The Steroid Catabolic Pathway of the Intracellular Pathogen Rhodococcus equi Is Important for Pathogenesis and a Target for Vaccine Development

Abstract

Rhodococcus equi causes fatal pyogranulomatous pneumonia in foals and immunocompromised animals and humans. Despite its importance, there is currently no effective vaccine against the disease. The actinobacteria R. equi and the human pathogen Mycobacterium tuberculosis are related, and both cause pulmonary diseases. Recently, we have shown that essential steps in the cholesterol catabolic pathway are involved in the pathogenicity of M. tuberculosis. Bioinformatic analysis revealed the presence of a similar cholesterol catabolic gene cluster in R. equi. Orthologs of predicted M. tuberculosis virulence genes located within this cluster, i.e. ipdA ( rv3551), ipdB ( rv3552), fadA6 and fadE30, were identified in R. equi RE1 and inactivated. The ipdA and ipdB genes of R. equi RE1 appear to constitute the α-subunit and β-subunit, respectively, of a heterodimeric coenzyme A transferase. Mutant strains RE1Δ ipdAB and RE1Δ fadE30, but not RE1Δ fadA6, were impaired in growth on the steroid catabolic pathway intermediates 4-androstene-3,17-dione (AD) and 3aα-H-4α(3′-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5α-hydroxy-methylhexahydro-1-indanone propionate; 5OH-HIP). Interestingly, RE1Δ ipdAB and RE1Δ fadE30, but not RE1Δ fadA6, also displayed an attenuated phenotype in a macrophage infection assay. Gene products important for growth on 5OH-HIP, as part of the steroid catabolic pathway, thus appear to act as factors involved in the pathogenicity of R. equi. Challenge experiments showed that RE1Δ ipdAB could be safely administered intratracheally to 2 to 5 week-old foals and oral immunization of foals even elicited a substantial protective immunity against a virulent R. equi strain. Our data show that genes involved in steroid catabolism are promising targets for the development of a live-attenuated vaccine against R. equi infections.

Author Summary

Rhodococcus equi causes fatal pyogranulomatous bronchopneumonia in young foals and is an emerging opportunistic pathogen of immunocompromised humans. Despite its importance, there is currently no safe and effective vaccine against R. equi infections. Like Mycobacterium tuberculosis, the causative agent of human tuberculosis, R. equi is able to infect, survive and multiply inside alveolar macrophages. Recently we have shown that essential steps in the cholesterol catabolic pathway (encoded by the rv3551, rv3552, fadE30 genes) are involved in the pathogenicity of M. tuberculosis. We hypothesized that the orthologous genes in the cholesterol catabolic gene cluster of R. equi also are essential for its virulence mechanism. Analysis of the respective R. equi strain RE1 mutants revealed that they were impaired in growth on intermediates of the steroid catabolic pathway and had attenuated phenotypes in a macrophage infection assay. Mutant RE1Δ ipdAB, carrying a deletion of the orthologs of rv3551 and rv3552, could be safely administered to 2–5 week-old foals intratracheally and oral immunization provided a substantial protection against infection by a virulent R. equi strain. Our data show that genes important for methylhexahydroindanone propionate degradation, part of the steroid catabolic pathway, are promising targets for the development of a live-attenuated vaccine against R. equi infections.