching for protein homologs, detection of genomic regions of difference (RODs), and calculating pan-genome-wide association statistics. Using these tools we were able to determine the prevalence of 18 autotransporters (ATs) in a large, phylogenetically heterogeneous strain panel and demonstrate that many AT proteins are not associated with E. coli pathotypes. According to multivariate analyses and statistics the distribution of AT variants is instead significantly dependent on phylogenetic lineages. As a consequence, ATs are not suitable to serve as pathotype markers (Zude et al., 2014). During the German Shiga toxin-producing E. coli (STEC) outbreak in 2011, the largest to date, we were one of the teams capable of analyzing the genomic features of two isolates. Based on MLST and detection of orthologous proteins to known E. coli reference genomes the close phylogenetic relationship and overall genome similarity to enteroaggregative E. coli (EAEC) 55989 was revealed. In particular, we identified VFs of both STEC and EAEC pathotypes, most importantly the prophage-encoded Shiga toxin (Stx) and the pAA-type plasmid harboring aggregative adherence fimbriae. As a result, we could show that the epidemic was caused by an unusual hybrid pathotype of the O104:H4 serotype. Moreover, we detected the basis of the antibiotic multi-resistant phenotype on an extended-spectrum beta-lactamase (ESBL) plasmid through comparisons to reference plasmids. With this information we proposed an evolutionary horizontal gene transfer (HGT) model for the possible emergence of the pathogen (Brzuszkiewicz et al., 2011). Similarly to ExPEC, E. coli isolates of bovine mastitis are genotypically and phenotypically highly diverse and many studies struggled to determine a positive association of putative VFs. Instead the general E. coli pathogen-associated molecular pattern (PAMP), lipopolysaccharide (LPS), is implicated as a deciding factor for intramammary inflammation. Nevertheless, a mammary pathogenic E. coli (MPEC) pathotype was proposed presumably encompassing strains more adapted to elicit bovine mastitis with virulence traits differentiating them from commensals. We sequenced eight E. coli isolates from udder serous exudate and six fecal commensals (Leimbach et al., 2016). Two mastitis isolate genomes were closed to a finished-grade quality (Leimbach et al., 2015). The genomic sequence of mastitis-associated E. coli (MAEC) strain 1303 was used to elucidate the biosynthesis gene cluster of its O70 LPS O-antigen. We analyzed the phylogenetic genealogy of our strain panel plus eleven bovine-associated E. coli reference strains and found that commensal or MAEC could not be unambiguously allocated to specific phylogroups within a core genome tree of reference E. coli. A thorough gene content analysis could not identify functional convergence of either commensal or MAEC, instead both have only very few gene families enriched in either pathotype. Most importantly, gene content and ecoli_VF_collection analyses showed that no virulence determinants are significantly associated with MAEC in comparison to bovine fecal commensals, disproving the MPEC hypothesis. The genetic repertoire of bovine-associated E. coli, again, is dominated by phylogenetic background. This is also mostly the case for large virulence-associated E. coli gene cluster previously associated with mastitis. Correspondingly, MAEC are facultative and opportunistic pathogens recruited from the bovine commensal gastrointestinal microbiota (Leimbach et al., 2017). Thus, E. coli mastitis should be prevented rather than treated, as antibiotics and vaccines have not proven effective. Although traditional E. coli pathotypes serve a purpose for diagnostics and treatment, it is clear that the current typing system is an oversimplification of E. coli's genomic plasticity. Whole genome sequencing (WGS) revealed many nuances of pathogenic E. coli, including emerging hybrid or heteropathogenic pathotypes. Diagnostic and public health microbiology need to embrace the future by implementing HTS techniques to target patient care and infection control more efficiently. N2 - Eines der definierenden Charakteristika intestinal pathogener E. coli (IPEC) Pathotypen ist ein spezifisches Repertoire an Virulenzfaktoren (VFs). Viele dieser IPEC VFs werden als Typisierungsmarker benutzt. Stattdessen sind Isolate extraintestinal pathogener E. coli (ExPEC) genotypisch vielfältig und beherbergen verschiedenartige VF Sets, welche in der Mehrheit auch als Fitnessfaktoren (FFs) für die gastrointestinale Kolonialisierung fungieren. Das Ziel dieser Dissertation war die genomische Charakterisierung pathogener und kommensaler E. coli in Bezug auf ihren Virulenz- und Antibiotikaresistenz-assoziierten Gengehalt sowie ihre phylogenetische Abstammung. Als Voraussetzung für die vergleichenden Analysen erstellte ich eine E. coli VF-Datenbank, ecoli_VF_collection, mit Fokus auf Virulenz-assoziierte Proteine von ExPEC (Leimbach, 2016b). Darüber hinaus programmierte ich mehrere Skripte und Pipelines zur Anwendung in der bakteriellen Genomik, bac-genomics-scripts (Leimbach, 2016a). Diese Sammlung beinhaltet Tools zur Unterstützung von Assemblierung und Annotation sowie komparativer Genomanalysen, wie Multilokus-Sequenztypisierung (MLST), Zuweisung von Clusters of Orthologous Groups (COG) Kategorien, Suche nach homologen Proteinen, Identifizierung von genomisch unterschiedlichen Regionen (RODs) und Berechnung Pan-genomweiter Assoziationsstatistiken. Mithilfe dieser Tools konnten wir die Prävalenz von 18 Autotransportern (ATs) in einer großen, phylogenetisch heterogenen Stammsammlung bestimmen und nachweisen, dass viele AT-Proteine nicht mit E. coli Pathotypen assoziiert sind. Multivariate Analysen und Statistik legten offen, dass die Verteilung von AT-Varianten vielmehr signifikant von phylogenetischen Abstammungslinien abhängt. Deshalb sind ATs nicht als Marker für Pathotypen geeignet (Zude et al., 2014). Während des bislang größten Ausbruchs von Shiga-Toxin-produzierenden E. coli (STEC) im Jahre 2011 in Deutschland waren wir eines der Teams, welches die