TY - JOUR A1 - Hennessen, Fabienne A1 - Miethke, Marcus A1 - Zaburannyi, Nestor A1 - Loose, Maria A1 - Lukežič, Tadeja A1 - Bernecker, Steffen A1 - Hüttel, Stephan A1 - Jansen, Rolf A1 - Schmiedel, Judith A1 - Fritzenwanker, Moritz A1 - Imirzalioglu, Can A1 - Vogel, Jörg A1 - Westermann, Alexander J. A1 - Hesterkamp, Thomas A1 - Stadler, Marc A1 - Wagenlehner, Florian A1 - Petković, Hrvoje A1 - Herrmann, Jennifer A1 - Müller, Rolf T1 - Amidochelocardin overcomes resistance mechanisms exerted on tetracyclines and natural chelocardin JF - Antibiotics N2 - The reassessment of known but neglected natural compounds is a vital strategy for providing novel lead structures urgently needed to overcome antimicrobial resistance. Scaffolds with resistance-breaking properties represent the most promising candidates for a successful translation into future therapeutics. Our study focuses on chelocardin, a member of the atypical tetracyclines, and its bioengineered derivative amidochelocardin, both showing broad-spectrum antibacterial activity within the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) panel. Further lead development of chelocardins requires extensive biological and chemical profiling to achieve favorable pharmaceutical properties and efficacy. This study shows that both molecules possess resistance-breaking properties enabling the escape from most common tetracycline resistance mechanisms. Further, we show that these compounds are potent candidates for treatment of urinary tract infections due to their in vitro activity against a large panel of multidrug-resistant uropathogenic clinical isolates. In addition, the mechanism of resistance to natural chelocardin was identified as relying on efflux processes, both in the chelocardin producer Amycolatopsis sulphurea and in the pathogen Klebsiella pneumoniae. Resistance development in Klebsiella led primarily to mutations in ramR, causing increased expression of the acrAB-tolC efflux pump. Most importantly, amidochelocardin overcomes this resistance mechanism, revealing not only the improved activity profile but also superior resistance-breaking properties of this novel antibacterial compound. KW - chelocardins KW - atypical tetracyclines KW - broad-spectrum antibiotics KW - clinical isolates KW - uropathogens KW - urinary tract infection (UTI) KW - resistance-breaking properties KW - mechanism of resistance KW - AcrAB-TolC efflux pump Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213149 SN - 2079-6382 VL - 9 IS - 9 ER - TY - JOUR A1 - Mühlberg, Eric A1 - Umstätter, Florian A1 - Domhan, Cornelius A1 - Hertlein, Tobias A1 - Ohlsen, Knut A1 - Krause, Andreas A1 - Kleist, Christian A1 - Beijer, Barbro A1 - Zimmermann, Stefan A1 - Haberkorn, Uwe A1 - Mier, Walter A1 - Uhl, Philipp T1 - Vancomycin-lipopeptide conjugates with high antimicrobial activity on vancomycin-resistant enterococci JF - Pharmaceuticals N2 - Multidrug-resistant bacteria represent one of the most important health care problems worldwide. While there are numerous drugs available for standard therapy, there are only a few compounds capable of serving as a last resort for severe infections. Therefore, approaches to control multidrug-resistant bacteria must be implemented. Here, a strategy of reactivating the established glycopeptide antibiotic vancomycin by structural modification with polycationic peptides and subsequent fatty acid conjugation to overcome the resistance of multidrug-resistant bacteria was followed. This study especially focuses on the structure–activity relationship, depending on the modification site and fatty acid chain length. The synthesized conjugates showed high antimicrobial potential on vancomycin-resistant enterococci. We were able to demonstrate that the antimicrobial activity of the vancomycin-lipopeptide conjugates depends on the chain length of the attached fatty acid. All conjugates showed good cytocompatibility in vitro and in vivo. Radiolabeling enabled the in vivo determination of pharmacokinetics in Wistar rats by molecular imaging and biodistribution studies. An improved biodistribution profile in comparison to unmodified vancomycin was observed. While vancomycin is rapidly excreted by the kidneys, the most potent conjugate shows a hepatobiliary