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.

Transcriptional Profiling of Mycobacterium tuberculosis Exposed toIn VitroLysosomal Stress

“…To support the relevance of our transcriptomics approach to study M. tuberculosis responses to the lysosomal environment, we selected one gene candidate, Rv1258c, which was found highly expressed under iLivE conditions and functionally relevant as a membrane Tap-like efflux pump (84)(85)(86). A previous study reported that M. tuberculosis transposon mutants of Rv1258c displayed reduced viability at 96 h postmacrophage infection (71).…”

“…Our results support a role for Rv1258c in M. tuberculosis survival during exposure to the lysosomal content. Previous literature proposed that the Tap efflux pump activity helps mycobacteria cope with the hostile environment by pumping out hostderived antimicrobial molecules (84)(85)(86). To test whether the Tap efflux pump activity is involved in the M. tuberculosis response to SF killing, the Ox-Rv1258c strain was incubated with SF in the presence of the proton motive force inhibitor CCCP.…”

WhiB7, an Fe-S-dependent Transcription Factor That Activates Species-specific Repertoires of Drug Resistance Determinants in Actinobacteria

“…Indeed, whiB7 is highly up-regulated in macrophages (11) and in the lungs of infected animals (12). WhiB7 activates expression of its own regulon that includes resistance genes involved in antibiotic efflux (tap, Rv1258c) (13), ribosome modification (erm, Rv1988) (14,15), modulation of the host immune responses (eis, Rv2416c, and erm) (16,17), and mycobacterial survival within macrophages (eis) (7).…”

Analysis of Mutations in Streptomycin-Resistant Strains Reveals a Simple and Reliable Genetic Marker for Identification of the Mycobacterium tuberculosis Beijing Genotype

“…In addition, the nucleotide sequence of the Rv1258c gene has been investigated as a potential STR-resistance determinant, since this gene encodes the drug efflux pump Tap in M. tuberculosis, which transports STR and other antibiotics (9)(10)(11). A nucleotide insertion in the coding sequence of the Rv1258c gene has been identified, which does not seem to be associated with resistance to this drug.…”