genomischen Eigenschaften zweier Isolate analysieren konnte. Basierend auf MLST und Detektion orthologer Proteine zu bekannten E. coli Referenzgenomen konnte ihre enge phylogenetische Verwandschaft und Ähnlichkeit des gesamten Genoms zum enteroaggregativen E. coli (EAEC) 55989 aufgedeckt werden. Im Detail identifizierten wir VFs von STEC und EAEC Pathotypen, vor allem das Prophagen-kodierte Shiga-Toxin (Stx) und ein Plasmid des pAA-Typs kodierend für aggregative Adhärenz-Fimbrien. Die Epidemie wurde demnach durch einen ungewöhnlichen Hybrid-Pathotyp vom O104:H4 Serotyp verursacht. Zusätzlich identifizierten wir die Grundlage für den multiresistenten Phänotyp dieser Ausbruchsstämme auf einem Extended-Spektrum-beta-Laktamase (ESBL) Plasmid über Vergleiche mit Referenzplasmiden. Mit diesen Informationen konnten wir ein horizontales Gentransfer-Modell (HGT) zum Auftreten dieses Pathogenen vorschlagen (Brzuszkiewicz et al., 2011). Ähnlich zu ExPEC sind E. coli Isolate boviner Mastitiden genotypisch und phänotypisch sehr divers, und viele Studien scheiterten am Versuch eine positive Assoziation vermeintlicher VFs nachzuweisen. Stattdessen gilt Lipopolysaccharid (LPS) als entscheidender Faktor zur intramammären Entzündung. Gleichwohl wurde ein mammärer pathogener E. coli (MPEC) Pathotyp vorgeschlagen, der mutmaßlich Stämme umfasst, welche eher geeignet sind eine bovine Mastitis auszulösen und über Virulenz-Merkmale von Kommensalen abgegrenzt werden können. Wir sequenzierten acht E. coli Isolate aus serösem Eutersekret und sechs fäkale Kommensale (Leimbach et al., 2016). Bei zwei Mastitisisolaten wurden die Genome vollständig geschlossen (Leimbach et al., 2015). Anhand der genomischen Sequenz des Mastitis-assoziierten E. coli (MAEC) Stamms 1303 wurde das Gencluster zur Biosynthese seines O70 LPS O-Antigens aufgeklärt. Wir analysierten die phylogenetische Abstammung unserer Stammsammlung plus elf bovin-assoziierter E. coli Referenzstämme, aber konnten weder MAEC noch Kommensale bestimmten Phylogruppen innerhalb eines Core-Genom Stammbaums aus Referenz-E. coli eindeutig zuordnen. Eine ausführliche Gengehalt-Analyse konnte keine funktionelle Konvergenz innerhalb von Kommensalen oder MAEC identifizieren. Stattdessen besitzen beide nur sehr wenige Genfamilien, die bevorzugt in einer der beiden Pathotypen vorkommen. Weder eine Gengehalt- noch eine ecoli_VF_collection-Analyse konnte zeigen, dass eine signifikante Assoziation von bestimmten Virulenzfaktoren mit MAEC, im Vergleich zu bovinen fäkalen Kommensalen, besteht. Damit wurde die MPEC Hypothese widerlegt. Auch das genetische Repertoire von Rinder-assoziierten E. coli wird durch die phylogenetische Abstammung bestimmt. Dies ist überwiegend auch bei großen Virulenz-assoziierten Genclustern der Fall, die bisher mit Mastitis in Verbindung gebracht wurden. Dementsprechend sind MAEC fakultative und opportunistische Pathogene, die ihren Ursprung als Kommensale in der bovinen gastrointestinalen Mikrobiota haben (Leimbach et al., 2017). Obwohl traditionelle E. coli Pathotypen in der Diagnostik und Behandlung einen Zweck erfüllen, ist es offensichtlich, dass das derzeitige Typisierungs-System die genomische Plastizität von E. coli zu sehr vereinfacht. Die Gesamtgenom-Sequenzierung (WGS) deckte viele Nuancen pathogener E. coli auf, einschließlich entstehender hybrider oder heteropathogener Pathotypen. Diagnostische und medizinische Mikrobiologie müssen einen Schritt in Richtung Zukunft gehen und HTS-Technologien anwenden, um Patientenversorgung und Infektionskontrolle effizienter zu unterstützen. KW - Escherichia coli KW - Autotransporter KW - STEC KW - Bovine Mastitis KW - high-throughput sequencing KW - virulence factors KW - pathotypes KW - phylogeny KW - ecoli_VF_collection KW - bac-genomics-scripts KW - autotransporter KW - entero-aggregative-haemorrhagic Escherichia coli (EAHEC) KW - mastitis-associated Escherichia coli (MAEC) Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-154539 ER - TY - JOUR A1 - Hickey, Scott F. A1 - Sridhar, Malathy A1 - Westermann, Alexander J. A1 - Qin, Qian A1 - Vijayendra, Pooja A1 - Liou, Geoffrey A1 - Hammond, Ming C. T1 - Transgene regulation in plants by alternative splicing of a suicide exon JF - Nucleic Acids Research N2 - Compared to transcriptional activation, other mechanisms of gene regulation have not been widely exploited for the control of transgenes. One barrier to the general use and application of alternative splicing is that splicing-regulated transgenes have not been shown to be reliably and simply designed. Here, we demonstrate that a cassette bearing a suicide exon can be inserted into a variety of open reading frames (ORFs), generating transgenes whose expression is activated by exon skipping in response to a specific protein inducer. The surprisingly minimal sequence requirements for the maintenance of splicing fidelity and regulation indicate that this splicing cassette can be used to regulate any ORF containing one of the amino acids Glu, Gln or Lys. Furthermore, a single copy of the splicing cassette was optimized by rational design to confer robust gene activation with no background expression in plants. Thus, conditional splicing has the potential to be generally useful for transgene regulation. KW - kingdom KW - pre-messenger RNA KW - gene expression KW - elements KW - decay KW - arabidopsis KW - eukaryotes KW - mechanisms Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134724 VL - 40 IS - 10 ER - TY - JOUR A1 - Afonso-Grunz, Fabian A1 - Hoffmeier, Klaus A1 - Müller, Sören A1 - Westermann, Alexander J. A1 - Rotter, Björn A1 - Vogel, Jörg A1 - Winter, Peter A1 - Kahl, Günter T1 - Dual 3'Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells JF - BMC Genomics N2 - Background: The interaction of eukaryotic host and prokaryotic pathogen cells is linked to specific changes in the cellular proteome, and consequently to infection-related gene expression patterns of the involved cells. To simultaneously assess the transcriptomes of both organisms during their interaction we developed dual 3'Seq, a tag-based sequencing protocol that allows for exact