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Abstract
Rhodococcus equi is an opportunistic facultative intracellular pathogen of macrophages that causes severe pyogranulomatous pneumonia in young foals and immunocompromised people. Virulence of this organism is dependent on a plasmid-encoded pathogenicity island (PAI) that houses a unique family of highly related proteins of unknown function termed the virulence-associated proteins (VapACDEFGHIX). VapA, a surface-localized lipoprotein, is essential but not sufficient for R. equi replication in macrophages and establishment of a chronic infection in the Severe Combined Immunodeficient (SCID) mouse infection model system. To determine the identity of other plasmid-derived genes necessary for intramacrophage replication, we constructed and analyzed a series of deletion mutant strains. The first assessed was a PAI deletion mutant which was used a molecular tool for narrowing the search for additional plasmid-derived virulence factors aside from vapA. Expression of vapA in the attenuated PAI mutant from a constitutive promoter, thus eliminating the requirement of the PAI-encoded vapA regulators, did not restore the capacity of the PAI strain to replicate intracellularly, indicating that additional virulence genes resided within the deleted PAI region. Subsequently, several more deletion strains were constructed and analyzed to establish the requirement of other PAI genes for intramacrophage growth, most notably, a multiple-deletion mutant (MDM) in which 14 out of 26 PAI-encoded genes were removed including every full length vap gene apart from vapA. Experimental assessment of the MDM mutant demonstrated that the collective deletion of these genes did not impair the intracellular growth capacity of R. equi. This finding indicated that VapA was the only Vap family member essential for intramacrophage growth, and that most of the PAI region was dispensable for the same. We subsequently hypothesized that the minimum virulence plasmid-encoded genes necessary for R. equi replication in macrophages was vapA and its regulators, virR and orf8. Expression of these three genes from their native promoters in an avirulent virulence plasmid-cured strain of R. equi (strain 103-) promoted intracellular replication in macrophages. This finding implied that the combined effect of the presence of VapA and regulator-induced alteration of chromosomal gene expression allows R. equi adaption to life within the restrictive macrophage environment.