excretion profile. In conclusion, these results demonstrate the potential of the structural modification of already established antibiotics to provide highly active compounds for tackling multidrug-resistant bacteria. KW - antibiotics KW - multidrug-resistant bacteria KW - enterococci KW - vancomycin KW - structural modification KW - fatty acids KW - polycationic peptides Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-205879 SN - 1424-8247 VL - 13 IS - 6 ER - TY - THES A1 - Wencker, Freya Dorothea Ruth T1 - The methionine biosynthesis operon in \(Staphylococcus\) \(aureus\): Role of concerted RNA decay in transcript stability and T-box riboswitch turnover T1 - Das Methioninbiosynthese-Operon in \(Staphylococcus\) \(aureus\): Der Einfluss von koordiniertem RNA Abbau auf Transkriptstabilität und T-Box-Riboswitch-Prozessierung N2 - Methionine is the first amino acid of every newly synthesised protein. In combination with its role as precursor for the vital methyl-group donor S-adenosylmethionine, methionine is essential for every living cell. The opportunistic human pathogen Staphylococcus aureus is capable of synthesising methionine de novo, when it becomes scarce in the environment. All genes required for the de novo biosynthesis are encoded by the metICFE-mdh operon, except for metX. Expression is controlled by a hierarchical network with a methionyl-tRNA-specific T-box riboswitch (MET-TBRS) as centrepiece, that is also referred to as met leader (RNA). T-box riboswitches (TBRS) are regulatory RNA elements located in the 5’-untranslated region (5’-UTR) of genes. The effector molecule of T-box riboswitches is uncharged cognate tRNA. The prevailing mechanism of action is premature termination of transcription of the nascent RNA in the absence of the effector (i.e. uncharged cognate tRNA) due to formation of a hairpin structure, the Terminator stem. In presence of the effector, a transient stabilisation of the alternative structure, the Antiterminator, enables transcription of the downstream genes (‘read-through’). Albeit, after the read-through the thermodynamically more stable Terminator eventually forms. The Terminator and the Antiterminator are two mutually exclusive structures. Previous work of the research group showed that in staphylococci the MET-TBRS ensures strictly methionine-dependent control of met operon expression. Uncharged methionyl-tRNA that activates the system is only present in sufficient amounts under methionine-deprived conditions. In contrast to other bacterial TBRS, the staphylococcal MET-TBRS has some characteristic features regarding its length and predicted secondary structure whose relevance for the function are yet unkown. Aim of the present thesis was to experimentally determine the structure of the met leader RNA and to investigate the stability of the met operon-specific transcripts in the context of methionine biosynthesis control. Furthermore, the yet unknown function of the mdh gene within the met operon was to be determined. In the context of this thesis, the secondary structure of the met leader was determined employing in-line probing. The structural analysis revealed the presence of almost all highly conserved T-box riboswitch structural characteristics. Furthermore, three additional stems, absent in all T-box riboswitches analysed to date, could be identified. Particularly remarkable is the above average length of the Terminator stem which renders it a potential target of the double-strand-specific endoribonuclease III (RNase III). The RNase III-dependent cleavage of the met leader could be experimentally verified by the use of suitable mutants. Moreover, the exact cleavage site within the Terminator was determined. The unusual immediate separation of the met leader from the met operon mRNA via the RNase III cleavage within the Terminator stem induces the rapid degradation of the met leader RNA and, most likely, that of the 5’-region of the met mRNA. The met mRNA is degraded from its 5’-end by the exoribonuclease RNase J. The stability of the met mRNA was found to vary over the length of the transcript with an instable 5’-end (metI and metC) and a longer half-life towards the 3’-end (metE and mdh). The varying transcript stability is reflected by differences in the