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Staphylococcus aureus is a pathogenic bacterium infecting the human host. It’s multifaced adaptation to various environmental conditions is mediated by a tight regulation of the virulence factors influencing the host’s immune system. In this thesis two regulators of gene expression were analysed: (i) the global influence of the two-component system SaePQRS and (ii) the regulation of superantigen gene expression by the alternative sigma factor σB. At the outset of this thesis, single target genes induced by SaeRS were known (hla, hlb, cap5, fnbA, coa). In order to get a general idea of the Sae-regulon, the influence of SaePQRS on gene-expression was analysed in two strain backgrounds by proteomics and transcriptomics aproaches. Recapitulatory, expression of at least 18 secreted and two covalently cell-wall bound proteins was decreased following inactivation of the Sae-system. Sae-dependently expressed were, amongst others, well decribed virulence factors like the y-hemolysins HlgA, HlgB, HlgC, LukM and LukF, the innate immune system modulating proteins Efb, CHIPS and SCIN-B as well as the enterotoxin SEB. SaeR acts as an activator of its target genes. Some proteins were detected in increased amounts in the extracellular proteome of the Sae-deficient strain. However, these changes did not occur at the transcriptional level. The expression of virulence factors is determined by other global regulators. No influence of SaePQRS on the transcription of five substancial regulators, namely the Agr-system and its effector molecule RNAIII, the alternative sigma factor σB, the two-component system ArlRS and the DNA-binding protein SarA, could be shown. In the second part of this thesis the issue was broached to the regulation of gene-expression of a subgroup of virulence factors, the superantigens (SAgs) of S. aureus by SaePQRS and σB. In contrast to their well described molecule structure and function, the regulation of their gene expression was largely unknown. Six different S. aureus strains (two laboratory strains and four clinical isolates) encoding one to seven SAg-genes each, were used for analysis of a total of twelve SAgs regarding their transcription and mitogenic activity. The transcriptional units were characterized using Northern-Blotting. The expression of SAgs could be correlated to the respective growth phase. While egc-SAgs were expressed mainly at low optical densities, seb was induced during late growth phase. In contrast, the transcription of sea, seh, sek, tst and sep remained constant and growth-phase independent. The transcriptional dataset was verified using T-cell proliferation assays. The expression of seh, tst and the egc-operon was dependent on σB. A potential σB-dependent promotor could be identified preceeding seo, the first gene of the egc-operon. In contrast, the expression of seb was increased in sigB-deficient background. This might be due to indirect effects. Expression of seb required SaePQRS. Transcriptional datasets were verified by Immuno-Blotting and T-cell-proliferation assays. In conclusion, the same mutation in sigB but in different strain backgrounds could result in opposite phenotypes with respect to their mitogenic activity. Besides well characterized virulence factors, some secreted proteins with so far unknown function belong to the Sae-regulon. Given that the influence of SaePQRS was restricted to virulence factors and induced especially modulators of the innate immune system, it can be assumed, that these proteins potentially play a role in virulence of S. aureus. In the third part of this thesis, one of these potential new virulence factors, namely SACOL0908, was analysed in detail. In cooperation with the group of Prof. Stehle, Tübingen, the crystal structure was solved. The protein folding of SACOL0908 is new with only minor similarities to described protein structures. Recombinantly expressed SACOL0908 binds to granulocytes. These cells belong to the innate immune system, incorporate bacteria by phagocytosis and kill them. The receptor for SACOL0908 on the surface of granulocytes could not be identified using immunoprecipitation, antibody-blocking assays and functional assays in cooperation with the group of Prof. Peschel, Tübingen. The gene encoding SACOL0908 was deleted in two S. aureus strain backgrounds (COL and Newman). These mutants are currently in use to characterize their phenotype in mouse-infection studies.
