title: Generation of attenuated Mycobacterium bovis strains by signature-tagged mutagenesis for discovery of novel vaccine candidates.
abstract: Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, has a particularly wide host range and causes tuberculosis in most mammals, including humans. A signature tag mutagenesis approach, which employed illegitimate recombination and infection of guinea pigs, was applied to M. bovis to discover genes important for virulence and to find potential vaccine candidates. Fifteen attenuated mutants were identified, four of which produced no lesions when inoculated separately into guinea pigs. One of these four mutants had nine deleted genes including mmpL4 and sigK and, in guinea pigs with aerosol challenge, provided protection against tuberculosis at least equal to that of M. bovis BCG. Seven mutants had mutations near the esxA (esat-6) locus, and immunoblot analysis of these confirmed the essential role of other genes at this locus in the secretion of EsxA (ESAT-6) and EsxB (CFP10). Mutations in the eight other attenuated mutants were widely spread through the chromosome and included pks1, which is naturally inactivated in clinical strains of M. tuberculosis. Many genes identified were different from those found by signature tag mutagenesis of M. tuberculosis by use of a mouse infection model and illustrate how the use of different approaches enables identification of a wider range of attenuating mutants.
authors.affiliation: AgResearch, Wallaceville Animal Research Centre, P.O. Box 40063, Upper Hutt, New Zealand. desmond.collins@agresearch.co.nz
authors.forename: Desmond M
authors.initials: DM
authors.lastname: Collins
authors.forename: Bronwyn
authors.initials: B
authors.lastname: Skou
authors.forename: Stefan
authors.initials: S
authors.lastname: White
authors.forename: Shalome
authors.initials: S
authors.lastname: Bassett
authors.forename: Lauren
authors.initials: L
authors.lastname: Collins
authors.forename: Raewyn
authors.initials: R
authors.lastname: For
authors.forename: Kathryn
authors.initials: K
authors.lastname: Hurr
authors.forename: Grant
authors.initials: G
authors.lastname: Hotter
authors.forename: Geoffrey W
authors.initials: GW
authors.lastname: de Lisle
mesh_terms: D000818:Animals; D001500:BCG Vaccine / Q000276:immunology*; D002417:Cattle; D006168:Guinea Pigs; D016296:Mutagenesis; D009163:Mycobacterium bovis / Q000235:genetics / Q000276:immunology; D011995:Recombination, Genetic; D014611:Vaccination; D014613:Vaccines, Attenuated / Q000276:immunology
chemical_list: D001500:BCG Vaccine; D014613:Vaccines, Attenuated