quantification of differentially expressed transcripts in interacting pro-and eukaryotic cells without prior fixation or physical disruption of the interaction. Results: Human epithelial cells were infected with Salmonella enterica Typhimurium as a model system for invasion of the intestinal epithelium, and the transcriptional response of the infected host cells together with the differential expression of invading and intracellular pathogen cells was determined by dual 3'Seq coupled with the next-generation sequencing-based transcriptome profiling technique deepSuperSAGE (deep Serial Analysis of Gene Expression). Annotation to reference transcriptomes comprising the operon structure of the employed S. enterica Typhimurium strain allowed for in silico separation of the interacting cells including quantification of polycistronic RNAs. Eighty-nine percent of the known loci are found to be transcribed in prokaryotic cells prior or subsequent to infection of the host, while 75% of all protein-coding loci are represented in the polyadenylated transcriptomes of human host cells. Conclusions: Dual 3'Seq was alternatively coupled to MACE (Massive Analysis of cDNA ends) to assess the advantages and drawbacks of a library preparation procedure that allows for sequencing of longer fragments. Additionally, the identified expression patterns of both organisms were validated by qRT-PCR using three independent biological replicates, which confirmed that RELB along with NFKB1 and NFKB2 are involved in the initial immune response of epithelial cells after infection with S. enterica Typhimurium. KW - complete genome sequence KW - secretion systems KW - RNA-Seq KW - deepSuperSAGE KW - transcriptome KW - gene expression KW - serovar Typhimurium KW - human macrophages KW - epithelial cells KW - infection KW - SuperSAGE KW - receptors KW - Dual 3'seq KW - MACE KW - tag based KW - simultaneous KW - genome wide KW - gene expression profiling KW - host pathogen interaction KW - Salmonella enterica Typhimurium strain SL1344 Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143230 VL - 16 IS - 323 ER - TY - JOUR A1 - Rodriguez, Héctor A1 - Rico, Sergio A1 - Yepes, Ana A1 - Franco-Echevarría, Elsa A1 - Antoraz, Sergio A1 - Santamaría, Ramón I. A1 - Díaz, Margerita T1 - The two kinases, AbrC1 and AbrC2, of the atypical two-component system AbrC are needed to regulate antibiotic production and differentiation in Streptomyces coelicolor JF - Frontiers in Microbiology N2 - Two-component systems (TCSs) are the most important sensing mechanisms in bacteria. In Streptomyces, TCSs-mediated responses to environmental stimuli are involved in the regulation of antibiotic production. This study examines the individual role of two histidine kinases (HKs), AbrC1 and AbrC2, which form part of an atypical TCS in Streptomyces coelicolor. gRT-PCR analysis of the expression of both kinases demonstrated that both are expressed at similar levels in NB and NMMP media. Single deletion of abrC1 elicited a significant increase in antibiotic production, while deletion of abrC2 did not have any clear effect. The origin of this phenotype, probably related to the differential phosphorylation ability of the two kinases, was also explored indirectly, analyzing the toxic phenotypes associated with high levels of phosphorylated RR. The higher the AbrC3 regulator phosphorylation rate, the greater the cell toxicity. For the first time, the present work shows in Streptomyces the combined involvement of two different HKs in the response of a regulator to environmental signals. Regarding the possible applications of this research, the fact that an abrC1 deletion mutant overproduces three of the S. coelicolor antibiotics makes this strain an excellent candidate as a host for the heterologous production of secondary metabolites. KW - halstedii JM8 KW - biosynthesis KW - expression mutants KW - domain genes A3(2) KW - two-component systems KW - Streptomyces KW - antibiotic production KW - histidine kinases KW - heterologous production KW - activation KW - response regulator KW - PCR Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143048 VL - 6 IS - 450 ER - TY - JOUR A1 - Nguyen, Minh Thu A1 - Kraft, Beatrice A1 - Yu, Wenqi A1 - Demicrioglu, Dogan Doruk A1 - Hertlein, Tobias A1 - Burian, Marc A1 - Schmaler, Mathias A1 - Boller, Klaus A1 - Bekeredjian-Ding, Isabelle A1 - Ohlsen, Knut A1 - Schittek, Birgit A1 - Götz, Friedrich T1 - The vSa\(\alpha\) Specific Lipoprotein Like Cluster (lpl) of S. aureus USA300 Contributes to Immune Stimulation and Invasion in Human Cells JF - PLoS Pathogens N2 - All Staphylococcus aureus genomes contain a genomic island, which is termed vSa\(\alpha\) and characterized by two clusters of tandem repeat sequences, i.e. the exotoxin (set) and 'lipoprotein-like' genes (lpl). Based on their structural similarities the vSa\(\alpha\) islands have been classified as type I to IV. The genomes of highly pathogenic and particularly epidemic S. aureus strains (USA300, N315, Mu50, NCTC8325, Newman, COL, JH1 or JH9) belonging to the clonal complexes CC5 and CC8 bear a type I vSa\(\alpha\) island. Since the contribution of the lpl gene cluster encoded in the vSa\(\alpha\) island to virulence is unclear to date, we deleted the entire lpl gene cluster in S. aureus USA300. The results showed that the mutant was deficient in the stimulation of pro-inflammatory cytokines in human monocytes, macrophages and keratinocytes. Purified lipoprotein Lpl1 was further shown to elicit a TLR2-dependent response. Furthermore, heterologous expression of the USA300 lpl cluster in other S. aureus strains enhanced their immune stimulatory activity. Most importantly, the lpl cluster contributed to invasion of S. aureus into human keratinocytes and mouse skin and the non-invasive S. carnosus expressing the lpl gene cluster became invasive. Additionally, in a murine kidney abscess model the bacterial burden in the kidneys was higher in wild type than in mutant mice. In this infection model the lpl cluster, thus, contributes to virulence. The present report is one of the first studies addressing the role of the