Adaptation, fitness and resistance in rifampicin-resistant M. tuberculosis

总结 传播仍然是全球耐药性结核病流行背后的驱动力。发生这种情况 尽管观察到获得耐药性对病原体具有适应性成本。健身费用 与rpoB突变相关，该突变赋予对利福平的耐药性。的生理基础 突变体rpoB诱导的适应性成本在很大程度上仍然未知，除了补偿性突变 被发现可以改善适应度成本，并与可传递性相关。当前 下一代RNA测序（RNA-seq）的进展使我们能够生成和比较 具有不同适应度水平的rpoB突变的转录组学概况。利用这项技术，我们的目标是阐明 不同的耐药rpoB突变如何改变RNA聚合酶的功能， 转录组，转录组如何随着增加补偿性rpoC突变而演变，以及转录组是否 这些事件的组合改变了适应性和分离株的倾向，不仅获得了额外的 耐药性，而且影响二线药物的药物敏感性。此外，为了阐明 适应性突变会影响它们各自的转录组，以确保它们也能在宿主环境中存活。 以确定宿主对突变的M的基因表达响应。结核病（MTB）。我们建议 通过以下三个目标来解决这些问题：1）确定如何组合不同的rpoB 具有或不具有补偿（rpoC）突变的突变影响MTB的转录组，2）确定 携带有或没有rpoC突变的不同适合性rpoB突变的MTB的体内转录组， 3)确定利福平耐药MTB的不良治疗结局是否与rpoB突变相关 影响二线抗结核药物的体内MIC。为了实现这些目标，我们将在体外选择rpoB 具有与结核病爆发相关的临床相关遗传背景的突变体， 易产生多药耐药性。将使用竞争适应性试验选择分离株 携带跨越体外生长适应性表型谱的rpoB突变。rpoC的突变将 将其工程化到选定的rpoB突变体中，并将使用RNA-seq来确定转录组谱。 将感染巨噬细胞，以确定压力如何改变细菌的转录组， 使用双重RNA-seq，这些突变体对巨噬细胞本身有影响。基因控制和补偿 与调节基因（如在初步数据中所见）一起的适应性将使用计算机模拟来识别。 最后，突变体将暴露于二线药物，以确定它们是否更快地获得额外的细胞因子。 耐药性，降低对二线药物的敏感性，从而导致治疗效果不佳， MDR菌株。了解适应性的致病性如何驱动抗性获得的进化， 利福平耐药菌株的传播，从而调整患者管理方案。

Summary Transmission remains the driving force behind the global drug resistant Tuberculosis (TB) epidemic. This occurs despite the observation that the acquisition of drug resistance has a fitness cost on the pathogen. Fitness costs have been associated with rpoB mutations which confer resistance to rifampicin. The physiological basis of the mutant rpoB induced fitness cost remains largely unknown with the exception that compensatory mutations have been found to ameliorate the fitness cost and have been associated with transmissibility. The current advancements in next generation RNA sequencing (RNA-seq) enables us to generate and compare the transcriptomic profiles of rpoB mutations with various levels of fitness. Using this technology, we aim to elucidate how different resistance-conferring rpoB mutations alter the function of RNA polymerase and thereby the transcriptome, how a transcriptome evolves with the addition of a compensatory rpoC mutation and whether the combination of these events alters fitness and the propensity of the isolate to not only acquire additional resistance but also to influence drug susceptibility to second-line drugs. Furthermore to elucidate how different fitness mutations influence their respective transcriptomes to ensure survive within the host environment as well as to determine the host gene expression response to the mutated M. tuberculosis (MTB). We propose to address these questions using the following three aims: 1) Determine how the combination of different rpoB mutations with or without a compensatory (rpoC) mutation influences the transcriptome of MTB, 2) Determine the in vivo transcriptome of MTB harbouring different fitness rpoB mutations with or without a rpoC mutation and 3) Determine whether poor treatment outcome of rifampicin-resistant MTB is related to rpoB mutations influencing the MIC of second-line anti-TB drugs in vivo. To achieve these aims we will select rpoB in vitro mutants with a clinically relevant genetic background which has been associated with TB outbreaks and a predisposition to develop multidrug resistance. Competition fitness assays will be used to select isolates harbouring rpoB mutations spanning the spectrum of in vitro growth fitness phenotypes. Mutations in rpoC will be engineered into the selected rpoB mutants, and RNA-seq will be used to determine the transcriptomic profiles. Macrophages will be infected to determine how stress changes the transcriptome of the bacteria and whether these mutants have an effect on the macrophage itself using dual RNA-seq. Genes governing and compensating for fitness together with regulatory genes (as seen in preliminary data) will be identified using a in silico modelling. Lastly, mutants will be exposed to second-line drug to determine whether they more rapidly acquire additional resistance and decreases susceptibility to second-line drugs thereby resulting in poor treatment outcome for MDR strains. Understanding how pathogenicity of fitness, drives the evolution of resistance acquisition and transmission of rifampicin resistance strains, thereby adapting patient management programs.