Drug tolerance, bacterial heterogeneity and adverse TB treatment outcomes

摘要--项目4 也许改善结核病(TB)治疗结果的最大障碍是六个月的多- 可靠地治愈活动性疾病患者所需的药物疗法。长期的治疗既昂贵又 导致依从性差。该项目旨在了解导致长时间结核病的细菌因素。 治疗、结核病治疗失败和复发。结核分枝杆菌(Mtb)是引起结核病的病原体。 具有极强的适应环境的能力。药物治疗可以导致 长期存在的“表型耐药”(即耐药)亚群，以及非典型的 极低水平的耐药突变株(即亚断点升高的MIC菌株)，最终可能复发 尽管接受了数月看似有效的药物治疗。我们压倒一切的假设是， 治疗结核病所需的费用和即使在适当治疗的情况下也可能出现的不良结核病结果是密切相关的 结核分枝杆菌可能存在的表型耐药和低水平遗传耐药机制 治疗前的亚群，或治疗期间出现的亚群。该项目将应用我们拥有的新工具 开发了一种机器人，包括分枝杆菌活阵列的单细胞倍增评估(ODELAM) Transwell耐受性和抗性系统--一个完整的转录因子诱导因子文库 以及耐受的glpK突变Mtb菌株，以研究其背后的机制 细菌状态以及耐药和低水平耐药在不同结核分枝杆菌中的异质性表达 亚种群。从定义明确的实验室菌株扩展到具有良好特性的多样化集合 临床结核分枝杆菌菌株与治愈或复发有关，我们将从机理上对其进行理解。 细菌表型及其在治疗结果中的作用仍然缺乏特征性。这些发现将导致 为开发靶向结核病治疗提供重要机会，以提高治疗成功率 缩短治疗时间。这项工作将在三个相关目标下进行：目标1.界定 结核分枝杆菌的异质性和表型耐药。目的2.确定Mtb位相变化在 群体异质性、耐药和新发耐药。目的3.评估基因的作用(S)， 具有明确的治疗相关表型的临床分离株的网络和群体异质性。

ABSTRACT - PROJECT 4 Perhaps the greatest barrier to improving tuberculosis (TB) treatment outcomes is the six months of multi- drug therapy that is required to reliably cure a patient with active disease. Lengthy therapy is both costly and leads to poor adherence. This project aims to understand the bacterial factors responsible for lengthy TB treatment, TB treatment failure, and relapse. Mycobacterium tuberculosis (Mtb), the causative agent of TB has developed an exquisite ability to adapt to its environment. Drug treatment can lead to the development of “phenotypically drug-resistant” (i.e. drug tolerant) subpopulations that persist for long periods, as well as atypical very-low level drug resistant mutants (i.e. elevated sub-breakpoint MIC strains) that can eventually relapse despite months of seemingly effective drug treatment. Our overriding hypothesis is that the length of time required to treat TB and the adverse TB outcomes that can occur even with adequate therapy are strongly linked to phenotypic drug resistance and low-level genetic drug resistance mechanisms that can be present in Mtb subpopulation pre-treatment, or that arise during treatment. This project will apply novel tools we have developed, including One-cell Doubling Evaluation of Living Arrays of Mycobacterium (ODELAM), a robotic Transwell Tolerance and Resistance (TTR) system, a complete library of transcription factor inducible (TFI) strains, and constitutively tolerant glpK mutant Mtb strains to investigate the mechanisms that underlie these bacterial states, as well as the heterogeneous expression of tolerance and low level resistance in different Mtb sub-populations. Expanding from well-defined laboratory strains to a diverse collection of well characterized clinical Mtb strains associated with either cure or relapse, we will develop a mechanistic understanding of these still poorly characterized bacterial phenotypes and their role in treatment outcome. These discoveries will lead to important opportunities for developing targeted TB treatments that increase therapeutic success while shortening treatment times. This work will be conducted in three related aims: Aim 1. Define the links between heterogeneity and phenotypic drug tolerance in Mtb. Aim 2. Determine the role of Mtb phase variation on population heterogeneity, drug tolerance and emergent drug resistance. Aim 3. Assess role(s) of genes, networks, and population heterogeneity in clinical isolates with defined treatment-related phenotypes.