vSa\(\alpha\) encoded lpl gene cluster in staphylococcal virulence. The finding that the lpl gene cluster contributes to internalization into non-professional antigen presenting cells such as keratinocytes high-lights the lpl as a new cell surface component that triggers host cell invasion by S. aureus. Increased invasion in murine skin and an increased bacterial burden in a murine kidney abscess model suggest that the lpl gene cluster serves as an important virulence factor. KW - resistant Staphylococcus-aureus KW - bacterial lipoproteins KW - internalization KW - evolution KW - fibronectin-binding protein KW - toll-like receptor 2 KW - epithelial cells KW - genome sequence KW - activation KW - mechanisms Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-151856 VL - 11 IS - 6 ER - TY - JOUR A1 - Espina, Laura A1 - Pagán, Rafael A1 - López, Daniel A1 - García-Gonzalo, Diego T1 - Individual Constituents from Essential Oils Inhibit Biofilm Mass Production by Multi-Drug Resistant Staphylococcus aureus JF - Molecules N2 - Biofilm formation by Staphylococcus aureus represents a problem in both the medical field and the food industry, because the biofilm structure provides protection to embedded cells and it strongly attaches to surfaces. This circumstance is leading to many research programs seeking new alternatives to control biofilm formation by this pathogen. In this study we show that a potent inhibition of biofilm mass production can be achieved in community-associated methicillin-resistant S. aureus (CA-MRSA) and methicillin-sensitive strains using plant compounds, such as individual constituents (ICs) of essential oils (carvacrol, citral, and (+)-limonene). The Crystal Violet staining technique was used to evaluate biofilm mass formation during 40 h of incubation. Carvacrol is the most effective IC, abrogating biofilm formation in all strains tested, while CA-MRSA was the most sensitive phenotype to any of the ICs tested. Inhibition of planktonic cells by ICs during initial growth stages could partially explain the inhibition of biofilm formation. Overall, our results show the potential of EOs to prevent biofilm formation, especially in strains that exhibit resistance to other antimicrobials. As these compounds are food additives generally recognized as safe, their anti-biofilm properties may lead to important new applications, such as sanitizers, in the food industry or in clinical settings. KW - Listeria monocytogenes KW - carvacrol KW - strains KW - essential oils KW - anti-biofilm KW - bacterial biofilms KW - food industry KW - antibacterial KW - inactivation KW - components KW - citrus KW - biofilms KW - Staphylococcus aureus KW - (+)-limonene KW - citral Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-151845 VL - 20 SP - 11357 EP - 11372 ER - TY - INPR A1 - Bartfeld, Sina T1 - Modeling infectious diseases and host-microbe interactions in gastrointestinal organoids T2 - Developmental Biology N2 - Advances in stem cell research have allowed the development of 3-dimensional (3D) primary cell cultures termed organoid cultures, as they closely mimic the in vivo organization of different cell lineages. Bridging the gap between 2-dimensional (2D) monotypic cancer cell lines and whole organisms, organoids are now widely applied to model development and disease. Organoids hold immense promise for addressing novel questions in host-microbe interactions, infectious diseases and the resulting inflammatory conditions. Researchers have started to use organoids for modeling infection with pathogens, such as Helicobacter pylori or Salmonella enteritica, gut- microbiota interactions and inflammatory bowel disease. Future studies will broaden the spectrum of microbes used and continue to establish organoids as a standard model for human host-microbial interactions. Moreover, they will increasingly exploit the unique advantages of organoids, for example to address patient-specific responses to microbes. KW - gastrointestinal disease KW - salmonella KW - microbiota KW - inflammatory bowel disease KW - organoid culture KW - helicobacter KW - rotavirus KW - norovirus Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-138788 UR - http://www.sciencedirect.com/science/article/pii/S0012160616304602 SN - 0012-1606 N1 - This is the accepted version of the following article: Bartfeld, Sina, Modeling infectious diseases and host-microbe interactions in gastrointestinal organoids, Developmental Biology, 2016, 420, 2, 262-270. http://dx.doi.org/10.1016/j.ydbio.2016.09.014 ER - TY - THES A1 - Sharan, Malvika T1 - Bio-computational identification and characterization of RNA-binding proteins in bacteria T1 - Bioinformatische Identifikation und Charakterisierung von RNA-bindenden Proteinen in Bakterien N2 - RNA-binding proteins (RBPs) have been extensively studied in eukaryotes, where they post-transcriptionally regulate many cellular events including RNA transport, translation, and stability. Experimental techniques, such as cross-linking and co-purification followed by either mass spectrometry or RNA sequencing has enabled the identification and characterization of RBPs, their conserved RNA-binding domains (RBDs), and the regulatory roles of these proteins on a genome-wide scale. These developments in quantitative, high-resolution, and high-throughput screening techniques have greatly expanded our understanding of RBPs in human and yeast cells. In contrast, our knowledge of number and potential diversity of RBPs in bacteria is comparatively poor, in part due to the technical challenges associated with existing global screening approaches developed in eukaryotes. Genome- and proteome-wide screening approaches performed in silico may circumvent these technical issues to obtain a broad picture of the RNA interactome of bacteria and identify strong RBP candidates for more detailed experimental study. Here, I report APRICOT (“Analyzing Protein RNA Interaction by Combined Output Technique”), a computational pipeline for the sequence-based identification and characterization of candidate RNA-binding proteins encoded in the genomes of all domains of life using RBDs