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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. Löffler 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.
This thesis contains results from transcriptome studies on different aspects of host-pathogen interactions. First, liver gene expression profiles from a murine chronic stress model served to elucidate aspects of the influence of stress on metabolism and immune response state. Chronic stress in female BALB/c mice was shown to lead to a hypermetabolic syndrome including induction of gluconeogenesis, hypercholesteremia, and loss of essential amino acids, to the induction of the acute phase response, but also of immune suppressive pathways and to the repression of hepatic antigen presentation. Increased leukocyte trafficking, increased oxidative stress together with counter-regulatory gene expression changes, and an induction of apoptosis were detected. The influence of intra-venous infection on the host kidney gene expression was analyzed in another murine model using the wild type strain Staphylococcus aureus RN1HG and its isogenic sigB mutant. Gene expression profiling indicated a highly reproducible host kidney response to infection. The comparison of infected with non-infected samples revealed a strong inflammatory reaction of kidney tissue, e. g. Toll-like receptor signaling, complement system, antigen presentation, interferon and IL-6 signaling. However, the results of this study did not provide any hints for differences in the pathomechanism of the S. aureus strains RN1HG and ΔsigB, since the host response did not differ between infections with the two strains analyzed. Effects of SigB might be transient, only apparent at earlier time points, or might also be compensated for in the in vivo infection by the interlaced pattern of other regulators. SigB might possess only to a lesser extent characteristics attributed to virulence factors and might act in vivo more like a virulence modulator and fine tune bacterial reactions. In addition to the analysis of tissue samples, different in vitro models were furthermore studied. The third part of this thesis focuses on bone-marrow derived macrophages (BMM) of the two mouse strains BALB/c and C57BL/6, which are described in literature to exhibit genetically determined differences in their reaction to infection. Expression profiling was performed on control and IFN-γ treated samples from a serum-free cultivation system and revealed mainly induction of gene expression after treatment of BMM with IFN-γ. Gene expression changes confirmed known IFN-γ effects like induction of immunoproteasome, antigen presentation, interferon signaling related genes, GTPase/GBPs, and inducible NO synthase. IFN-γ dependent gene expression changes were highly similar in BALB/c and C57BL/6 BMM. Considering gene expression differences between BMM of both strains, a similar expression trend was visible on the level of untreated controls as well as after IFN-γ treatment. Differentially expressed genes between BMM of both strains included immune-relevant genes as well as genes linked to cell death, but the coverage of functional groups was limited. The bronchial epithelial cell line S9 was used as an in vitro model system for the infection with S. aureus RN1HG. The fourth chapter in this thesis includes S9 cell gene expression signatures 2.5 h and 6.5 h after start of infection. At the early time point, only 40 genes were differentially expressed, which nevertheless indicated a beginning pro-inflammatory response, e. g. induction of cytokines (IL-6, IFN-β, LIF) or prostaglandin-endoperoxide synthase 2 (PTGS2), but also counter-regulatory processes, e. g. induction of CD274. The host cell response was dramatically aggravated at the later 6.5 h time point. Differential expression was detected for 1196 genes. These included induced cytokines, pattern recognition receptor signaling, antigen presentation, and genes involved in immune defense (e. g. GBPs, MX, APOL). Negative effects on growth and proliferation were even more enhanced in comparison to the early time point, and signs for apoptotic processes were revealed. Finally, the last chapter addresses amongst others the pathogen’s expression profile in the S9 cell in vitro infection model at the two time points 2.5 h and 6.5 h after start of infection by tiling array gene expression analysis. The pathogen expression profiling revealed the activity of the SaeRS two-component system in internalized staphylococci. Partly dependent on SaeRS, the induction of adhesins (e. g. fnbAB, clfAB), toxins (hlgBC, lukDE, hla), and immune evasion genes (e. g. chp, eap) was observed. Furthermore, expression changes of metabolic genes were recorded (gene induction of amino acid biosynthesis, TCA cycle, gluconeogenesis; gene repression of glycolysis, purine biosynthesis, tRNA synthetases). Expression analysis recorded a distinct bacterial expression program, which supported literature results of a specific, bacterial strain and host cell line dependent transcriptional adaptation of the pathogen.
