Evolution of XDR-TB and the associated proteome

[摘要] ENGLISH SUMMARY: Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis, carries a substantial health burden worldwide and in South Africa. Efforts towards prevention and effective treatment of this disease is essential to meet the END TB goals. The disease is exacerbated by HIV co-infection and drug resistance. Multidrug-resistant (MDR) TB (resistance to at least rifampicin and isoniazid) places enormous resource constraints on TB control programs with poorer treatment outcomes. More recently, extensively drug-resistant (XDR) TB (MDR-TB with additional resistance to a fluoroquinolone and an injectable) has become a global concern. XDR-TB develops through the acquisition of mutations in the gyrA and rrs genes. The aim of this work was to investigate aspects of the evolution and physiology of XDR-TB, in particular acquisition of mutations and their impact on protein abundance. Assessment of the impact of nucleoside reverse transcriptase inhibitors on the mutation rate of Mycobacterium smegmatis indicated no significant effect, suggesting that antiretroviral treatment does not contribute to the overlap of HIV infection and drug-resistant TB. Analysis of spontaneous ofloxacin-resistant mutants indicated that a Beijing clinical isolate acquired high level drug resistance mutations more readily than H37Rv, providing a possible reason for the association of Beijing with drug resistance. Analysis of spontaneous moxifloxacin resistant mutants showed that gyrA mutations at codons 88 and 94 were associated with resistance (defined as minimum inhibitory concentration (MIC) of ≥ 2 μg/ml). Despite the presence of gyrA mutations, moxifloxacin significantly impeded bacterial growth, supporting its continued use for the treatment of ofloxacin-resistant M. tuberculosis.In an attempt to determine whether the fluoroquinolone MIC could be increased we selected spontaneous mutants from a fluoroquinolone resistant clone on higher concentrations of the fluoroquinolone. Sequencing of gyrA and gyrB identified additional mutations suggesting that double mutations are responsible for increasing the MIC. We could not find any involvement of efflux pump activity in modulating the MIC.To determine the influence of the gyrA Asp94Gly and rrs A1401G mutation on the physiology of the pathogen we assessed their effect on protein abundance. A strong signature of differentially abundant proteins common to both clones, expressed from the ESX-5 cluster, suggested either the presence of a common unknown genetic variant (in unmapped genomic regions) or common physiological changes related to drug resistance. The gyrA mutant uniquely demonstrated decreased abundance in transport proteins, suggesting decreased cell wall permeability and increased drug tolerance. Changed abundance of proteins involved in transcription/translation was also observed, suggesting impaired functionality of the mutated gyrase. The rrs mutant displayed lowered abundance of stress proteins belonging to the DosR/DevR regulon, potentially impacting the mutant's response to dormancy. Ofloxacin treatment of the gyrA mutant resulted in increased abundance of proteins involved in iron acquisition and differential abundance of proteins indicating decreased cell division and growth. We hypothesize that increased abundance of iron acquisition proteins relates to chelation of iron by ofloxacin. Amikacin treatment of the rrs mutant decreased ribosomal protein abundance and increased proteins involved in tRNA-related processes. We hypothesise that this relates to increased degradation of ribosomes and a mechanism to compensate for reduced translational fidelity.This study improved our understanding of the physiological factors contributing to the emergence of XDR-TB. Furthermore, our results suggest that the physiology of XDR M. tuberculosis differs from susceptible strains. These changes in physiology could inform further research on drug targets and optimal treatment regimens.

[发布机构] Stellenbosch University