Virulence plasmid encoded transcriptional regulators of the intracellular pathogen Rhodococcus equi

Rhodococcus equi is an intracellular pathogen of macrophages, which often causes life-threatening foal pneumonia. The intracellular replication of R. equi is dependent on a virulence plasmid containing a pathogenicity island (PAI). The vapA, virR, virS genes located in the PAI are essential and sufficient for intracellular growth of R. equi. VapA is the key virulence factor, which functions as a pH-neutralizing factor during phagocytosis. The LysR-type transcriptional regulator VirR and the two-component system orphan response regulator VirS are required for transcription of vapA and mediate crosstalk with the R. equi chromosome for host-specific niche adaptation. VirR and VirS are products of the virR operon. Transcription of the virR operon is driven by the PvirR promoter located upstream of virR and by an inducible promoter Porf5 located within the virR gene which is regulated by both temperature and pH in a synergistic manner. Temperature is the primary environmental signal activating Porf5. During growth at high temperatures, Porf5 is more actively responding to pH signals. Although VirS was considered as a promising candidate involved in pH regulation, deletion of virS does not affect pH regulated activity of Porf5. Surprisingly, a frameshift mutation in virR abolished pH regulation of Porf5, which could be restored by complementation with a trans-acting VirR. This demonstrated that the effect of the virR frameshift mutation on Porf5 activity was not due to disruption of Porf5 but by the inactivation of the virR gene. VirR is therefore required for pH regulation of Porf5 and hence expression of virS. Transcriptome analysis of different R. equi strains grown at high and low pH revealed that R. equi has at least two pH regulons: a general pH regulon and a VirR-mediated virulence-associated pH regulon. The former contains 423 genes involved in transportation, nitrogen metabolism and amino acid metabolism. The latter only contains two chromosomal genes (REQ01110 and REQ08750), both of which are homologues of icgA, a gene located immediately downstream of virR and encoding major facilitator superfamily transporters. Considering the high similarity between icgA, REQ01110, and REQ08750, and the fact that they are all regulated by pH in a VirR-dependent manner, they potentially play a role in VirR-mediated pH regulation. Mutation of a conserved aspartate residue (D57A) abolished VirS-dependent transcription of vapA, suggesting phosphorylation is required for activation of VirS. The phosphorylation of VirS is highly likely mediated by a sensor kinase. Sensor kinases including VirX, VirY, MprB, REQ45230, and KdpD were chosen as candidates involved in the activation of VirS. However, multiple deletions of these sensor kinase genes did not affect VirS-dependent gene transcription. The result suggests three possible scenarios of activation of VirS: 1. The sensor kinase responsible for the activation of VirS is not chosen in this study, which requires further investigation. 2. VirS is phosphorylated in a sensor kinase-independent manner, potentially, by a low molecular weight phosphodonor. 3. VirS is a stand-alone response regulator whose activity is independent of phosphorylation. KdpD/KdpE is the most upregulated two-component system in the presence of VirR and VirS during the growth under VapA inducing growth conditions, and therefore may play a role in pathogenicity. The KdpD/KdpE in R. equi regulates the transcription of the high-affinity potassium transporting system KdpFABC. Deletion of kdpD leads to increased transcription of the kdp genes, suggesting KdpD is a transcriptional repressor of the Kdp system. However, the deletion of kdpD did not affect the intracellular replication of R. equi in the observed period. The result suggests that kdpD is not required for intracellular growth, which may be due to the increased expression of the kdpFABC genes in the absence of KdpD.