Identification of mechanisms regulating the intra cellular concentration of rifampicin in Mycobacterium Tuberculosis

[摘要] ENGLISH ABSTRACT: Rifampicin resistance in clinical isolates of Mycobacterium tuberculosis develops through selection of bacterial variants harbouring mutations in the rpoB gene. These mutations infer a fitness-cost in the absence of antibiotic pressure, however, fitness-levels of rifampicin-resistant strains can be restored by compensatory mutations in rpoA and rpoC. This study was the first to investigate the epidemiological relevance of these compensatory mutations in clinical M. tuberculosis isolates collected in South Africa. Through targeted DNA sequencing, we demonstrated a strong association between rpoC mutations and transmission, and the rpoB S531L mutation. Our study emphasises the epidemiological relevance of compensatory evolution in response to the emergence of rifampicin resistance, and illustrates how compensatory mutations may be selected as a function of epistatic interactions.Recently a hypothesis has been developed which suggests that the activation of efflux systems through exposure to rifampicin may explain the observed spectrum of rifampicin resistance phenotypes. To elucidate whether rifampicin dependent activation of efflux systems also increases energy production, the RNA expression profiles of candidate energy metabolism genes were investigated. This study demonstrated that rifampicin exposure induced an overall increase in the expression of energy metabolism genes. Our findings suggest that the response to rifampicin is not universal and may depend on other genomic mutations. From these results we conclude that the stress response induced by exposure to rifampicin increases the energy production which fuels efflux activity thereby enabling the cell to extrude rifampicin in an energy dependent manner. This also provides a platform to explain the mechanism by which the newly developed drug, TMC207, increases the rate of culture conversion when used in combination with second-line anti-TB drugs. We propose that inhibition of ATP synthesis by TMC207 will deprive the efflux pumps and transporter genes of energy, which will result in the accumulation of second-line anti-TB drugs within the bacilli, leading to more efficient binding of the second-line drugs to their targets and ultimately to cell death.To identify the genetic basis governing the level of rifampicin resistance, we sequenced the genomes of MDR clinical isolates and in vitro generated rifampicin resistant mutants. Only minor genetic changes in addition to the rpoB mutation were identified in the genomes of in vitro rifampicin resistant mutants which displayed varying levels of resistance. This suggests that these mutants may either use alternative regulatory mechanisms or have acquired SNPs outside the genetic regions investigated in this study to modulate rifampicin resistance levels. In contrast, the genomes of clinical MDR isolates from the Low Copy Clade showed considerable variability in genes involved in cell wall, cellular processes and lipid metabolism, while the genomes from the Beijing Clade displayed variability in genes known to confer drug resistance and compensatory mechanisms. These results suggest that the structure and processes of the cell wall, as well as lipid metabolism plays a critical role in determining the intra-cellular concentration of rifampicin. Finally, this study illustrated the complexity in the physiology of M. tuberculosis resistant to rifampicin, whereby multiple mechanisms are employed by the bacteria to modulate its resistance levels.

[发布机构] Stellenbosch University