available cellular protein levels. The obtained data suggest that programmed mRNA degradation is another level of regulation in the complex network of staphylococcal de novo methionine biosynthesis control. In addition, the MET-TBRS was studied with regard to a future use as a drug target for novel antimicrobial agents. To this end, effects of a dysregulated methionine biosynthesis on bacterial growth and survival were investigated in met leader mutants that either caused permanent transcription of the met operon (‘ON’) or prevented operon transcription (‘OFF’), irrespective of the methionine status in the cell. Methionine deprivation turned out to be a strong selection pressure, as ‘OFF’ mutants acquired adaptive mutations within the met leader to restore met operon expression that subsequently re-enabled growth. The second part of the thesis was dedicated to the characterisation of the Mdh protein that is encoded by the last gene of the met operon and whose function is unknown yet. At first, co-transcription and -expression with the met operon could be demonstrated. Next, the Mdh protein was overexpressed and purified and the crystal structure of Mdh was solved to high resolution by the Kisker research group (Rudolf-Virchow-Zentrum Würzburg). Analysis of the structure revealed the amino acid residues crucial for catalytic activity, and zinc was identified as a co-factor of Mdh. Also, Mdh was shown to exist as a dimer. However, identification of the Mdh substrate was, in the context of this thesis, (still) unsuccessful. Nevertheless, interactions of Mdh with enzymes of the met operon could be demonstrated by employing the bacterial two-hybrid system. This fact and the high conservation of mdh/Mdh on nucleotide and amino acid level among numerous staphylococcal species suggests an important role of Mdh within the methionine metabolism that should be a worthwhile subject of future research. N2 - Methionin ist die erste Aminosäure in jedem neu gebildeten Protein. Zusammen mit seiner Funktion als Vorläufermolekül für die Synthese des essenziellen Methylgruppendonors S-Adenosylmethionin ist Methionin damit für jede lebende Zelle unverzichtbar. Staphylococcus aureus, ein opportunistisches Humanpathogen, ist in der Lage, Methionin de novo zu synthetisieren, wenn es nicht in ausreichender Menge in der Umgebung vorhanden ist. Mit Ausnahme von MetX sind alle für die Methioninsynthese benötigten Enzyme im metICFE-mdh-Operon kodiert. Die Expression des Operons wird durch ein komplexes hierarchisches Netzwerk reguliert, dessen zentrales Steuerelement ein Methionyl-tRNA-spezifischer T-Box-Riboswitch (MET-TBRS) ist, der auch als met-leader (RNA) bezeichnet wird. T-Box Riboswitches (TBRS) sind regulatorische RNA-Elemente, die in der untranslatierten Region am 5'-Ende (5'-UTR) ihrer zu kontrollierenden Gene liegen. Sie nutzen unbeladene tRNAs als Effektormoleküle. Die Funktionsweise der meisten TBRS beruht auf dem vorzeitigen Abbruch der Transkription der naszierenden mRNA, der durch die Ausbildung einer Haarnadelstruktur (Terminator) im Transkript herbeigeführt wird, wenn das Effektormolekül (i.e. unbeladene tRNA) fehlt. Sobald passende unbeladene tRNA verfügbar ist und bindet, wird eine alternative Struktur, der Antiterminator, kurzzeitig stabilisiert, der die Transkription und damit ein "Durchlesen" in die stromabwärtsliegenden Gene ermöglicht. Terminator und Antiterminator sind zwei sich gegenseitig ausschließende Strukturen, wobei der Terminator die thermodynamisch deutlich stabilere Struktur des TBRS ist, die sich dementsprechend auch in den vollständigen Transkripten erneut ausbildet. Bisherige Vorarbeiten der Arbeitsgruppe zeigten, dass in Staphylokokken der MET-TBRS die Kontrolle der Methioninsynthese in strikter Abhängigkeit von Methionin gewährleistet. Unbeladene Methionyl-tRNA, die nur unter Methioninmangelbedingungen in ausreichenden Konzentrationen vorliegt, aktiviert das System. Im Unterschied zu anderen bakteriellen TBRS weist der Staphylokokken-MET-TBRS (met-leader) hinsichtlich seiner Länge und vorhergesagten Struktur einige Besonderheiten auf, deren Bedeutung für die Funktion bislang unklar sind. Ziel der vorliegenden Arbeit war es daher, die Struktur der met-leader-RNA experimentell zu bestimmen und die