Single-cell factors of tuberculosis drug tolerance during adaptation to environmental stressors

项目摘要 结核病（TB）是由结核分枝杆菌（Mtb）感染引起的。结核病需要长期的多药治疗 由于宿主中的Mtb存在相当大的药物耐受性，因此仍然难以治疗。到 合理设计结核病的药物治疗方案，我们需要了解Mtb如何创造和维持一个 人口结构，产生不同的药物敏感性的个人。分枝杆菌表现出细胞间 它们细胞生理学基本特征的异质性，由确定性、不对称生长引起， 和分裂模式。这种独特的生长模式造成细胞大小、生长速率和细胞内蛋白质分配的变化。 细胞成分。分枝杆菌细胞大小提供了对细胞生理学的重要见解，因为细胞大小是 与抗生素敏感性密切相关。MTB在不同的环境中改变其细胞大小分布 在宿主中遇到的应激源。尽管在致病性微生物与病原微生物的相互作用中， 结核病与宿主，单细胞生长，细胞周期进程，细胞大小控制和药物敏感性的研究 已经在营养充足的生长条件下在非致病性非Mtb分枝杆菌中进行。缺乏 了解环境特异性控制结核分枝杆菌细胞大小和细胞群的细节 结构是一个关键的实验空白，必须弥合这一空白，才能进入设计结核病疗法的新阶段 针对耐药亚群的药物。为了弥合这一差距，我们试图了解结核分枝杆菌细胞的生长和 复制过程是在宿主组织中遇到的各种环境条件下介导的， 这些特征决定了抗生素的敏感性。系统的、定量的方法是关键， 了解分枝杆菌如何耐受抗生素压力，将使我们能够合理地设计更有效的 结核病治疗方案。在这个项目中，我们将研究结核分枝杆菌适应细胞大小控制的过程，以不同的 生长环境中遇到的主机感染，并定量表征详细的不同 耐药结核分枝杆菌亚群我们将使用活细胞显微镜，荧光标记物， 图像分析我们将把这些定量分析整合到数学模型中， 细胞生长和分裂的策略。我们的多学科方法将量化细胞之间的关系， 大小和细胞周期进展与药物敏感性在不同的元素应力条件下的主机 环境为了将结核分枝杆菌的生长特征和变异与人类的治疗结果联系起来，我们的实验 将集中于从治愈或复发的患者中分离出的临床菌株。我们预计这些模型将形成 创建针对新出现的耐药亚群的优化治疗方案的基础 使用现有抗生素的不同组合。

Project Summary Tuberculosis (TB) is caused by infection with Mycobacterium tuberculosis (Mtb). TB requires a lengthy multidrug treatment and remains difficult to treat because there is considerable drug tolerance among Mtb in the host. To rationally design drug regimens for tuberculosis, we need to understand how Mtb creates and maintains a population structure that generates individuals with diverse drug sensitivities. Mycobacteria exhibit cell-to-cell heterogeneity in fundamental features of their cell physiologies, arising from a deterministic, asymmetric growth and division pattern. This unique growth pattern creates variation in cell size, growth rate, and partitioning of cellular components. Mycobacterial cell size provides critical insight into cell physiology because cell size is tightly connected to antibiotic sensitivity. Mtb alter their cell size distributions under different environmental stressors that are encountered in the host. Despite the key role of stresses during the interactions of pathogenic Mtb and the host, studies of single-cell growth, cell cycle progression, cell size control, and drug susceptibility have been conducted in non-pathogenic, non-Mtb mycobacteria under nutrient-replete growth conditions. A lack of understanding of the details involved in environment-specific control of Mtb cell size and cell population structure is a critical experimental gap that must be bridged to enter into a new phase of designing TB therapies that target drug tolerant subpopulations. To bridge this gap, we seek to understand how Mtb cell growth and replication processes are mediated in various environmental conditions encountered in host tissues and how these characteristics determine antibiotic susceptibility. A systematic, quantitative approach is key to understanding how mycobacteria tolerate antibiotic stress and will allow us to rationally design more effective TB regimens. In this project, we will investigate the process by which Mtb adapt cell size control to different growth environments encountered during host infection and quantitatively characterize in detail the distinct subpopulations of drug-tolerant Mtb. We will use a combination of live-cell microscopy, fluorescent markers, and image analysis. We will integrate these quantitative analyses into mathematical models to rigorously test cellular strategies of cell growth and division. Our multidisciplinary approach will quantify the relationships between cell size and cell cycle progression with drug susceptibility in distinct elemental stress conditions of the host environment. To connect Mtb growth features and variation to treatment outcome in humans, our experiments will focus on clinical isolates from patients that were cured or relapsed. We anticipate that these models will form the basis from which to create optimized treatment regimens that target emerging drug-tolerant subpopulations using different combinations of existing antibiotics.