Identification and mutagenesis by allelic exchange of choE, encoding a cholesterol oxidase from the intracellular pathogen Rhodococcus equi

The virulence mechanisms of the facultative intracellular parasite Rhodococ cus equi remain largely unknown. Among the candidate virulence factors of t his pathogenic actinomycete is a secreted cholesterol oxidase, a putative m embrane-damaging toxin. We identified and characterized the gene encoding t his enzyme, the choE monocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified in Brevibacterium steroli cum and Streptomyces spp. ChoE also exhibits significant similarities to pu tative cholesterol oxidases encoded by Mycobacterium tuberculosis and Mycob acterium leprae. Genetic tools for use with R. equi are poorly developed. H ere we describe the first targeted mutagenesis system available for this ba cterium. It is based on a suicide plasmid, a selectable marker (the aacC4 a pramycin resistance gene from Salmonella), and homologous recombination. Th e choE allele was disrupted by insertion of the aacC4 gene, cloned in pUC19 and introduced by electroporation in R. equi. choE recombinants were isola ted at frequencies between 10(-2), and 10(-3). Twelve percent of the recomb inants were double-crossover choE mutants. The choE mutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-produ cing bacteria (Listeria ivanovii). Functional complementation was achieved by expression of choE from pVK173-T, a pAL5000 derivative conferring hygrom ycin resistance. Our data demonstrate that ChoE is an important cytolytic f actor for R. equi. The highly efficient targeted mutagenesis procedure that we used to generate choE isogenic mutants will be a valuable tool for the molecular analysis of R. equi virulence.