Studies on regulatory networks governing virulence gene transcription in Staphylococcus aureus

Abstract

Staphylococcus aureus pathogenicity is dependent on the coordinated action of a number of virulence factors and the expression of these virulence factors is determined by several global regulators. The main regulator seems to be agr but there are several additional regulators (mostly sarA homologues) involved that mainly act downstream of agr. Some of these regulators control virulence gene expression directly but they also regulate each other forming complex regulatory networks. The work described in this thesis aims at better understanding of the function of the agr system and how different regulators act together in controlling transcription of virulence genes.

Most virulence factors in S. aureus are expressed in a growth phasedependent manner governed by the auto-inducible quorum sensing system agr. Activation of agr results in rapid increase of the regulator RNAIII and occurs in response to accumulation of the auto-inducing peptide (AIP). In order to activate the agr system a low basal transcription of the agr operon must be assumed. This basal activity of the operon is stimulated by sarA. To be able to study how SarA would affect activation of agr, a mathematical model of the agr system was set up. The model predicted that the agr system is hysteretic, meaning that activation of agr occurs in a switch-like manner at a specific concentration of AIP, whereas it is inactivated at a specific lower concentration of AIP. According to the model, SarA does not seem essential for the function of the agr switch but alters the concentration of AIP (cell density) at which agr is activated. This was supported by Northern blot analysis of RNAIII in S. aureus mutants with different levels of sarA expression.

To determine how agr and the other regulators act together in controlling transcription of virulence genes, we studied the regulation of one gene (spa) that is negatively regulated by agr and the genes encoding extracellular proteases (aur and sspA), which are positively regulated by agr. To analyze the general principles of how each component in a regulatory system contributes to expression of a virulence gene, a mathematical model of the regulation of spa (protein A) transcription was developed.

Parameter values in this mathematical model were determined by fitting the output of the model to quantitative Northern blot data from various S. aureus regulatory mutants using a gradient search method. The model was validated by correctly predicted spa expression levels in different regulatory mutants not included in the parameter value search. The mathematical model revealed that Rot and SarS act synergistically to stimulate spa expression and that sarA and sarS seem to balance each other in a way that when the activating impact of sarS is small, e.g. in the wild type, the repressive impact of sarA is small, while in a agr-deficient background, when the impact of sarS is maximal, the repressive effect of sarA is close to its maximum.

Previous studies have shown that SarR down-regulates transcription of sarA, which is a repressor of the aur and sspA transcription. This means that inactivation of sarR would result in decreased transcription of aur and sspA, which was confirmed by mRNA analysis using quantitative real-time PCR. However, we also found that sarR acted as a direct stimulator of aur and sspA transcription and that sarR is required for maximal transcription of aur and sspA.