The vapA co-expressed virulence plasmid gene vcgB (orf10) of the intracellular actinomycete Rhodococcus equi.

The virulence plasmid of the pathogenic actinomycete Rhodococcus equi is essential for proliferation of this pathogen in macrophages and the development of disease. The pathogenicity island of this plasmid encodes a family of virulence-associated proteins (Vap), one of which (VapA) is a virulence factor. This paper describes the vcgAB operon (vapA co-expressed gene), located upstream of the vapA operon. Transcription of the vcgAB operon gave rise to transcripts with a half-life similar to those determined for other virulence plasmid genes (1.8 min). Transcription started at a promoter similar to the vapA promoter, and proceeded through an inefficient terminator into the downstream vcgC gene. In addition, vcgC is also transcribed from a promoter downstream of vcgB. The vcgAB and vapA operons were coordinately regulated by temperature and pH in a synergistic manner. The latter parameter only affected transcription at higher growth temperatures, indicating that temperature is the dominant regulatory signal. Transcription of the vcgAB operon increased 10-fold during the late exponential and stationary growth phases. Transcription was also upregulated during the initial hours following phagocytosis by phagocytic cells. In contrast to vcgA and vcgC, the vcgB gene is conserved in the porcine VapB-encoding plasmid, as well as in pathogenic mycobacteria. The coordinated regulation of vcgB and vapA, transcription of vcgB following phagocytosis and conservation of vcgB in pathogenic mycobacteria indicate a role for vcgB and the vcg genes in the virulence of R. equi.

Differential mRNA stability of the vapAICD operon of the facultative intracellular pathogen Rhodococcus equi.

The gene encoding virulence associated protein A (VapA) is clustered with three vapA homologues (vapICD) within the pathogenicity island of the virulence plasmid of Rhodococcus equi. Northern blot analysis showed a vapA transcript of c. 700 nucleotides (nt) suggesting that vapA is a monocistronic transcript. However, using the more sensitive RT-PCR, it was shown that vapA is cotranscribed with the downstream vapICD genes forming a 2.3-kb operon. This initial transcript is subsequently processed to give rise to a 700 nt vapA transcript with a half-life of 7.5 min. In contrast, the vapI, vapC and vapD transcripts have an average half-life of 1.8 min, identical to that of the five cistronic virR operon located upstream of the vapA operon. It is speculated that the need for differential gene expression arises from the different localisation of the Vap proteins. VapA is tethered to the surface of the cell wall, whereas VapC and VapD are secreted, diffusable proteins. The intercistronic region between vapC and vapD harbours two short ORFs (OrfA, OrfB). These ORFs are translationally coupled to vapC and vapD in which the start codon overlaps the stop codon of the preceding gene.

Isocitrate lyase of the facultative intracellular pathogen Rhodococcus equi.

Isocitrate lyase is the first enzyme of the glyoxylate shunt which is required for the assimilation of fatty acids and acetate. The intracellular pathogen Rhodococcus equi contains high activities of this enzyme following growth on acetate and lactate, indicating that it plays an important role in the metabolism of these substrates. The gene encoding isocitrate lyase (aceA) was cloned and sequenced. It specifies a 46846 Da protein, which was shown to be functional by expressing it in Escherichia coli. A gene similar to fadB, encoding 3-hydroxyacyl-CoA dehydrogenase, was located 90 bp downstream from aceA. Northern hybridization and RT-PCR experiments showed that aceA and fadB are cotranscribed into a 2.8 kb transcript. A smaller 1.6 kb aceA transcript was also observed which was 2.5-fold more abundant than the aceA-fadB transcript. It is proposed that a stable hairpin structure with a free energy (DeltaG) of -28.5 kcal x mol(-1) and located in the 90 bp aceA-fadB intergenic region is involved in stabilizing the aceA transcript.

The LysR-type transcriptional regulator VirR is required for expression of the virulence gene vapA of Rhodococcus equi ATCC 33701.

The virulence of the intracellular pathogen Rhodococcus equi in foals is dependent on the presence of an 81-kb virulence plasmid encoding the virulence protein VapA. Expression of this protein is induced by exposure to oxidative stress, high temperatures, and low pHs, which reflect the conditions encountered by R. equi when it enters the host environment. The aim of this study was to determine whether the LysR-type transcriptional regulator VirR, which is encoded by the virulence plasmid, is required for the expression of vapA. It was shown that the virR gene is cotranscribed with four downstream genes, one of which encodes a two-component response regulator. The expression of VapA, as monitored by Western blotting, was completely dependent on the presence of virR. Maximal expression was observed when vapA was present together with the complete virR operon, suggesting that at least one of the virR operon genes, in addition to virR, is required for the expression of vapA to wild-type levels. The transcriptional start site of vapA was determined to be a cytidine located 226 bp upstream from the vapA initiation codon. His-tagged VirR protein was expressed in Escherichia coli and purified by nickel affinity chromatography. DNA binding studies showed that purified VirR binds to a DNA fragment containing the vapA promoter. We therefore conclude that VirR is required for the activation of vapA transcription.