Stabilität met-Operon-spezifischer Transkripte im Kontext der Methioninbiosynthesekontrolle zu untersuchen. Ebenso sollte die bisher unbekannte Funktion des mdh-Genes im Operon aufgeklärt werden. Im Rahmen dieser Doktorarbeit wurde die Sekundärstruktur der met-leader-RNA mit Hilfe des so genannten In-line Probings bestimmt. Die Sekundärstruktur weist neben fast allen hochkonservierten Strukturmerkmalen eines T-Box-Riboswitches auch drei zusätzliche Haarnadelstrukturen auf, die bisher in keinem anderen T-Box-Riboswitch gefunden wurden. Besonders auffällig ist die überdurchschnittliche Länge des met-leader-Terminators, der dadurch zur potentiellen Zielstruktur für die Doppelstrang-spezifische Endoribonuklease RNase III wird. Mittels geeigneter Mutanten konnte die RNase III-abhängige Prozessierung der met-leader-RNA experimentell bewiesen werden. Ebenso wurde die exakte Schnittstelle im Terminator bestimmt. Die ungewöhnliche Prozessierung des Terminators durch die RNase III spaltet die met-leader-RNA von der met-mRNA ab, was den raschen weiteren Abbau der met-leader-RNA und sehr wahrscheinlich auch den der met-mRNA einleitet. So wird die met-mRNA durch die Exoribonuklease RNase J vom 5'-Ende her abgebaut, wobei die Stabilität bezogen auf die Gesamtheit des Moleküls stark variiert: Das 5'-Ende mit den Genen metI und metC wird äußerst schnell degradiert, während das 3'-Ende mit metE und mdh deutlich stabiler ist. Die variierende mRNA-Stabilität spiegelt sich auch in Unterschieden hinsichtlich der verfügbaren zellulären Proteinmengen wider. Die Daten legen daher nahe, dass programmierte mRNA-Degradation eine weitere Ebene im komplexen Kontrollnetzwerk darstellt, durch die in Staphylokokken die Methioninbiosynthese sehr exakt den jeweiligen Bedürfnissen angepasst wird. Des Weiteren wurde der MET-TBRS im Hinblick auf eine zukünftige Nutzung als Angriffspunkt für neue antibakterielle Wirkstoffe untersucht. Dazu wurden die Auswirkungen einer dysregulierten Methioninbiosynthese auf das bakterielle Wachstum und Überleben mit Hilfe von met-leader-Mutanten analysiert, die entweder zu einer permanenten Aktivierung („ON“) oder Deaktivierung („OFF“) der met-Operon-Transkription, unabhängig vom Methioninstatus in der Zelle, führten. Es zeigte sich, dass Methioninmangel einen starken Selektionsdruck darstellt, da die „OFF“-Mutanten in der Lage waren, durch den Erwerb von adaptiven Mutationen innerhalb der met-leader-Sequenz, das met-Operon erneut zu aktivieren und wieder zu wachsen. Der zweite Teil dieser Arbeit widmete sich der Charakterisierung des Mdh-Proteins, das im letzten Gen des met-Operons kodiert ist und dessen Funktion derzeit gänzlich unbekannt ist. Zunächst konnte die Kotranskription und -expression von mdh mit dem met-Operon gezeigt werden. In Zusammenarbeit mit der Arbeitsgruppe Kisker (Rudolf-Virchow-Zentrum Würzburg) wurden anhand von Kristallstrukturanalysen die Aminosäuren identifiziert, die entscheidend für die katalytische Aktivität des Mdh-Enzyms sind, wobei Zink als ein Kofaktor fungiert. Ebenso zeigte sich, dass Mdh als Dimer vorliegt. Allerdings ist die Identifizierung des Mdh-Substrates im Rahmen dieser Arbeit (noch) nicht gelungen. Mittels eines bakteriellen Zwei-Hybridsystems wurde jedoch nachgewiesen, dass Mdh mit den anderen Enzymen des met-Operons interagiert. Dies und die hohe Konservierung von mdh/Mdh auf Nukleotid- und Aminosäureebene in verschiedenen Staphylokokkenarten legt eine wichtige Funktion von Mdh im Methioninstoffwechsel nahe, die lohnenswerter Gegendstand weiterer Untersuchungen sein sollte. KW - Staphylococcus aureus KW - RNA Abbau KW - Methioninbiosynthese KW - MET-T-box riboswitch KW - riboswitch KW - methionine biosynthesis KW - RNA decay Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-207124 ER - TY - JOUR A1 - Wencker, Freya D. R A1 - Marincola, Gabriella A1 - Schoenfelder, Sonja M. K. A1 - Maaß, Sandra A1 - Becher, Dörte A1 - Ziebuhr, Wilma T1 - Another layer of complexity in Staphylococcus aureus methionine biosynthesis control: unusual RNase III-driven T-box riboswitch cleavage determines met operon mRNA stability and decay JF - Nucleic Acids Research N2 - In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5′ untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5′-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3′-end. The obtained data suggest that coordinated RNA decay represents another checkpoint in a complex regulatory network that adjusts costly methionine biosynthesis to current metabolic requirements. KW - allelic replacement KW - expression KW - translation KW - mechanism KW - acid KW - endoribonuclease KW - antitermination KW - transcription KW - proteins KW - geometry Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259029 VL - 49 IS - 4 ER - TY - JOUR A1 - Gerova, Milan A1 - Wicke, Laura A1 - Chihara, Kotaro A1 - Schneider, Cornelius A1 - Lavigne, Rob A1 - Vogel, Jörg T1 - A grad-seq view of RNA and protein complexes in Pseudomonas aeruginosa under standard and bacteriophage predation conditions JF - mbio N2 - The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell. IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser. KW - Grad-seq KW - Pseudomonas KW - UKZ KW - bacteriophage KW - infection KW - Pseudomonas aeruginosa KW - RNA-binding proteins KW - noncoding RNA KW - phage Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259054 VL - 12 IS - 1 ER - TY - JOUR A1 - Seethaler, Marius A1 - Hertlein, Tobias A1 - Wecklein, Björn A1 - Ymeraj, Alba A1 - Ohlsen, Knut A1 - Lalk, Michael A1 - Hilgeroth, Andreas T1 - Novel small-molecule antibacterials against Gram-positive pathogens of Staphylococcus and Enterococcus species JF - Antibiotics N2 - Defeat of the antibiotic resistance of pathogenic bacteria is one great challenge today and for the future. In the last century many classes of effective antibacterials have been developed, so that upcoming resistances could be met with novel drugs of various compound classes. Meanwhile, there is a certain lack of research of the pharmaceutical companies, and thus there are missing developments of novel antibiotics. Gram-positive bacteria are the most important cause of clinical infections. The number of novel antibacterials in clinical trials is strongly restricted. There is an urgent need to find novel antibacterials. We used synthetic chemistry to build completely novel hybrid molecules of substituted indoles and benzothiophene. In a simple one-pot reaction, two novel types of thienocarbazoles were yielded. Both indole substituted compound classes have been evaluated as completely novel antibacterials against the Staphylococcus and Enterococcus species. The evaluated partly promising activities depend on the indole substituent type. First lead compounds have been evaluated within in vivo studies. They confirmed the in vitro results for the new classes of small-molecule antibacterials. KW - antibacterial activity KW - synthesis KW - substituent KW - structure-activity KW - inhibition Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193130 SN - 2079-6382 VL - 8 IS - 4 ER - TY - THES A1 - Matera, Gianluca T1 - Global mapping of RNA-RNA interactions in \(Salmonella\) via RIL-seq T1 - Globale Analyse der RNA-RNA-Interaktionen in \(Salmonella\) mittels RIL-seq N2 - RNA represents one of the most abundant macromolecules in both eukaryotic and prokaryotic cells. Since the discovery that RNA could play important gene regulatory functions in the physiology of a cell, small regulatory RNAs (sRNAs) have been at the center of molecular biology studies. Functional sRNAs can be independently transcribed or derived from processing of mRNAs and other non-coding regions and they often associate with RNA-binding proteins (RBPs). Ever since the two major bacterial RBPs, Hfq and ProQ, were identified, the way we approach the identification and characterization of sRNAs has drastically changed. Initially, a single sRNA was annotated and its function studied with the use of low-throughput biochemical techniques. However, the development of RNA-seq techniques over the last decades allowed for a broader identification of sRNAs and their functions. The process of studying a sRNA mainly focuses on the characterization of its interacting RNA partner(s) and the consequences of this binding. By using RNA interaction by ligation and sequencing (RIL-seq), the present thesis aimed at a high-throughput mapping of the Hfq-mediated RNA-RNA network in the major human pathogen Salmonella enterica. RIL-seq was at first performed in early