known from experimental studies. The pipeline identifies functional motifs in protein sequences of an input proteome using position-specific scoring matrices and hidden Markov models of all conserved domains available in the databases and then statistically score them based on a series of sequence-based features. Subsequently, APRICOT identifies putative RBPs and characterizes them according to functionally relevant structural properties. APRICOT performed better than other existing tools for the sequence-based prediction on the known RBP data sets. The applications and adaptability of the software was demonstrated on several large bacterial RBP data sets including the complete proteome of Salmonella Typhimurium strain SL1344. APRICOT reported 1068 Salmonella proteins as RBP candidates, which were subsequently categorized using the RBDs that have been reported in both eukaryotic and bacterial proteins. A set of 131 strong RBP candidates was selected for experimental confirmation and characterization of RNA-binding activity using RNA co-immunoprecipitation followed by high-throughput sequencing (RIP-Seq) experiments. Based on the relative abundance of transcripts across the RIP-Seq libraries, a catalogue of enriched genes was established for each candidate, which shows the RNA-binding potential of 90% of these proteins. Furthermore, the direct targets of few of these putative RBPs were validated by means of cross-linking and co-immunoprecipitation (CLIP) experiments. This thesis presents the computational pipeline APRICOT for the global screening of protein primary sequences for potential RBPs in bacteria using RBD information from all kingdoms of life. Furthermore, it provides the first bio-computational resource of putative RBPs in Salmonella, which could now be further studied for their biological and regulatory roles. The command line tool and its documentation are available at https://malvikasharan.github.io/APRICOT/. N2 - RNA-bindende Proteine (RBPs) wurden umfangreich in Eukaryoten erforscht, in denen sie viele Prozesse wie RNA-Transport, -Translation und -Stabilität post-transkriptionell regulieren. Experimentelle Methoden wie Cross-linking and Koimmunpräzipitation mit nachfolgedener Massenspektromentrie / RNA-Sequenzierung ermöglichten eine weitreichende Charakterisierung von RBPs, RNA-bindenden Domänen (RBDs) und deren regulatorischen Rollen in eukaryotischen Spezies wie Mensch und Hefe. Weitere Entwicklungen im Bereich der hochdurchsatzbasierten Screeningverfahren konnten das Verständnis von RBPs in Eukaryoten enorm erweitern. Im Gegensatz dazu ist das Wissen über die Anzahl und die potenzielle Vielfalt von RBPs in Bakterien dürftig. In der vorliegenden Arbeit präsentiere ich APRICOT, eine bioinformatische Pipeline zur sequenzbasierten Identifikation und Charakterisierung von Proteinen aller Domänen des Lebens, die auf RBD-Informationen aus experimentellen Studien aufbaut. Die Pipeline nutzt Position Specific Scoring Matrices und Hidden-MarkovModelle konservierter Domänen, um funktionelle Motive in Proteinsequenzen zu identifizieren und diese anhand von sequenzbasierter Eigenschaften statistisch zu bewerten. Anschließend identifiziert APRICOT mögliche RBPs und charakterisiert auf Basis ihrer biologischeren Eigenschaften. In Vergleichen mit ähnlichen Werkzeugen übertraf APRICOT andere Programme zur sequenzbasierten Vorhersage von RBPs. Die Anwendungsöglichkeiten und die Flexibilität der Software wird am Beispiel einiger großer RBP-Kollektionen, die auch das komplette Proteom von Salmonella Typhimurium SL1344 beinhalten, dargelegt. APRICOT identifiziert 1068 Proteine von Salmonella als RBP-Kandidaten, die anschließend unter Nutzung der bereits bekannten bakteriellen und eukaryotischen RBDs klassifiziert wurden. 131 der RBP-Kandidaten wurden zur Charakterisierung durch RNA co-immunoprecipitation followed by high-throughput sequencing (RIP-seq) ausgewählt. Basierend auf der relativen Menge an Transkripten in den RIP-seq-Bibliotheken wurde ein Katalog von angereicherten Genen erstellt, der auf eine potentielle RNA-bindende Funktion in 90% dieser Proteine hindeutet. Weiterhin wurden die Bindungstellen einiger dieser möglichen RBPs mit Cross-linking and Co-immunoprecipitation (CLIP) bestimmt. Diese Doktorarbeit beschreibt die bioinformatische Pipeline APRICOT, die ein globales Screening von RBPs in Bakterien anhand von Informationen bekannter RBDs ermöglicht. Zudem enthält sie eine Zusammenstellung aller potentieller RPS in Salmonella, die nun auf ihre biologsche Funktion hin untersucht werden können. Das Kommondozeilen-Programm und seine Dokumentation sind auf https://malvikasharan.github.io/APRICOT/ verfügbar. KW - Bioinformatics Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-153573 ER - TY - JOUR A1 - Masic, Anita A1 - Hurdayal, Ramona A1 - Nieuwenhuizen, Natalie E. A1 - Brombacher, Frank A1 - Moll, Heidrun T1 - Dendritic Cell-Mediated Vaccination Relies on Interleukin-4 Receptor Signaling to Avoid Tissue Damage after Leishmania major Infection of BALB/c Mice JF - PLoS Neglected Tropical Diseases N2 - Prevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor \(\alpha\) (IL-4R \(\alpha\))-deficient (CD11c\(^{cre}\)IL-4R \(\alpha^{-/lox}\) BALB/c mice were given either wt or IL-4R \(\alpha\)-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2x10\(^5\) stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4R alpha-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4R alpha-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11c\(^{cre}\)IL-4R \(\alpha^{-/lox}\) mice immunized with CpG ODN-exposed LmAg-loaded IL-4R \(\alpha\)-deficient DC, indicating the influence of IL-4R \(\alpha\)-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4R \(\alpha\) signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms. KW - cytokines KW - necrosis-factor-alpha KW - T helper cell KW - visceral leishmaniasis KW - intracellular pathogen KW - interferon-gamma KW - IL-12 production KW - deficient mice KW - resistance KW - responses Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133869 VL - 6 IS - 7 ER - TY - THES A1 - Hagmann [geb. Kischkies], Laura Violetta T1 - Stringent response regulation and its impact on ex vivo survival in the commensal pathogen \(Neisseria\) \(meningitidis\) T1 - Regulation der stringenten Kontrolle und ihre Auswirkungen auf das ex vivo Überleben des kommensalen Erregers \(Neisseria\) \(meningitidis\) N2 - Neisseria meningitidis is a commensal bacterium which sometimes causes serious disease in humans. Recent studies in numerous human pathogenic bacteria have shown that the stringent response contributes to bacterial virulence. Therefore, this study analyzed the regulation of the stringent response in meningococci and in particular of RelA as well as its contribution to ex vivo fitness in a strain- and condition- dependent manner by using the carriage strain α522 and the hyperinvasive strain MC58 in different in vitro and ex vivo conditions. Growth experiments revealed that both wild-type strains were almost indistinguishable in their ex vivo phenotypes. However, quantitative real time PCR (qRT-PCR) found differences in the gene expression of relA between both strains. Furthermore, in contrast to the MC58 RelA mutant strain α522 deficient in RelA was unable to survive in human whole blood, although both strains showed the same ex vivo phenotypes in saliva and cerebrospinal fluid. Moreover, strain α522 was depended on a short non-coding AT-rich repeat element (ATRrelA) in the promoter region of relA to survive in human blood. Furthermore, cell culture experiments with human epithelial cells revealed that in both strains the deletion of relA resulted in a significantly decreased invasion rate while not significantly affecting adhesion. In order to better understand the conditional lethality of the relA deletion, computational and experimental analyses were carried out to unravel differences in amino acid biosynthetic pathways between both strains. Whereas strain MC58 is able to synthesize all 20 amino acids, strain α522 has an auxotrophy for cysteine and glutamine. In addition, the in vitro growth experiments found that RelA is required for growth in the absence of external amino acids in both strains. Furthermore, the mutant strain MC58 harboring an ATRrelA in its relA promoter region showed improved growth in minimal medium supplemented with L-cysteine and/or L-glutamine compared to the wild-type strain. Contrary, in strain α522 no differences between the wild-type and the ATRrelA deletion mutant were observed. Together this indicates that ATRrelA interferes with the complex regulatory interplay between the stringent response pathway and L-cysteine as well as L-glutamine metabolism. It further suggests that meningococcal virulence is linked to relA in a strain- and condition- depended manner. In conclusion, this work highlighted the role of the stringent response and of non-coding regulatory elements for bacterial virulence and indicates that virulence might be related to the way how meningococci accomplish growth within the host environments. N2 - Neisseria meningitidis ist ein kommensal lebendes, fakultativ pathogenes Bakterium, welches unter nicht vollständig verstandenen Umständen lebensbedrohliche Krankheitsbilder bei Menschen verursacht. Aktuelle Studien haben gezeigt, dass die stringente Antwort einen Einfluss auf die bakterielle Virulenz haben kann. Aus diesem Grund untersucht diese Arbeit die Regulation der stringenten Antwort, insbesondere die Rolle von RelA, sowie den Einfluss der stringenten Antwort auf die Ex-vivo-Fitness der Meningokokken. Die Ergebnisse wurden für den Trägerstamm α522 und den hyperinvasiven Stamm MC58 erhoben und miteinander verglichen. Wachstumsexperimente zeigten, dass sich beide Wildtyp-Stämme in ihren Ex-vivo-Phänotypen nicht unterscheiden. Jedoch wurden mittels quantitativer Echtzeit-PCR (qRT-PCR) Unterschiede zwischen beiden Stämmen in der Genexpression von relA gefunden. Zudem war die α522 relA Mutante im Gegensatz zu der MC58 relA Mutante nicht in der Lage, in menschlichem Vollblut zu überleben. Allerdings zeigten in Saliva und Liquor beide Stämme den gleichen Phänotyp. Außerdem war der Trägerstamm auf eine kurze, nicht-codierende AT-reiche Region (ATRrelA) in der Promotorregion von relA angewiesen, um im menschlichen Blut überleben zu können. Darüber hinaus zeigten Zellkulturexperimente mit humanen Epithelzellen, dass die Deletion relA die Invasionsrate in beiden Stämmen signifikant verringerte, obwohl die Adhäsionsrate durch die Deletion unbeeinflusst blieb. Um besser verstehen zu können, weshalb die Deletion von relA unter bestimmten Bedingungen letal ist, wurden mit In-silico- und experimentellen Analysen nach Unterschieden in den Aminosäurebiosynthesewegen beider Stämme gesucht. Es zeigte sich, dass Stamm MC58 in der Lage ist alle 20 Aminosäuren zu synthetisieren, während Stamm α522 eine Auxotrophie für Cystein und Glutamin aufweist. Ferner zeigten die In-vitro-Wachstumsversuche, dass RelA bei Aminosäuremangel essentiell für beide Stämme ist. Darüber hinaus zeigte eine MC58 Mutante mit einer ATRrelA –Kopie in der relA Promotorregion ein im Vergleich zum Wildtyp-Stamm verbessertes Wachstum in mit L-Cystein und/oder L-Glutamin angereichertem Minimalmedium. Gegensätzlich dazu zeigte der Stamm α522 keine Unterschiede im Wachstum zwischen dem Wildtyp und einer ATRrelA Deletions-Mutante. Dies deutet darauf hin, dass ATRrelA an dem komplexen regulatorischen Zusammenspiel der stringenten Antwort und dem L-Cystein- beziehungsweise dem L-Glutamin-Metabolismus beteiligt ist. Es lässt sich vermuten, dass RelA zu der Virulenz von Meningokokken in einer stamm- und umgebungsspezifischen Weise beiträgt. Abschließend hebt diese Arbeit die Rolle von kleinen regulatorischen Elementen für die bakterielle Virulenz hervor und postuliert, dass die Virulenz der Meningokokken auf ihrer Fähigkeit basiert, sich der durch den Wirt gegebenen Umgebung anzupassen. KW - Neisseria meningitidis KW - Stringente Kontrolle KW - Virulenzfaktor KW - Genregulation KW - Transposon KW - Stringent response KW - RelA KW - MITE KW - Stringente Antwort Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144352 ER - TY - JOUR A1 - Schmidtke, Cornelius