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6个月的多- 可靠地治愈患有活动性疾病的患者所需的药物治疗。长期治疗既昂贵， 导致粘附性差。该项目旨在了解导致长期结核病的细菌因素 治疗、结核病治疗失败和复发。结核分枝杆菌（Mtb），结核病的病原体， 进化出了适应环境的精妙能力药物治疗可导致 长期存在的“表型耐药”（即耐药）亚群，以及非典型耐药亚群。 最终可能复发的极低水平耐药突变株（即亚折点MIC菌株升高） 尽管几个月的药物治疗看似有效。我们最重要的假设是 治疗结核病所需的药物和即使采用适当治疗也可能发生的结核病不良结局之间存在密切联系 表型耐药和低水平的遗传耐药机制，可以存在于结核分枝杆菌 治疗前或治疗期间出现的亚群。这个项目将应用我们现有的新工具 开发，包括单细胞倍增评估活阵列的分枝杆菌（ODELAM），机器人 Transwell耐受和抗性（TTR）系统，一个完整的转录因子诱导（TFI）文库， 菌株，和组成型耐受glpK突变Mtb菌株，以研究这些机制的基础 细菌状态，以及不同Mtb中耐受性和低水平抗性的异质性表达 亚种群。从定义明确的实验室菌株扩展到各种特征明确的 与治愈或复发相关的临床Mtb菌株，我们将对这些菌株的机制进行了解。 细菌表型和它们在治疗结果中的作用的特征仍然很差。这些发现将导致 开发靶向结核病治疗的重要机会， 缩短治疗时间。这项工作将在三个相关目标下进行：目标1。定义之间的链接 结核分枝杆菌异质性和表型耐药性目标2.确定Mtb相位变化对 群体异质性、药物耐受性和新出现的耐药性。目标3：评估基因的作用， 网络和具有确定的治疗相关表型的临床分离株的群体异质性。

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.