The accessory gene regulator, agr, a system for coordinate control of virulence genes in Staphylococcus aureus

Abstract

Bacterial pathogenicity is generally multifactorial, and genes encoding virulence factors are often subject to coordinate regulation. The regulating systems are supposed to adapt the production of virulence factors in response to changes in the environment during the process of infection. Understanding of the molecular mechanisms involved is therefore essential for a proper understanding of the pathogenesis of infectious diseases. The Gram positive bacterium Staphylococcus aureus is a major human pathogen causing a variety of diseases such as wound infections, abscesses, septicaemia, endocarditis, food poisoning and toxic shock syndrome.

The virulence of S. aureus is associated with a wide range of extracellular toxins, enzymes and cell-surface proteins that are believed to contribute in a co-operative way to increase the invasive powers of the pathogen and to help it escape clearance by the immune system of the host. Many of the genes encoding these virulence factors have been shown to be coordinately controlled by a chromosomal locus called agr, accessory gene regulator. The agr locus was defined by an insertion of the transposon, Tn551, at a single site on the chromosome that resulted in depressed production of at least 15 extracellular proteins, including alpha-toxin and proteinases, and a simultaneous increase in the production of the cell bound proteins, i. e. protein A and coagulase. This thesis describes the cloning of the agr locus and shows that it is responsible for regulation of the virulence genes. Subsequent work by others, revealed that the agr locus consists of two divergent transcription units, one polycistronic transcript, encoding four genes; agrB, agrD, agrC, and agrA, and 510 nt RNA, RNAIII, which is the effector of the regulation of the virulence genes. The products of the agr locus constitute an autoregulated, cell density dependent, signal transduction system, whose main function seems to be to activate transcription of the RNAIII gene.

In this thesis, the synthesis of RNAIII and the function of RNAIII as a regulator of virulence gene expression have been studied. Transcription of the RNAIII gene requires the activated form of the response regulator, AgrA, which is synthesised from the agr operon, and an additional factor encoded by sar. The structure and function of the RNAIII promoter has been studied in detail. We have shown that SarA, but not AgrA, binds to multiple sites within the RNAIII promoter region. It has been indicated that RNAIII regulates its target genes both at the level of transcription initiation and translation. By analysing the transcription of the alpha-toxin gene in different strains of S. aureus, we could identify sequences upstream of the hla promoter that are needed for RNAIII dependent regulation. It was also shown that additional factors are needed together with RNAIII to activate transcription. RNAIII also stimulates translation of the alpha-toxin mRNA. We have shown that the ribosome binding site of the alpha-toxin mRNA is blocked by intramolecular base-pairing. Hybridisation with RNAIII prevents this base-pairing and makes the alpha-toxin mRNA accessible for translation initiation. This findings makes RNAIII to the first example of an antisense RNA that stimulates translation of its target mRNA.