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

耐利福平结核分枝杆菌的适应、适应度和耐药性

• 批准号: 10407527
• 负责人: Elizabeth Maria Streicher
• 金额: $ 13.69万
• 依托单位: STELLENBOSCH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-20 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10407527
• 关键词: Address; Affect; Amino Acids; Bacillus; Bacteria; Biological Assay; Compensation; Complex; DNA-Directed RNA Polymerase; Data; Disease Outbreaks; Drug resistance in tuberculosis; Drug resistant Mycobacteria Tuberculosis; Engineering; Ensure; Environment; Epidemic; Event; Evolution; Exposure to; Gene Expression; Gene Expression Profile; Generations; Genes; Genetic; Genetic Transcription; Goals; Growth; In Vitro; Infection; Infectious Diseases Research; Knowledge; Macrophage; Maps; Modeling; Multi-Drug Resistance; Mutate; Mutation; Mycobacterium tuberculosis; Pathogenicity; Patients; Pharmaceutical Preparations; Phenotype; Physiological; Physiology; Point Mutation; Population; Predisposition; Regulator Genes; Reporting; Research; Resistance; Resolution; Rifampicin resistance; Rifampin; Sigma Factor; Signal Transduction; Stress; Technology; Treatment outcome; Tuberculosis; Universities; acquired drug resistance; bacterial fitness; biological adaptation to stress; clinically relevant; cost; driving force; fitness; in silico; in vivo; insight; mutant; next generation; pathogen; progenitor; programs; promoter; resistant strain; response; stressor; transcriptome; transcriptome sequencing; transcriptomics; transmission process; tuberculosis drugs

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.

摘要 传播仍然是全球耐药结核病(TB)流行的驱动力。这种情况会发生 尽管观察到，获得抗药性对病原体的适应成本是有的。健身费用 与rpoB突变有关，这种突变使人对利福平产生耐药性。精神障碍的生理基础 突变的rpoB诱导的适应成本在很大程度上仍然未知，除了代偿性突变 已被发现可以改善健身成本，并与传播性有关。海流 下一代RNA测序(rna-seq)的进步使我们能够生成和比较 不同适合度的rpoB基因突变的转录图谱。利用这项技术，我们的目标是阐明 不同的抗药性rpoB突变如何改变RNA聚合酶的功能，从而改变 转录组，转录组是如何随着补偿的rpoC突变的增加而进化的，以及 这些事件的组合改变了适应性和隔离倾向，不仅获得了额外的 耐药性不仅影响药物对二线药物的敏感性。进一步阐明不同之处 适合性突变影响它们各自的转录本，以确保在宿主环境中也能生存 以确定宿主对结核分枝杆菌突变株(MTB)的基因表达反应。我们建议 通过以下三个目标解决这些问题：1)确定不同的rpoB如何组合 带有或不带有代偿性(RpoC)突变的突变影响结核分枝杆菌的转录组，2)决定 携带不同适合度rpoB突变的结核分枝杆菌体内转录组 3)确定耐利福平的结核分枝杆菌治疗效果差是否与rpoB突变有关 对二线抗结核药物体内MIC的影响。为了达到这些目的，我们将在体外选择rpoB 具有临床相关遗传背景的突变与结核病暴发和 容易产生多药耐药。将使用竞争适应度分析来选择分离物 携带rpoB突变，跨越体外生长适应表型的范围。RpoC基因突变将 被工程到选定的rpoB突变体中，RNA-seq将用于确定转录图谱。 巨噬细胞将被感染，以确定应激如何改变细菌的转录组以及是否 这些突变体使用双重RNA-seq对巨噬细胞本身产生影响。基因控制和补偿 对于适合性和调节基因(如在初步数据中所见)，将使用电子计算机建模来识别。 最后，突变者将接触二线药物，以确定他们是否更快地获得了额外的 耐药并降低对二线药物的敏感性，从而导致治疗结果不佳 多药耐药菌株。了解适应性的致病性如何驱动抗性获得和进化 传播利福平耐药菌株，从而适应患者管理计划。