Studies on global regulators involved in virulence gene expression in Staphylococcus aureus

Sammanfattning: Staphylococcus aureus is a leading cause of skin, soft tissue, respiratory and endovascular diseases. Staphylococcal pathogenicity depends on the coordinated action of at least 40 different virulence factors, including cytolytic toxins (hla, hlb, luk) enterotoxins (ent), several proteases (ssp, aur, scp) and a large number of cell wall-associated proteins, such as protein A (spa), and fibronectin binding protein (FnBPs). The regulation of virulence genes in S. aureus is complex and involves several global regulators (sarA, sarA-homologues, agr, arl, rot, sae) that interact to determine the level of production of different virulence factors. It has been reported that the global regulators agr (accessory gene regulator) and sarA (staphylococcal accessory regulator) can modulate virulence gene expression both negatively and positively. The reason for this diverse effect was studied by looking for additional regulators. By affinity purification using promoter elements from four differently regulated genes, hla (alpha-hemolysin), hld (RNAIII), spa (protein A) and ssp (serine protease) a new global regulator, SarH1 (renamed SarS), was identified. Transcription analysis revealed that sarS was strongly repressed by both sarA and agr, and that the up-regulation of spa in agr and sarA mutant cells, was mediated by SarS. It was also showed that sarA-dependent activation of hla was mediated by SarS. In spite of increased transcription of spa in sarA mutant cells, no protein A was detected. Furthermore, sarA mutants produced less FnBPs than wild type cells, though fnb mRNA levels were the same. Using global protease inhibitors, it was shown that the lack of FnBPs in sarA mutant cells, was due to the high levels of extracellular proteases. Inactivation of the major protease genes, sspA, sspB, aur and scp, confirmed that both FnBPs and protein A were degraded by the secreted proteases, in particular by the serine protease (SspA). Considering the role of FnBPs and protein A in staphylococcal virulence, 92 clinical S. aureus strains were analysed for the expression of extracellular proteases. All strains possessed the major protease genes, although only 23% were protease positive on casein agar plates. Transcription analysis of the protease negative isolates revealed that expression of the protease genes was suppressed due to high stress sigma factor B (SigB)-dependent expression of the protease repressor sarA. Other SigB-dependent traits such as pigmentation and expression of asp 23 were also increased in protease-negative compared to protease-positive strains. The reason for the apparent SigB deficiency in strains with high protease production was investigated. Cloning and sequencing of the SigB operon in three protease and a-hemolysin hyper-producing strains revealed mutations that could explain the SigB deficiency. The role of sigmaB in regulation of protease and hla was further investigated. At a low sigmaB activity sarA seemed to be an activator of hla via suppression of sarS, while at high sigmaB levels inactivation of sarA had no apparent effect on hla expression, probably because of high levels of the SigB-dependent sarS expression. However, inactivation of sarS did not increase the level of hla expression, probably because RNAIII was completely down-regulated in the sarS mutant. The results indicate that SigB activates an as yet unknown repressor of RNAIII, which is partially counteracted by sarA and sarS.