stationary phase growing bacteria, which enabled the identification of ~1,800 unique interactions. In- depth analysis of such complex network was performed with the aid of a newly implemented RIL-seq browser. The interactome revealed known and new interactions involving sRNAs and genes part of the envelope regulon. A deeper investigation led to the identification of a new RNA sponge of the MicF sRNA, namely OppX, involved in establishing a cross-talk between the permeability at the outer membrane and the transport capacity at the periplasm and the inner membrane. Additionally, RIL-seq was applied to Salmonella enterica grown in SPI-2 medium, a condition that mimicks the intracellular lifestyle of this pathogen, and finally extended to in vivo conditions during macrophage infection. Collectively, the results obtained in the present thesis helped unveiling the complexity of such RNA networks. This work set the basis for the discovery of new mechanisms of RNA-based regulation, for the identification of a new physiological role of RNA sponges and finally provided the first resource of RNA interactions during infection conditions in a major human pathogen. N2 - RNA ist eines der am häufigsten vorkommenden Makromoleküle sowohl in eukaryontischen als auch in prokaryontischen Zellen. Seit der Entdeckung, dass RNA wichtige genregulatorische Funktionen in der Physiologie einer Zelle spielen könnte, stehen kleine regulatorische RNAs (sRNAs) im Mittelpunkt molekularbiologischer Studien. Funktionelle sRNAs können alleinstehend von nicht-codierenden oder codierenden Bereichen des Genoms transkribiert werden, aber sie können auch durch die Prozessierung einer mRNA entstehen. Des Weiteren sind sRNAs häufig mit RNA- bindenden Proteinen (RBPs) assoziiert. Seitdem die beiden wichtigsten bakteriellen RBPs, Hfq und ProQ, identifiziert wurden, hat sich die Art und Weise, wie wir an die Identifizierung und Charakterisierung von sRNAs herangehen, drastisch verändert. Ursprünglich wurden sRNAs annotiert und anschließend für einzelne sRNAs die Funktion mit biochemischen Techniken untersucht. Die Entwicklung von RNA-seq-Techniken in den letzten Jahrzehnten ermöglichte nun jedoch eine globale Identifizierung von sRNAs und ihren Funktionen. Der Prozess der Untersuchung einer sRNA konzentriert sich hauptsächlich auf die Charakterisierung ihrer interagierenden RNA-Partner und die Folgen dieser Bindung. Mit Hilfe der RNA-Interaktion durch Ligation und Sequenzierung (RIL-seq) wurde in der vorliegenden Arbeit eine Hochdurchsatzkartierung des Hfq-vermittelten RNA-RNA-Netzwerks in dem wichtigen humanen Krankheitserreger Salmonella enterica durchgeführt. RIL-seq wurde zunächst in Bakterien in der frühen stationären Wachstumsphase durchgeführt, was die Identifizierung von ~1.800 einzigartigen Interaktionen ermöglichte. Mit Hilfe eines neu implementierten RIL-seq-Browsers wurde daraufhin eine eingehende Analyse dieses komplexen Netzwerks durchgeführt. Das Interaktom enthüllte bekannte und neue Interaktionen zwischen sRNAs und mRNAs, die Teil des Zellwand-Regulons sind. Eine tiefergehende Untersuchung führte zur Identifizierung eines neuen RNA-Schwammes, OppX, welcher mit der sRNA MicF bindet und so die Herstellung eines Cross-Talks zwischen der Permeabilität an der äußeren Membran und der Transportkapazität am Periplasma und der inneren Membran ermöglicht. Darüber hinaus wurde RIL-seq für Salmonella enterica angewandt, welche in SPI-2-Medium gewachsen waren, wobei diese Bedingung, die den intrazellulären Lebensstil dieses Erregers nachahmt. Durch die Infektion von Makrophagen mit dem Bakterium, wurde das RIL-seq Protokoll des Weiteren unter in vivo Bedingungen getestet. Insgesamt trugen die in dieser Arbeit erzielten Ergebnisse dazu bei, die Komplexität solcher RNA- Netzwerke zu enthüllen. Diese Arbeit bildete die Grundlage für die Entdeckung neuer Mechanismen der RNA-basierten Regulierung als auch für die Identifizierung einer neuen physiologischen Rolle von RNA- Schwämmen und lieferte letztendlich die erste Untersuchung für RNA- Interaktionen unter Infektionsbedingungen in einem wichtigen menschlichen Krankheitserreger. KW - Small RNA KW - RNA KW - infection biology KW - Salmonella KW - MicF Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-268776 