@article{ElMoualiGerovacMineikaitėetal.2021, author = {El Mouali, Youssef and Gerovac, Milan and Mineikaitė, Raminta and Vogel, J{\"o}rg}, title = {In vivo targets of Salmonella FinO include a FinP-like small RNA controlling copy number of a cohabitating plasmid}, series = {Nucleic Acids Research}, volume = {49}, journal = {Nucleic Acids Research}, number = {9}, doi = {10.1093/nar/gkab281}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-261072}, pages = {5319-5335}, year = {2021}, abstract = {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.}, language = {en} } @article{MottolaRamirezZavalaHuenningeretal.2021, author = {Mottola, Austin and Ram{\´i}rez-Zavala, Bernardo and H{\"u}nninger, Kerstin and Kurzai, Oliver and Morschh{\"a}user, Joachim}, title = {The zinc cluster transcription factor Czf1 regulates cell wall architecture and integrity in Candida albicans}, series = {Molecular Microbiology}, volume = {116}, journal = {Molecular Microbiology}, number = {2}, doi = {10.1111/mmi.14727}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259583}, pages = {483-497}, year = {2021}, abstract = {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.}, language = {en} } @article{SvenssonSharma2022, author = {Svensson, Sarah L. and Sharma, Cynthia M.}, title = {Small RNAs that target G-rich sequences are generated by diverse biogenesis pathways in Epsilonproteobacteria}, series = {Molecular Microbiology}, volume = {117}, journal = {Molecular Microbiology}, doi = {10.1111/mmi.14850}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259602}, pages = {215-233}, year = {2022}, abstract = {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.}, language = {en} } @article{PrezzaRyanMaedleretal.2022, author = {Prezza, Gianluca and Ryan, Daniel and M{\"a}dler, Gohar and Reichardt, Sarah and Barquist, Lars and Westermann, Alexander J.}, title = {Comparative genomics provides structural and functional insights into Bacteroides RNA biology}, series = {Molecular Microbiology}, volume = {117}, journal = {Molecular Microbiology}, number = {1}, doi = {10.1111/mmi.14793}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259594}, pages = {67-85}, year = {2022}, abstract = {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.}, language = {en} } @article{MasotaVoggOhlsenetal.2021, author = {Masota, Nelson E. and Vogg, Gerd and Ohlsen, Knut and Holzgrabe, Ulrike}, title = {Reproducibility challenges in the search for antibacterial compounds from nature}, series = {PLoS One}, volume = {16}, journal = {PLoS One}, number = {7}, doi = {10.1371/journal.pone.0255437}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-260239}, year = {2021}, abstract = {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.}, language = {en} } @article{WallaschekReuterSilkenatetal.2021, author = {Wallaschek, Nina and Reuter, Saskia and Silkenat, Sabrina and Wolf, Katharina and Niklas, Carolin and {\"O}zge, Kayisoglu and Aguilar, Carmen and Wiegering, Armin and Germer, Christoph-Thomas and Kircher, Stefan and Rosenwald, Andreas and Shannon-Lowe, Claire and Bartfeld, Sina}, title = {Ephrin receptor A2, the epithelial receptor for Epstein-Barr virus entry, is not available for efficient infection in human gastric organoids}, series = {PLoS Pathogens}, volume = {17}, journal = {PLoS Pathogens}, number = {2}, doi = {10.1371/journal.ppat.1009210}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259206}, pages = {e1009210}, year = {2021}, abstract = {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.}, language = {en} } @article{CorreiaSantosBischlerWestermannetal.2021, author = {Correia Santos, Sara and Bischler, Thorsten and Westermann, Alexander J. and Vogel, J{\"o}rg}, title = {MAPS integrates regulation of actin-targeting effector SteC into the virulence control network of Salmonella small RNA PinT}, series = {Cell Reports}, volume = {34}, journal = {Cell Reports}, number = {5}, doi = {10.1016/j.celrep.2021.108722}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259134}, pages = {108722}, year = {2021}, abstract = {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.}, language = {en} } @phdthesis{ReuterWeissenberger2022, author = {Reuter-Weissenberger, Philipp}, title = {The role of a fungal-specific transcription regulator on vacuolar biology and host interaction in \(Candida\) \(albicans\)}, doi = {10.25972/OPUS-25928}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259287}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {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.}, subject = {Vakuole}, language = {en} } @article{MarincolaLiongSchoenetal.2021, author = {Marincola, Gabriella and Liong, Olivia and Schoen, Christoph and Abouelfetouh, Alaa and Hamdy, Aisha and Wencker, Freya D. R. and Marciniak, Tessa and Becker, Karsten and K{\"o}ck, Robin and Ziebuhr, Wilma}, title = {Antimicrobial Resistance Profiles of Coagulase-Negative Staphylococci in Community-Based Healthy Individuals in Germany}, series = {Frontiers in Public Health}, volume = {9}, journal = {Frontiers in Public Health}, issn = {2296-2565}, doi = {10.3389/fpubh.2021.684456}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-240881}, year = {2021}, abstract = {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.}, language = {en} } @article{MarincolaJaschkowitzKieningeretal.2021, author = {Marincola, Gabriella and Jaschkowitz, Greta and Kieninger, Ann-Katrin and Wencker, Freya D.R. and Feßler, Andrea T. and Schwarz, Stefan and Ziebuhr, Wilma}, title = {Plasmid-Chromosome Crosstalk in Staphylococcus aureus: A Horizontally Acquired Transcription Regulator Controls Polysaccharide Intercellular Adhesin-Mediated Biofilm Formation}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {11}, journal = {Frontiers in Cellular and Infection Microbiology}, issn = {2235-2988}, doi = {10.3389/fcimb.2021.660702}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-232903}, year = {2021}, abstract = {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.}, language = {en} }