Staphylococcus aureus is a commensal colonizing 20-30% of the population as well as a pathogen causing diverse diseases ranging from skin infections via toxin mediated diseases to life threatening conditions. In its interplay with the human host, this microorganism resorts to an extensive repertoire of both membrane-bound and secreted virulence factors facilitating adhesion to, invasion of, and spreading into various host tissues. Among the numerous virulence factors produced by S. aureus are the staphylococcal superantigens (SAgs). They directly cross-link conserved regions of the T cell-receptor with MHC class II molecules (outside the peptide-binding cleft) on antigen presenting cells. This results in a strong stimulation of up to 20% of all T cells which respond with proliferation and massive cytokine release. Recently, the enterotoxin gene cluster (egc) located on a pathogenicity island was described. The egc-genes are the most prevalent SAg genes in commensal and invasive S. aureus isolates. However, they appear to cause toxic shock only very rarely and their presence is negatively correlated with severity of S. aureus sepsis. Therefore it was suggested that SAgs might differ in their pro-inflammatory potential. In addition to their superantigenicity, SAgs also act as conventional antigens and induce a specific antibody response. In contrast to non-egc SAgs, despite the high prevalence of egc SAgs, neutralizing antibodies against egc SAgs are very rare, even among carriers of egc-positive S. aureus strains. In order to find an explanation for this “egc-gap”, we have tested two non-exclusive hypotheses: (i) egc and non-egc SAgs have unique intrinsic properties and drive the immune response into different directions and (ii) egc and non-egc SAgs are released by S. aureus under different conditions, which shape the immune response to them. To test these hypotheses, we compared the effects of egc and non-egc SAgs on human blood cells. Their T cell-mitogenic potencies, the elicited cytokine profiles as well as their impact on gene expression were highly similar. Both egc and non-egc SAgs induced a very strong pro-inflammatory response. In contrast, the regulation of SAg release by S. aureus differed markedly between egc and non-egc SAgs. Egc-encoded proteins were secreted by S. aureus during exponential growth, while non-egc SAgs were released in the stationary phase. We conclude that the distinct biological behavior of egc and non-egc SAgs is not due to their intrinsic properties, which are very similar, but is caused by their differential release by S. aureus. Traditionally, S. aureus has not been considered as an intracellular pathogen but strong evidence emerged indicating that staphylococci can invade and persist in various cell types. Internalization might constitute a bacterial strategy to evade the host’s defense reactions and the action of antibiotics. The intracellular niche might thus constitute a reservoir for chronic or relapsing infections. Contrary to their potential importance, genome-wide functional genomics analyses of the adaptation reactions of S. aureus to the host cell environment are rare and so far confined to gene expression profiling. Investigations addressing the proteome of internalized S. aureus are still lacking due to the challenge of obtaining a sufficient number of infecting bacteria. The proteome of other pathogens such as Francisella tularensis has been characterized by classical 2-DE approaches. However, the number of bacteria required for such a 2-DE based approach is often exceeding the numbers available from in vivo infection models. Furthermore, this approach does not allow monitoring of time-dependent quantitative changes in protein levels. Here, a workflow allowing time-resolved analysis of internalized S. aureus by combining pulse-chase stable isotope labeling by amino acids in cell culture with high capacity cell sorting, on-membrane digestion, and high-sensitivity mass spectrometry is presented. This workflow permits detection and quantitative monitoring of several hundred staphylococcal proteins from as little as a few million internalized S. aureus cells. This approach has been used to reveal time-resolved changes in levels of proteins in S. aureus RN1HG upon internalization by human bronchial epithelial cells. Proteins involved in stress adaptation as well as protein folding and some components of the phosphotransferase system were upregulated in internalized staphylococci, whereas proteins of the purine biosynthesis pathway and tRNA aminoacylation were downregulated. Furthermore, regulatory adaptive responses of internalized S. aureus to the intracellular milieu were shown as global regulators displayed increased protein abundance levels compared to non-internalized bacteria. Taken together, we observed changes in levels of proteins with functions in protection against oxidative damage and adaptation of cell wall synthesis in internalized S. aureus.