ER - TY - JOUR A1 - Stelzner, Kathrin A1 - Boyny, Aziza A1 - Hertlein, Tobias A1 - Sroka, Aneta A1 - Moldovan, Adriana A1 - Paprotka, Kerstin A1 - Kessie, David A1 - Mehling, Helene A1 - Potempa, Jan A1 - Ohlsen, Knut A1 - Fraunholz, Martin J. A1 - Rudel, Thomas T1 - Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells JF - PLoS Pathogens N2 - Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection. Author summary Staphylococcus aureus is an antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium can asymptomatically colonize the upper respiratory tract and skin of humans and take advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus was not regarded as intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death of epithelial cells mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A. KW - Staphylococcus aureus KW - Staphylococcal infection KW - host cells KW - HeLa cells KW - cytotoxicity KW - intracellular pathogens KW - apoptosis KW - epithelial cells Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-263908 VL - 17 IS - 9 ER - TY - JOUR A1 - Kayisoglu, Özge A1 - Schlegel, Nicolas A1 - Bartfeld, Sina T1 - Gastrointestinal epithelial innate immunity-regionalization and organoids as new model JF - Journal of Molecular Medicine N2 - The human gastrointestinal tract is in constant contact with microbial stimuli. Its barriers have to ensure co-existence with the commensal bacteria, while enabling surveillance of intruding pathogens. At the centre of the interaction lies the epithelial layer, which marks the boundaries of the body. It is equipped with a multitude of different innate immune sensors, such as Toll-like receptors, to mount inflammatory responses to microbes. Dysfunction of this intricate system results in inflammation-associated pathologies, such as inflammatory bowel disease. However, the complexity of the cellular interactions, their molecular basis and their development remains poorly understood. In recent years, stem cell-derived organoids have gained increasing attention as promising models for both development and a broad range of pathologies, including infectious diseases. In addition, organoids enable the study of epithelial innate immunity in vitro. In this review, we focus on the gastrointestinal epithelial barrier and its regional organization to discuss innate immune sensing and development. KW - regionalization and organoids KW - immunity KW - gastrointestinal tract Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265220 VL - 99 IS - 4 ER - TY - JOUR A1 - Pernitzsch, Sandy R. A1 - Alzheimer, Mona A1 - Bremer, Belinda U. A1 - Robbe-Saule, Marie A1 - De Reuse, Hilde A1 - Sharma, Cynthia M. T1 - Small RNA mediated gradual control of lipopolysaccharide biosynthesis affects antibiotic resistance in Helicobacter pylori JF - Nature Communications N2 - The small, regulatory RNA RepG (Regulator of polymeric G-repeats) regulates the expression of the chemotaxis receptor TlpB in Helicobacter pylori by targeting a variable G-repeat in the tlpB mRNA leader. Here, we show that RepG additionally controls lipopolysaccharide (LPS) phase variation by also modulating the expression of a gene (hp0102) that is co-transcribed with tlpB. The hp0102 gene encodes a glycosyltransferase required for LPS O-chain biosynthesis and in vivo colonization of the mouse stomach. The G-repeat length defines a gradual (rather than ON/OFF) control of LPS biosynthesis by RepG, and leads to gradual resistance to a membrane-targeting antibiotic. Thus, RepG-mediated modulation of LPS structure might impact host immune recognition and antibiotic sensitivity, thereby helping H. pylori to adapt and persist in the host. The small RNA RepG modulates expression of chemotaxis receptor TlpB in Helicobacter pylori by targeting a length-variable G-repeat in the tlpB mRNA. Here, Pernitzsch et al. show that RepG also gradually controls lipopolysaccharide biosynthesis, antibiotic susceptibility, and in-vivo colonization of the stomach, by regulating a gene that is co-transcribed with tlpB. KW - bacterial genetics KW - bacterial immune evasion KW - pathogens KW - small RNAs Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-261536 VL - 12 IS - 1 ER - 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 -