Identification and Mutagenesis by Allelic Exchange ofchoE, Encoding a Cholesterol Oxidase from the Intracellular PathogenRhodococcus equi

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Abstract
The virulence mechanisms of the facultative intracellular parasiteRhodococcus equiremain 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, thechoEmonocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified inBrevibacterium sterolicumandStreptomycesspp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded byMycobacterium tuberculosisandMycobacterium leprae. Genetic tools for use withR. equiare poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (theaacC4apramycin resistance gene fromSalmonella), and homologous recombination. ThechoEallele was disrupted by insertion of theaacC4gene, cloned in pUC19 and introduced by electroporation inR. equi. choErecombinants were isolated at frequencies between 10−2and 10−3. Twelve percent of the recombinants were double-crossoverchoEmutants. ThechoEmutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression ofchoEfrom pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor forR. equi. The highly efficient targeted mutagenesis procedure that we used to generatechoEisogenic mutants will be a valuable tool for the molecular analysis ofR. equivirulence.