TY - THES A1 - Groma, Michaela T1 - Identification of a novel LysR-type transcriptional regulator in \(Staphylococcus\) \(aureus\) T1 - Identifizierung eines neuen Transkriptionsregulators vom LysR-Typ in \(Staphylococcus\) \(aureus\) N2 - Staphylococcus aureus is a facultative pathogen which causes a variety of infections. The treatment of staphylococcal infections is complicated because the bacteria is resistant to multiple common antibiotics. S. aureus is also known to express a variety of virulence factors which modulate the host’s immune response in order to colonize and invade certain host cells, leading to the host cell’s death. Among the virulence factors is a LysR-type transcriptional regulator (lttr) which is required for efficient colonization of secondary organs. In a recent report, which used transposon screening on S. aureus-infected mice, it was found that the amount of a novel lttr852 mutant bacteria recovered from the kidneys was significantly lower compared to the wildtype strains. This doctoral thesis therefore focused on phenotypical and molecular characterization of lttr852. An assessment of the S. aureus biofilm formation and the hemolysis revealed that lttr852 was not involved in the regulation of these virulence processes. RNA-sequencing for potential target genes of lttr852 identified differentially expressed genes that are involved in branched chain amino-acid biosynthesis, methionine sulfoxide reductase and copper transport, as well as a reduced transcription of genes encoding urease and of components of pyrimidine nucleotides. Promoter fusion with GFP reporters as as well as OmniLog were used to identify conditions under which the lttr852 was active. The promoter studies showed that glucose and high temperatures diminish the lttr852 promoter activity in a time-dependent manner, while micro-aerobic conditions enhanced the promoter activity. Copper was found to be a limiting factor. In addition, the impact on promoter activity of the lttr852 was tested in the presence of various regulators, but no central link to the genes involved in virulence was identified. The present work, thus, showed that lttr852, a new member of the class of LysR-type transcriptional regulators in S. aureus, has an important role in the rapid adaptation of S. aureus to the changing microenvironment of the host. N2 - Staphylococcus aureus ist ein fakultativer Erreger, der eine Vielzahl von Infektionen verursacht. Die Behandlung von Staphylokokken-Infektionen ist aufgrund des Auftretens einer Resistenz gegen mehrere gängige Antibiotika kompliziert. Es ist bekannt, dass S. aureus eine Vielzahl von Virulenzfaktoren exprimiert, um die Immunantwort des Wirts zu umgehen, und so in bestimmte Wirtszellen einzudringen und diese zu kolonisieren, was zum Tod von Wirtszellen führen kann. Unter den Virulenzfaktoren befindet sich ein Transkriptions-regulator vom LysR-Typ (lttr), der für eine effiziente Besiedlung von Sekundärorganen erforderlich ist. In einem kürzlich durchgeführten Transposon-Screen, bei dem Mäuse mit S. aureus infiziert wurden, wurde ein neuartiger lttr, der lttr852 identifiziert, bei dem aus den Nieren gewonnenen Bakterien signifikant dezimiert waren. Diese Doktorarbeit befasste sich mit der phänotypischen und molekularen Charakterisierung von lttr852. Die Auswertung der Biofilmbildung und der Hämolyse von S. aureus ergab, dass lttr852 nicht an der Regulation dieser Virulenzprozesse beteiligt war. Die RNA-Sequenzierung für potenzielle Zielgene von lttr852 identifizierte sowohl eine erhöhte Expression von Genen, die in der Aminosäuren-Biosynthese, Methionin-Sulfoxid-Reduktase und dem Kupfertransport involviert sind, als auch eine verringerte Transkription von codierenden Genen der Urease und Komponenten der Pyrimidin Nukleotide. Die Promotorfusion mit dem GFP-Reporter sowie das OmniLog System wurden verwendet, um Bedingungen zu identifizieren unter denen das lttr852 aktiv ist. Die Promotorstudien ergaben, dass die Anwesenheit von Glucose und eine erhöhte Temperatur die Promotoraktivität von lttr852 zeitabhängig herabsetzt, wobei die Aktivität durch mikroaerobe Bedingungen begünstigt wird. Kupfer wurde als limitierender Faktor identifiziert. Außerdem wurde der Einfluss diverser Regulatoren auf die transkriptionelle Regulation von lttr852 kontrolliert, jedoch keine zentrale Rolle in der Regulation von Virulenzgenen zugewiesen. Damit konnte innerhalb der vorliegenden Arbeit gezeigt werden, dass lttr852, ein neues Mitglied der Klasse der LysR-Typ Transkriptionsregulatoren in S. aureus, eine wichtige Rolle in der schnellen Adaption von S. aureus an die wechselnde Mikroumgebungen des Wirts hat. KW - Staphylococcus aureus KW - transcriptional regulation KW - metabolic adaptation KW - secondary site infection KW - LysR-type Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246757 ER - TY - JOUR A1 - Krones, David A1 - Rühling, Marcel A1 - Becker, Katrin Anne A1 - Kunz, Tobias C. A1 - Sehl, Carolin A1 - Paprotka, Kerstin A1 - Gulbins, Erich A1 - Fraunholz, Martin T1 - Staphylococcus aureus α-Toxin Induces Acid Sphingomyelinase Release From a Human Endothelial Cell Line JF - Frontiers in Microbiology N2 - Staphylococcus aureus (S. aureus) is well known to express a plethora of toxins of which the pore-forming hemolysin A (α-toxin) is the best-studied cytolysin. Pore-forming toxins (PFT) permeabilize host membranes during infection thereby causing concentration-dependent effects in host cell membranes ranging from disordered ion fluxes to cytolysis. Host cells possess defense mechanisms against PFT attack, resulting in endocytosis of the breached membrane area and delivery of repair vesicles to the insulted plasma membrane as well as a concurrent release of membrane repair enzymes. Since PFTs from several pathogens have been shown to recruit membrane repair components, we here investigated whether staphylococcal α-toxin is able to induce these mechanisms in endothelial cells. We show that S. aureus α-toxin induced increase in cytosolic Ca2+ in endothelial cells, which was accompanied by p38 MAPK phosphorylation. Toxin challenge led to increased endocytosis of an extracellular fluid phase marker as well as increased externalization of LAMP1-positive membranes suggesting that peripheral lysosomes are recruited to the insulted plasma membrane. We further observed that thereby the lysosomal protein acid sphingomyelinase (ASM) was released into the cell culture medium. Thus, our results show that staphylococcal α-toxin triggers mechanisms in endothelial cells, which have been implicated in membrane repair after damage of other cell types by different toxins. KW - acid sphingomyelinase KW - staphylococcal alpha-toxin KW - sphingomyelinase release KW - lysosomal recruitment KW - Staphylococcus aureus Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-244843 SN - 1664-302X VL - 12 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 - 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 -