Transcriptome Reprogramming by Plasmid-Encoded Transcriptional Regulators Is Required for Host Niche Adaption of a Macrophage Pathogen

Abstract

bRhodococcus equi is a facultative intracellular pathogen of macrophages, relying on the presence of a conjugative virulence plasmid harboring a 21-kb pathogenicity island (PAI) for growth in host macrophages. The PAI encodes a family of 6 virulence-associated proteins (Vaps) in addition to 20 other proteins. The contribution of these to virulence has remained unclear. We show that the presence of only 3 virulence plasmid genes (of 73 in total) is required and sufficient for intracellular growth. These include a single vap family member, vapA, and two PAI-located transcriptional regulators, virR and virS. Both transcriptional regulators are essential for wild-type-level expression of vapA, yet vapA expression alone is not sufficient to allow intracellular growth. A whole-genome microarray analysis revealed that VirR and VirS substantially integrate themselves into the chromosomal regulatory network, significantly altering the transcription of 18% of all chromosomal genes. This pathoadaptation involved significant enrichment of select gene ontologies, in particular, enrichment of genes involved in transport processes, energy production, and cellular metabolism, suggesting a major change in cell physiology allowing the bacterium to grow in the hostile environment of the host cell. The results suggest that following the acquisition of the virulence plasmid by an avirulent ancestor of R. equi, coevolution between the plasmid and the chromosome took place, allowing VirR and VirS to regulate the transcription of chromosomal genes in a process that ultimately promoted intracellular growth. Our findings suggest a mechanism for cooption of existing chromosomal traits during the evolution of a pathogenic bacterium from an avirulent saprophyte. The genus Rhodococcus comprises a large number of metabolically diverse species that have attracted considerable biotechnological interest because of their ability to metabolize a wide variety of substrates, which finds applications in bioremediation and in the synthesis of precursors of pharmaceutical compounds (1). The genus contains only two pathogenic species: the plant pathogen Rhodococcus fascians and the animal pathogen Rhodococcus equi (2). Although the latter species was initially isolated from young foals, it has subsequently been isolated from a wide range of animals and humans (3). Disease in foals and immunosuppressed humans usually presents as pyogranulomatous pneumonia, although other manifestations, including osteomyelitis, may also occur. In pigs and cattle, R. equi is usually associated with submandibular lymphadenitis (3). In addition to having a pathogenic lifestyle, R. equi grows readily as a saprophyte in soils, as well as in the equine intestinal tract (3).R. equi is a parasite of macrophages, which prevents killing by the host cell through inhibition of phagosomal maturation, resulting in the formation of R. equi-containing vacuoles devoid of lysosomal markers, including cathepsin D and the proton-pumping vacuolar ATPase (vATPase) complex (4-6). The growth of...

Virulence‐associated protein A fromRhodococcus equiis an intercompartmental pH‐neutralising virulence factor

“…Additional factors encoded by the virulence plasmid (Coulson et al, 2015) are likely needed to boost VapA production by intracellular rhodococci and VapA may have activities beyond those presented here. Neutralisation of lysosome and phagosome pH increases bacterial viability and allows virulent, that is VapA-producing R. equi, to utilise intraphagosomal contents as a source of nutrients.…”

“…This explains why rVapA can turn a plasmid-less, avirulent R. equi isolate into a pathogen for macrophages. Additional factors encoded by the virulence plasmid (Coulson et al, 2015) are likely needed to boost VapA production by intracellular rhodococci and VapA may have activities beyond those presented here. To our knowledge, this is the first example of a Gram-positive pathogen whose membraneactive protein produced in one compartment is transferred to another compartment where it increases membrane permeability.…”

Effect of Macrolide and Rifampin Resistance on the Fitness of Rhodococcus equi

“…the expression of ϳ18% of chromosomal genes, impacting the cell's regulatory network to permit adaption to the macrophage environment (32). It is of relevance to note that all strains of each isogenic set carry the virulence plasmid, and the interactions between the virulence plasmid and chromosomal regulation might contribute to differences observed in in vitro growth of macrolide-resistant R. equi isolates in BHI compared to MM.…”

Tetracycline alters gene expression in Salmonella strains that harbor the Tn10 transposon

“…A relevant aspect of plasmid biology that has hitherto been studied in less detail corresponds to the crosstalk that the resident plasmid establishes with the host regulatory networks (Coulson et al ., ; Yun et al ., ). In this context, we have shown that plasmid R27 significantly influences Salmonella transcriptome, but especially when cells grow at low temperature (25°C) and enter the stationary phase (Paytubi et al ., ).…”