Staphylococcus (S.) aureus is the most common cause of nosocomial infections and the species is becoming increasingly resistant to antibiotics. In contrast, about 35% of the healthy population are colonized with S. aureus in the anterior nares. The genetic make-up of this species is highly diverse. Mobile genetic elements comprise about 15% of the S. aureus genome. They encode many virulence factors like the 21 different known staphylococcal superantigens (SAgs), highly potent activators of T lymphocytes. Besides their well known causative role in food poisoning and toxic shock syndrome, information about SAg involvement in pathogenesis is limited. On the other hand, the human host and its immune response are also highly diverse. This study focuses on SAgs, because they are potent virulence factors that are highly diverse and therefore mirror of the variability of the species S. aureus. The goals of this work were (i) to identify virulence determinants by comparing the prevalence of SAg genes and phages among colonizing and invasive S. aureus isolates and to correlate it with the clonal background, (ii) to determine the prevalence and the development of anti-SAg antibodies in healthy S. aureus carriers and noncarriers as well as in bacteremia patients, and (iii) to elucidate the reasons for the selective lack of neutralizing serum antibodies specific for a subgroup of SAgs, the egc SAgs. In search for a molecular-epidemiological associations between SAgs and different diseases caused by S. aureus we investigated the distribution of SAg genes and/ or bacteriophages and correlated this with the clonal background, determined by spa genotyping. The analysis of more than 700 S. aureus isolates from nasal colonization, bacteremia or furunculosis revealed that SAg-encoding mobile genetic elements and bacteriophages were strongly associated with the clonal background. As a consequence, each clonal lineage was characterized by a typical SAg gene and phage repertoire. Therefore, we suggest that the simultaneous assessment of virulence gene profiles and the genetic background strongly increases the discriminatory power of genetic investigations into the mechanisms of S. aureus pathogenesis. However, we found no association of SAg genes with bacteremia or furunculosis. While functional neutralization assays closely mimic the protective action of anti-SAg antibodies in vivo, they are labor-intensive and time-consuming. A fast and easy method for the simultaneous quantification of antibody binding to multiple staphylococcal antigens is the Luminex® technology. Using serum samples from persistent carriers and noncarriers we showed a strong correlation between antibody binding and neutralizing capacity against the SAg TSST-1. This assay confirmed the astonishing lack of antibodies against egc SAgs in healthy carriers and noncarriers, which was previously described by Holtfreter and coworkers. Since colonization is probably not sufficient to induce a robust antibody response as revealed by experimental colonization with S. aureus, we propose that (minor) infections are required to induce the high titers of non-egc SAg-neutralizing antibodies in healthy adults. To test this, we investigated whether SAgs elicit a neutralizing antibody response during S. aureus bacteremia. At the acute phase of the disease most patients already had neutralizing antibodies against non-egc SAgs, and antibody titers frequently increased during infection. Notably, egc SAgs did not elicit a boost or de novo generation of specific antibodies. The “egc gap” in the antibody response, which has now been shown in healthy adults, as well as following systemic infection with S. aureus, is astonishing. After all, egc SAgs are by far the most prevalent SAgs. In search for an explanation, the intrinsic properties of three recombinant egc (SEI, SElM, SElO) and non-egc SAgs (SEB, SElQ, TSST-1) were compared in depth. Egc and non-egc SAgs were very similar with regard to induced T cell proliferation, cytokine profiles, and gene expression of human immune cells. However, there was a striking difference in the regulation of the two groups of SAgs by S. aureus in bacterial culture. We conclude that the differential regulation of egc and non-egc SAg has an impact on the immune response. But how are SAgs regulated by S. aureus during its interaction with the host? Up until now most research on regulation of virulence factors has been performed in vitro. The immune response can help to shed light on this problem, because it is an exquisitely specific sensor for the exposure to different antigens. The high prevalence of neutralizing serum antibodies against non-egc SAgs indicates that most healthy adults have been exposed to these toxins during their encounters with S. aureus. For egc SAgs this remains an open question. However, initial data indicate that the egc SAg genes are transcribed during nasal colonization.
