@phdthesis{Pfoertner2016,
  author    = {Henrike Pf{\"o}rtner},
  title     = {Comparative proteome analyses of Staphylococcus aureus strains and their isogenic mutants in vitro and in host-pathogen interactions},
  journal    = {Vergleichende Proteom-Studien von Staphylococcus aureus St{\"a}mmen und ihren isogenen Mutanten in vitro und im Zusammenspiel von Wirt und Erreger},
  url       = {https://nbn-resolving.org/urn:nbn:de:gbv:9-002516-5},
  year      = {2016},
  abstract  = {Staphylococcus aureus can be a harmless colonizer of the human body, which colonizes about 20-30\% of the population. If S. aureus overcomes the outer physical barrier of the body, comprised of the skin and mucous surfaces, it can also cause severe diseases such as endocarditis, pneumonia, or sepsis. S. aureus possesses a variety of secreted and surface bound virulence factors to mediate attachment and invasion into the host, to disseminate an infection and to modulate and evade the immune system. But not only the huge amount of virulence factors turn S. aureus into a dangerous human pathogen, also its resistances to a broad spectrum of commonly used antibiotics make infections hard to treat. During the last years it became apparent that S. aureus can be internalized by as well as replicate and persist in professional and non-professional phagocytic cells. It is suggested that the intracellular compartment protects S. aureus from antibiotic treatment and the immune system. To accomplish the adaptation to the intracellular compartment, S. aureus needs to regulate its gene expression by regulatory systems. One of these regulators is the alternative sigma factor SigB, which directly and indirectly regulates the expression of about 200 genes in vitro. However, the stimuli leading to the activation of SigB in S. aureus are barely known and also its role during an infection varies, depending on the S. aureus strain and infection model used. Therefore, the importance of SigB during the early adaption of S. aureus to the intracellular environment should be elucidated using a cell culture infection model. First, the existing cell culture infection workflow had to be modified to improve the data analysis and to increase the yield of identified proteins to comparatively monitor the adaption reaction of S. aureus HG001 and its isogenic ΔsigB mutant to the intracellular milieu of S9 human bronchial epithelial cells. The proteome analysis in conjunction with RT-qPCR analysis of the wild type and the ΔsigB mutant revealed a fast and transient activation of SigB directly after internalization. Quantitative analysis of the intracellular bacterial titer demonstrated a requirement of SigB for intracellular replication. Differences in the proteome composition of the ΔsigB mutant in comparison to the wild type after internalization reflected the different growth rates, resistance to antibiotics and toxic compounds, adaptation to oxidative stress, and protein quality control mechanisms. The accessory gene regulator (Agr) is like SigB also a global regulator of gene expression in S. aureus. To elucidate possible benefits in the intracellular survival of the co-occurrence of S. aureus wild type and Δagr mutant cells, like it can be found in sites of an infection, a co-infection assay was established. With the co-infection assay the simultaneous and competitive intracellular survival in comparison to the individual intracellular survival was followed for three days post-infection (p.i.). The single and the co-infection revealed that the wild type was able to replicate more efficiently during the first hours p.i. than the Δagr mutant, but the mutant was able to survive more efficiently. The extracellular proteome of S. aureus represents the key compartment for virulence factors. Virulence factors are secreted or bound to the surface of the S. aureus cell. With the infection workflow applied in this study, secreted proteins are lost during the enrichment of the intracellular bacteria for proteome analysis. Therefore, no information about the levels or the regulation of virulence factor expression can be acquired in the cell culture infection model using cell sorting approaches. Hence, the extracellular proteome of S. aureus was analyzed in vitro from shake flask experiments. To get a comprehensive overview of the regulatory impact of different global regulators onto the secretome, S. aureus LS1 mutants lacking the global regulators Agr, SarA and SigB were compared to the respective wild type. Additionally the protein level of the secretome of the well characterized and frequently used S. aureus strains 6850, CowanI, HG001, LS1, SH1000, and USA300 was comparatively analyzed. This project was performed in collaboration with the group of Prof. L{\"o}ffler from the Institute of Medical Microbiology in Jena. The data of the extracellular proteome generated in this thesis were combined with phenotypic and toxicity data to explain strain differences in invasiveness, cytotoxicity, phagosomal escape, and intracellular persistence in infection experiments.},
  language  = {en}
}