A1 - Findeiß, Sven A1 - Sharma, Cynthia M. A1 - Kuhfuss, Juliane A1 - Hoffmann, Steve A1 - Vogel, Jörg A1 - Stadler, Peter F. A1 - Bonas, Ulla T1 - Genome-wide transcriptome analysis of the plant pathogen Xanthomonas identifies sRNAs with putative virulence functions JF - Nucleic Acids Research N2 - The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is an important model to elucidate the mechanisms involved in the interaction with the host. To gain insight into the transcriptome of the Xcv strain 85-10, we took a differential RNA sequencing (dRNA-seq) approach. Using a novel method to automatically generate comprehensive transcription start site (TSS) maps we report 1421 putative TSSs in the Xcv genome. Genes in Xcv exhibit a poorly conserved -10 promoter element and no consensus Shine-Dalgarno sequence. Moreover, 14% of all mRNAs are leaderless and 13% of them have unusually long 5'-UTRs. Northern blot analyses confirmed 16 intergenic small RNAs and seven cis-encoded antisense RNAs in Xcv. Expression of eight intergenic transcripts was controlled by HrpG and HrpX, key regulators of the Xcv type III secretion system. More detailed characterization identified sX12 as a small RNA that controls virulence of Xcv by affecting the interaction of the pathogen and its host plants. The transcriptional landscape of Xcv is unexpectedly complex, featuring abundant antisense transcripts, alternative TSSs and clade-specific small RNAs. KW - SUBSP carotovora KW - regulatory RNA KW - gene-cluster KW - campestris PV vesicatoria KW - escherichia coli KW - determines pathgenicity KW - hypersensitive response KW - ralstonia solanacearum KW - extracellular enzymes KW - secretion systems KW - transcription initiation site KW - RNA sequence analyses KW - messanger RNA KW - plants KW - libraries KW - genome KW - genes KW - gene expression profiling KW - genetic transcription KW - northern blotting KW - untranslated regions KW - xanthomonas KW - xanthomonas campestris KW - bacteria KW - virulence KW - pathogenetic organism KW - RNA KW - small RNA KW - pathogenicity KW - type III secretion system pathways KW - maps KW - consesus KW - host (organism) KW - type III protein secretion system complex Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131781 VL - 40 IS - 5 SP - 2020 EP - 2031 ER - TY - JOUR A1 - Pils, Stefan A1 - Kopp, Kathrin A1 - Peterson, Lisa A1 - Tascon, Julia Delgado A1 - Nyffenegger-Jann, Naja J. A1 - Hauck, Christof R. T1 - The Adaptor Molecule Nck Localizes the WAVE Complex to Promote Actin Polymerization during CEACAM3-Mediated Phagocytosis of Bacteria JF - PLoS One N2 - Background: CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. Principal Findings: In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Conclusions: Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment. KW - activation KW - neisseria gonorrhoeae KW - human pathogens KW - T cell KW - signal transduction KW - escherichia coli KW - epithelial cells KW - tyrosine kinase KW - receptor KW - adhesion Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131747 VL - 7 IS - 3 ER - TY - JOUR A1 - Ramachandran, Vinoy K. A1 - Shearer, Neil A1 - Jacob, Jobin J. A1 - Sharma, Cynthia M. A1 - Thompson, Arthur T1 - The architecture and ppGpp-dependent expression of the primary transcriptome of Salmonella Typhimurium during invasion gene expression JF - BMC Genomics N2 - Background: Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium (S. Typhimurium) requires expression of the extracellular virulence gene expression programme (STEX), activation of which is dependent on the signalling molecule guanosine tetraphosphate (ppGpp). Recently, next-generation transcriptomics (RNA-seq) has revealed the unexpected complexity of bacterial transcriptomes and in this report we use differential RNA sequencing (dRNA-seq) to define the high-resolution transcriptomic architecture of wildtype S. Typhimurium and a ppGpp null strain under growth conditions which model STEX. In doing so we show that ppGpp plays a much wider role in regulating the S. Typhimurium STEX primary transcriptome than previously recognised. Results: Here we report the precise mapping of transcriptional start sites (TSSs) for 78% of the S. Typhimurium open reading frames (ORFs). The TSS mapping enabled a genome-wide promoter analysis resulting in the prediction of 169 alternative sigma factor binding sites, and the prediction of the structure of 625 operons. We also report the discovery of 55 new candidate small RNAs (sRNAs) and 302 candidate antisense RNAs (asRNAs). We discovered 32 ppGpp-dependent alternative TSSs and determined the extent and level of ppGpp-dependent coding and non-coding transcription. We found that 34% and 20% of coding and non-coding RNA transcription respectively was ppGpp-dependent under these growth conditions, adding a further dimension to the role of this remarkable small regulatory molecule in enabling rapid adaptation to the infective environment. Conclusions: The transcriptional architecture of S. Typhimurium and finer definition of the key role ppGpp plays in regulating Salmonella coding and non-coding transcription should promote the understanding of gene regulation in this important food borne pathogen and act as a resource for future research. KW - legionella pneumophila KW - growth rate control KW - escherichia coli K-12 KW - pathogenicity island 2 KW - bacterial signal molecule KW - enterica serovar typhimurium KW - messenger RNA KW - protein synthesis KW - sationary phase KW - environmental regulation Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130625 VL - 13 IS - 25 ER - TY - JOUR A1 - Wang, Huiqiang A1 - Chen, Nanhai G. A1 - Minev, Boris R. A1 - Szalay, Aladar A. T1 - Oncolytic vaccinia virus GLV-1h68 strain shows enhanced replication in human breast cancer stem-like cells in comparison to breast cancer cells JF - Journal of Translational Medicine N2 - Background: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. Methods: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44\(^+\)CD24\(^+\)ESA\(^+\) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. Results: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44\(^+\)CD24\(^+\)ESA\(^+\) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44\(^+\)CD24\(^-\)ESA\(^+\) cells. Conclusions: Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors. KW - tumors KW - therapy KW - metastasis KW - identification KW - lines KW - gene expression KW - in-vitro propagation KW - acute myeloid leukemia KW - epithelial-mesenchymal transition KW - subpopulation Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130019 VL - 10 IS - 167 ER - TY - JOUR A1 - Gentschev, Ivaylo A1 - Adelfinger, Marion A1 - Josupeit, Rafael A1 - Rudolph, Stephan A1 - Ehrig, Klaas A1 - Donat, Ulrike A1 - Weibel, Stephanie A1 - Chen, Nanhai G. A1 - Yu, Yong A. A1 - Zhang, Qian A1 - Heisig, Martin A1 - Thamm, Douglas A1 - Stritzker, Jochen A1 - MacNeill, Amy A1 - Szalay, Aladar A. T1 - Preclinical Evaluation of Oncolytic Vaccinia Virus for Therapy of Canine Soft Tissue Sarcoma JF - PLoS One N2 - Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS. KW - breast-tumors KW - animal-model KW - nude-mice KW - cell-line KW - in-vitro KW - glv-1h68 KW - cancer KW - virotherapy KW - dogs KW - neutrophils Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129998 VL - 7 IS - 5 ER - TY - JOUR A1 - Sass, Andrea M. A1 - Van Acker, Heleen A1 - Förstner, Konrad U. A1 - Van Nieuwerburgh, Filip A1 - Deforce, Dieter A1 - Vogel, Jörg A1 - Coenye, Tom T1 - Genome-wide transcription start site profiling in biofilm-grown Burkholderia cenocepacia J2315 JF - BMC Genomics N2 - Background: Burkholderia cenocepacia is a soil-dwelling Gram-negative Betaproteobacterium with an important role as opportunistic pathogen in humans. Infections with B. cenocepacia are very difficult to treat due to their high intrinsic resistance to most antibiotics. Biofilm formation further adds to their antibiotic resistance. B. cenocepacia harbours a large, multi-replicon genome with a high GC-content, the reference genome of strain J2315 includes 7374 annotated genes. This study aims to annotate transcription start sites and identify novel transcripts on a whole genome scale. Methods: RNA extracted from B. cenocepacia J2315 biofilms was analysed by differential RNA-sequencing and the resulting dataset compared to data derived from conventional, global RNA-sequencing. Transcription start sites were annotated and further analysed according to their position relative to annotated genes. Results: Four thousand ten transcription start sites were mapped over the whole B. cenocepacia genome and the primary transcription start site of 2089 genes expressed in B. cenocepacia biofilms were defined. For 64 genes a start codon alternative to the annotated one was proposed. Substantial antisense transcription for 105 genes and two novel protein coding sequences were identified. The distribution of internal transcription start sites can be used to identify genomic islands in B. cenocepacia. A potassium pump strongly induced only under biofilm conditions was found and 15 non-coding small RNAs highly expressed in biofilms were discovered. Conclusions: Mapping transcription start sites across the B. cenocepacia genome added relevant information to the J2315 annotation. Genes and novel regulatory RNAs putatively involved in B. cenocepacia biofilm formation were identified. These findings will help in understanding regulation of B. cenocepacia biofilm formation. KW - persistence KW - genomic islands KW - pathogen KW - identification KW - bacteria KW - small RNAs KW - translation initiation KW - cepedia complex KW - global gene expression KW - SEQ KW - resistance KW - burkholderia cenocepacia KW - biofilms KW - dRNA-Seq KW - transcription start site KW - antisense RNA Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139748 VL - 16 IS - 775 ER - TY - JOUR A1 - von Bohl, Andreas A1 - Kuehn, Andrea A1 - Simon, Nina A1 - Nkwouano Ngongang, Vanesa A1 - Spehr, Marc A1 - Baumeister, Stefan A1 - Przyborski, Jude M. A1 - Fischer, Rainer A1 - Pradel, Gabriele T1 - A WD40-repeat protein unique to malaria parasites associates with adhesion protein complexes and is crucial for blood stage progeny JF - Malaria Journal N2 - Background During development in human erythrocytes, Plasmodium falciparum parasites display a remarkable number of adhesive proteins on their plasma membrane. In the invasive merozoites, these include members of the PfMSP1 and PfAMA1/RON complexes, which facilitate contact between merozoites and red blood cells. In gametocytes, sexual precursor cells mediating parasite transmission to the mosquito vector, plasma membrane-associated proteins primarily belong to the PfCCp and 6-cys families with roles in fertilization. This study describes a newly identified WD40-repeat protein unique to Plasmodium species that associates with adhesion protein complexes of both merozoites and gametocytes. Methods The WD40-repeat protein-like protein PfWLP1 was identified via co-immunoprecipitation assays followed by mass spectrometry and characterized using biochemical and immunohistochemistry methods. Reverse genetics were employed for functional analysis. Results PfWLP1 is expressed both in schizonts and gametocytes. In mature schizonts, the protein localizes underneath the merozoite micronemes and interacts with PfAMA1, while in gametocytes PfWLP1 primarily accumulates underneath the plasma membrane and associates with PfCCp1 and Pfs230. Reverse genetics failed to disrupt the pfwlp1 gene, while haemagglutinin-tagging was feasible, suggesting a crucial function for PfWLP1 during blood stage replication. Conclusions This is the first report on a plasmodial WD40-repeat protein associating with cell adhesion proteins. Since WD40 domains are known to mediate protein–protein contact by serving as a rigid scaffold for protein interactions, the presented data suggest that PfWLP1 supports the stability of adhesion protein complexes of the plasmodial blood stages. KW - PfCCp protein KW - Pfs230 KW - PfAMA1 KW - WD40 KW - gametocyte KW - microneme KW - merozoite KW - plasmodium falciparum KW - malaria Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139728 VL - 14 IS - 435 ER -