Identification and Mutagenesis by Allelic Exchange of choE, Encoding a Cholesterol Oxidase from the Intracellular Pathogen Rhodococcus equi

ABSTRACT

The virulence mechanisms of the facultative intracellular parasiteRhodococcus equi remain largely unknown. Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin. We identified and characterized the gene encoding this enzyme, thechoE monocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified inBrevibacterium sterolicum and Streptomyces spp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded by Mycobacterium tuberculosis andMycobacterium leprae. Genetic tools for use with R. equi are poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (the aacC4 apramycin resistance gene from Salmonella), and homologous recombination. The choE allele was disrupted by insertion of the aacC4 gene, cloned in pUC19 and introduced by electroporation in R. equi. choE recombinants were isolated at frequencies between 10⁻² and 10⁻³. Twelve percent of the recombinants were double-crossover choE mutants. The choEmutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression of choE from pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor for R. equi. The highly efficient targeted mutagenesis procedure that we used to generate choEisogenic mutants will be a valuable tool for the molecular analysis ofR. equi virulence.