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

Investigation published in Journal of Bacteriology

August 1st, 2001

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




Navas J., Gonzalez-Zorn B., Ladron N., Garrido P. and Vazquez-Boland JA.




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

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



Participants:

Universidad ComplutenseDepartamento de Sanidad Animal. Facultad de Veterinaria. Universidad Complutense (UCM).

Universidad ComplutenseServicio de Zoonosis de Transmisión Alimentaria y Resistencia a Antimicrobianos (ZTA). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM).

Universidad de CantabriaDepartamento de Biología Molecular. Facultad de Medicina. Universidad de Cantabria (UC).

Universidad ComplutenseGrupo de Patogénesis Molecular Bacteriana. Facultad de Veterinaria. Universidad Complutense (UCM).







Journal of Bacteriology
FACTOR YEAR Q
3.984 2001

NLMID: 2985120R

PMID: 11466283

ISSN: 0021-9193



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


JOURNAL: J Bacteriol


NUMERACIÓN: 183(16):4796-805


AÑO: 2001


PUBLISHER: American Society for Microbiology


AUTHORS: Navas J., Gonzalez-Zorn B., Ladron N., Garrido P. and Vazquez-Boland JA.


Bruno González Zorn

DOI: https://doi.org/10.1128/JB.183.16.4796-4805.2001


CITE THIS PUBLICATION:

Navas J., Gonzalez-Zorn B., Ladron N., Garrido P. and Vazquez-Boland JA. Identification and mutagenesis by allelic exchange of choE, encoding a cholesterol oxidase from the intracellular pathogen Rhodococcus equi. Journal of Bacteriology. 183(16):4796-805. 2001. (A). ISSN: 0021-9193. DOI: 10.1128/JB.183.16.4796-4805.2001


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