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 parasite Rhodococcus 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, the choE monocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified in Brevibacterium sterolicum and Streptomyces spp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded by Mycobacterium tuberculosis and Mycobacterium 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 choE mutation 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 choE isogenic mutants will be a valuable tool for the molecular analysis of R. equi virulence.

Alzheimer disease β-amyloid activity mimics cholesterol oxidase

“…Actual cholesterol oxidase is a bacterial flavoenzyme that is implicated in the ability of bacteria to induce cellular lysis upon infection of their hosts and to induce thrombus (23). Sequence similarity studies have failed to identify a mammalian counterpart, and cholesterol oxidase is generally regarded as unique to bacteria (24).…”

Cholesterol Oxidase: Source,Properties and Applications.

“…CHOx can be an interesting pharmaceutical target for treating bacterial infections. In vitro data suggested that during R. equi infection of the host cell, membrane lysis is facilitated by the induction of extracellular CHOx along with other candidate virulence factors [13]. The membranedamaging activity of R. equi requires the presence of bacterial sphingomyelinase C, thus indicating that the CHOx substrate is not directly accessible to the enzyme in intact membranes [99].…”

“…In most of microorganisms, the CHOx is employed in the initial step of cholesterol metabolism. While in case of pathogenic bacteria CHOx acts as membrane-damaging factors and therefore contributes as a virulence factor in the pathogenicity of these bacteria [5,13]. In addition, Streptomyces natalensis produces a cholesterol oxidase (PimE) which acts as a signaling protein for the biosynthesis of an antifungal antibiotic, polyene macrolide pimaricin [14,15].…”

Rhodococcus equi

“…This highly efficient system was used to isolate an attenuated mutant of R. equi, underscoring the usefulness of transposon mutagenesis [4]. Navas et al [50] developed a highly efficient system based on homologous recombination to disrupt the choE gene encoding cholesterol oxidase of R. equi. The development of random and site directed mutagenesis procedures for R. equi was a major development which will facilitate a detailed analysis of R. equi virulence.…”