SUMMARY To date, Staphylococcus aureus is the most common cause of nosocomial infections and the species is becoming increasingly resistant to antibiotics. Beyond this, S. aureus colonises the nasal mucosa of circa 35% of the healthy population, so-called carriers. Importantly, S. aureus nasal carriage is a major risk factor for the development of S. aureus infections, which are commonly caused by the colonising strain. This underlines the importance of host factors for the outcome of S. aureus-host interactions. Despite the clinical importance of nasal carriage, little is known about humoral immune responses triggered by colonisation. Therefore, this thesis was focussed on the anti-staphylococcal antibody responses of S. aureus carriers and noncarriers. Staphylococcal superantigens (SAgs) served as indicator antigens for our studies. SAgs are virulence factors with extraordinary variability in the species S aureus and act as extremely potent T cell mitogens. To date, 19 different SAg gene loci are known in the species S. aureus, but molecular-epidemiological studies on the distribution of these genes are limited. Therefore, we established five multiplex PCRs for the detection of all known SAgs. With this robust and high-throughput technique we analysed the SAg gene patterns of more than 300 isolates, including 107 nasal isolates of S. aureus carriers and 88 blood culture isolates of hospital patients from Western Pomerania. The SAg gene patterns were highly heterogeneous, which can be explained by their localisation on mobile genetic elements (MGE), such as genomic islands, pathogenicity islands, phages and plasmids. Most isolates (~80%) harboured SAg genes, on average five to six, and SAgs of the enterotoxin gene cluster (egc) were by far the most prevalent. Additionally, we observed a strict correlation between the presence of SAg genes and the T cell mitogenic potency of clinical isolates. SAg-encoding MGEs can be distributed by two distinct mechanisms: horizontal transfer by bacteriophages and vertical transmission to daughter cells. To investigate the distribution of SAg genes within the S. aureus population, we determined the clonal relationship of our isolates by spa genotyping. Interestingly, SAg-gene encoding MGEs were not randomly distributed, but rather closely linked to clonal lineages. Each clonal lineage was characterised by defined combinations of SAg genes. These data suggest that the simultaneous assessment of virulence gene profiles and the genetic background strongly enhances the discriminatory power of genetic investigations into the mechanisms of S. aureus virulence. Indeed, the comparison of virulence genes within each clonal complex indicated a role in invasiveness for some MGEs, e.g. the exfoliative toxin D-encoding pathogenicity island, while rendering it unlikely for SAgs. It is known that neutralising serum antibodies against the SAgs SEA, SEB, SEC, SED and TSST-1 are frequently present in healthy individuals. However, the neutralising antibody profiles against more recently described SAgs or complex SAg cocktails as secreted by clinical isolates had not been determined so far. Therefore, we screened more than 100 sera for their SAg neutralising capacity with a neutralisation assay. We observed a marked heterogeneity and surprisingly large “gaps” in the neutralising capacity. Interestingly, the egc SAgs were inhibited only rarely (5-10%), whereas between 32 and 86% of the tested sera neutralised “classical” SAgs. This “egc gap” in the SAg-neutralising antibody profiles of healthy individuals was unexpected, since egc SAgs are by far the most prevalent SAgs. We could demonstrate that the “egc gap” is probably not due to different T cell activating properties of egc SAgs compared to classical SAgs, but rather to a differential regulation of SAg gene expression. S. aureus carriers have an increased risk of developing an S. aureus bacteraemia, which is in most cases caused by the colonising strain. Intriguingly, a large prospective clinical trial revealed a considerably higher mortality in noncarriers with invasive S. aureus strains compared to carriers with invasive disease. To explain these paradoxical findings, we hypothesised that in carriers partial immunity against the colonising strain may contribute to their improved outcome. We used SAgs as strain-specific indicator antigens. Importantly, sera from persistent carriers neutralised SAgs of their colonising strain with significantly higher efficiency than sera from noncarriers. This antibody response was strain-specific, since the antibody response of carriers against other SAgs did not differ from that of noncarriers. Thus, colonisation with S. aureus confers a strong and strain-specific antibody response against staphylococcal SAgs. We suggest that in carriers neutralising antibodies directed against SAgs and other staphylococcal virulence factors confer partial protection during systemic infections. This could explain the better prognosis of carriers with S. aureus bacteraemia compared to noncarriers. Moreover, our data imply that the key to understanding the pathogenesis of S. aureus disease may lie in the identification of host factors rather than bacterial factors. Such host factors could be the immune status and gene polymorphisms that contribute to colonisation, susceptibility to infection and outcome of infection. Finally, while the treatment of S. aureus bacteraemia with pooled immunoglobulins was performed in the past without significant success, our findings on strain-specific antibody profiles suggest that therapies with customised cocktails of monoclonal antibodies could have a higher efficacy.