AbstractEfflux pumps play an integral role in the bacterial physiology and drug tolerance of M. tuberculosis. Their function allows the transport of important macromolecules such as cell wall components, siderophores, and virulence factors, outside of the bacterial cell. In addition, antimycobacterial drugs used in TB therapy are also substrates of M. tuberculosis efflux pumps, which can decrease the antibiotics’ effectiveness. There are many efflux pumps in M. tuberculosis where their role in the physiology in M. tuberculosis, or their contribution to drug efflux, is not well characterised. Therefore, this project aimed to select efflux pumps in M. tuberculosis which are thought to play a role in the efflux of drug or in an unknown function in M. tuberculosis.
Out of the sixty-four efflux pumps in M. tuberculosis, the efflux pumps MmpL7, JefA, and EfpA were identified as efflux pumps which had potential for further characterisation in this study. Loss of function of MmpL7 and JefA was investigated using M. tuberculosis transposon mutants. The disruption of mmpL7 was not found to increase the susceptibility of M. tuberculosis to isoniazid-exposure. However, when JefA function was disrupted, this led to a decreased susceptibility to ethambutol exposure which was not due to the induction of other efflux pumps. In addition, the transcriptomic response of an M. tuberculosis jefA mutant resulted in the induction of many virulence genes and genes which are involved in the acquisition of iron, potentially implying that JefA fulfils a function during macrophage infection.
The knockdown in expression of the essential efflux pump, EfpA, was achieved using CRISPR interference in M. tuberculosis. The growth dynamics of diminished efpA expression were assessed to reveal a reduction in viability, turbidity, and bacterial count in culture accompanied by an elongated cell morphology. The knockdown in efpA expression prolonged the early bactericidal effect of isoniazid-exposure in M. tuberculosis, which resulted in a substantial loss of bacterial viability in M. tuberculosis culture and a lack of detection of any viable bacteria after 45 days. It is also possible that the repression of efpA may increase the efficacy of other drugs to treat M. tuberculosis, including those used in multi-drug resistant therapies.
In this study, the impact that efflux pumps have on the susceptibility of M. tuberculosis to drug treatment and their functional role in M. tuberculosis was explored with a focused approach on three specific efflux pumps.
|Date of Award
|Simon Waddell (Supervisor) & Dr Joanna Bacon (Supervisor)