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Functional and genetic characterization of the tap efflux pump in Mycobacterium bovis BCG.

ABSTRACT: Efflux pumps extrude a wide variety of chemically unrelated compounds conferring multidrug resistance and participating in numerous physiological processes. Mycobacterium tuberculosis possesses many efflux pumps, and their roles in drug resistance and physiology are actively investigated. In this work we found that tap mutant cells showed changes in morphology and a progressive loss of viability upon subcultivation in liquid medium. Transcriptome analysis in Mycobacterium bovis BCG revealed that disruption of the Rv1258c gene, encoding the Tap efflux pump, led to an extensive change in gene expression patterns during stationary phase, with no changes during exponential growth. In stationary phase, Tap inactivation triggered a general stress response and led to a general repression of genes involved in cell wall biosynthesis, in particular the formation of the peptidoglycan; this suggested the accumulation of an unknown Tap substrate that reaches toxic concentrations during stationary phase. We also found that both disruption and overexpression of tap altered susceptibility to many clinically approved antibiotics in M. bovis BCG. Acriflavine and tetracycline accumulation assays and carbonyl cyanide m-chlorophenylhydrazone (CCCP) potentiation experiments demonstrated that this phenotype was due to an active efflux mechanism. These findings emphasize the important role of the Tap efflux pump in bacterial physiology and intrinsic drug resistance.

SUBMITTER: Ramon-Garcia S

PROVIDER: S-EPMC3318366 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Functional and genetic characterization of the tap efflux pump in Mycobacterium bovis BCG.

Ramón-García Santiago S Mick Virginie V Dainese Elisa E Martín Carlos C Thompson Charles J CJ De Rossi Edda E Manganelli Riccardo R Aínsa José A JA

Antimicrobial agents and chemotherapy 20120109 4

Efflux pumps extrude a wide variety of chemically unrelated compounds conferring multidrug resistance and participating in numerous physiological processes. Mycobacterium tuberculosis possesses many efflux pumps, and their roles in drug resistance and physiology are actively investigated. In this work we found that tap mutant cells showed changes in morphology and a progressive loss of viability upon subcultivation in liquid medium. Transcriptome analysis in Mycobacterium bovis BCG revealed that ...[more]

PMID: 22232275

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Project description:The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.

Dataset Information

Functional and genetic characterization of the tap efflux pump in Mycobacterium bovis BCG.

Publications

Functional and genetic characterization of the tap efflux pump in Mycobacterium bovis BCG.

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