Hantaviruses (family Bunyaviridae) are enveloped viruses with a segmented RNA genome of negative polarity. They can cause two different diseases in humans, the hemorrhagic fever with renal syndrome in Europe and Asia and the hantavirus cardiopulmonary syndrome in America. The transmission to humans is mainly indirect by inhalation of aerosolized virus-contaminated rodent excreta. In contrast to the initial assumption that hantaviruses are mainly carried by rodents, during the last years many novel hantaviruses were detected in shrews, moles and recently in bats. These findings raise important questions about the evolutionary history of hantaviruses, their host association and adaptation, the role and frequency of spillover infections and host switch events. This study aims to prove the presence, geographical distribution and host association of the rodent-borne Tula virus (TULV) and the shrew-associated Seewis virus (SWSV) in Central Europe. For this purpose, novel laboratory techniques for molecular and serological hantavirus detection were developed. Initially, a broad-spectrum molecular assay to identify small mammal species from Central Europe was developed. This novel assay is based on PCR amplification using degenerated primers targeting the cytochrome b (cyt b) gene, nucleotide sequence analysis of the amplified cyt b gene portion and followed by pairwise sequence comparison to published sequences using the BLAST function of GenBank. Different small mammal species prevalent in Central Europe could be determined by this new approach, including not only representatives of various Rodentia and Soricomorpha, but also representatives of the orders Erinaceomorpha, Lagomorpha, Carnivora and Chiroptera. For characterization of insectivore-borne hantavirus Thottapalayam virus (TPMV), specific monoclonal antibodies were generated that detect native virus in infected mammalian cells. For the detection of TPMV-specific antibodies, Asian house shrew Suncus murinus immunoglobulin G (IgG)-specific antibodies were produced in laboratory mice and rabbit. Using this anti-shrew IgG and recombinant TPMV nucleocapsid (N) protein, an indirect enzyme-linked immunosorbent assay (ELISA) was developed allowing the detection of TPMV N protein-specific antibodies in immunized and experimentally TPMV infected shrews. A Pan-Hantavirus SYBR-Green RT-qPCR was developed for the search to novel hantaviruses. By this novel RT-qPCR and other conventional RT-PCR approaches, TULV infections were identified for the first time in the Eurasian water vole Arvicola amphibius from different regions in Germany and Switzerland. The phylogenetic analyses of the different partial TULV small (S)-, medium (M)- and large (L)-genome segment sequences from A. amphibius, with those of Microtus arvalis- and M. agrestis-derived TULV lineages, revealed a geographical, but host-independent clustering and may suggest multiple TULV spillover or a potential host switch from M. arvalis or M. agrestis to A. amphibius. In a further comprehensive study, different shrew species (Sorex araneus, S. minutus, S. coronatus, and S. alpinus) were collected in Germany, Czech Republic, and Slovakia and screened by another L-segment-targeting Pan-Hantavirus RT-PCR approach. This screening revealed hantavirus L-segment sequences in a large number of S. araneus and a few S. minutus indicating a broad geographical distribution of this hantavirus. For detailed analyses, S-segment sequences were obtained, from S. araneus and S. minutus. The sequences demonstrated their similarity to SWSV sequences from Hungary, Finland, Austria and Germany. A detailed phylogenetic analysis showed low intra-cluster sequence variability, but high inter-cluster divergence suggesting a long-term SWSV evolution in local shrew populations. In conclusion, the investigations demonstrated a broad geographical distribution and multiple spillover infections of rodent-borne TULV and shrew-borne SWSV in Europe. The finding of putative spillover transmissions described here and in other studies underline the current problem of the hantavirus reservoir host definition. In contrast to the hypothesis of a long-standing hantavirus–rodent (small mammal) host coevolution, the investigations support a more dynamic evolutionary history of hantavirus diversification including spillover infections and host-switch events. In future in vitro and in vivo infection studies as well as field studies has to define factors determining the host specificity of these hantaviruses.