Regulation of virulence gene expression in Staphylococcus aureus

Abstract: The pathogenic bacterium Staphylococcus aureus has the ability to cause a wide variety of human diseases, ranging from superficial abscesses and wound infections to deep and systemic infections such as osteomyelitis, endocarditis and septicaemia. The ability to cause disease has been attributed to a large number of toxins and digesting enzymes as well as to proteins at the bacterial surface that bind various host molecules. These so-called virulence factors are accessory, and are supposed to be synthesised in response to the specific needs during the course of the infectious process. The work described in this thesis aims at a better understanding of the mechanisms that regulate the expression of virulence factors. Two interacting regulatory systems, agr (accessory gene regulator) and sar (staphylococcal accessory regulator), are involved in this regulation. The agr locus, which encodes a two-component signal transduction system responding to cell density, controls the expression of at least 25 different virulence factors. The effector molecule of the agr system is a regulatory RNA molecule, named RNAIII. The sar locus has been shown to regulate several staphylococcal virulence genes by modulating the activity of agr, but also via agr-independent mechanisms. The effector of the sar locus is a 14.7 kDa DNA binding protein, SarA. In animal models of infection both agr and sarA have been shown to affect virulence. How RNAIII and sarA function at the molecular level is, however, poorly understood. The structure and function of the RNAIII promoter have been studied in detail showing that SarA, which regulates the synthesis of RNAIII under certain growth conditions, binds to multiple sites within the RNAIII promoter region. It has also been shown that a region of 93 bp upstream of the transcription start point is sufficient for agr-dependent regulation of RNAIII synthesis. The RNAIII genes of several coagulase negative staphylococcal species S. epidermidis, S. simulans and S. warneri have been identified and analysed. The RNAIII molecules from the coagulase negative staphylococci were able to partially complement an RNAIII deficient S. aureus mutant. By the construction of hybrid RNAIII molecules it has also been demonstrated that highly conserved primary and secondary structures in both the 5´- and the 3´-half of the RNAIII molecule are required for regulation of virulence genes, and that separate parts of the molecule were involved in regulation of different target genes. Several genes known to be regulated by RNAIII have been demonstrated to be regulated by the sarA locus, independent of its effect on expression of RNAIII. By electrophoresis mobility shift experiments and DNase footprinting, SarA has been found to bind in a very similar way to the promoter regions of genes that are either activated or repressed by sarA. SarA does not appear to recognise a conserved DNA sequence motif but rather binds to AT-rich sequences. New potential regulators of agr (RNAIII), hla (alpha-hemolysin), ssp (serine protease) and spa (protein A) have been searched for using specific promoter DNA linked to magnetic beads. Of several new candidate regulators, one protein with a high degree of similarity to SarA, named SarH1 (Sar Homologue 1) has been characterised and found to be part of the agr-sarA regulatory network controlling virulence gene expression. By computer searches in the unfinished S. aureus genome databases four additional Sar homologues have been found, some of which may also be involved in this regulatory network.