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

项目摘要 结核病是由结核分枝杆菌感染引起的。结核病需要长时间的多种药物 而且仍然很难治疗，因为在宿主中结核杆菌之间存在相当大的耐药性。至 合理设计治疗结核病的药物方案，我们需要了解结核分枝杆菌是如何产生和维持 产生具有不同药物敏感性的个体的种群结构。分枝杆菌呈现细胞间传播 细胞生理的基本特征的异质性，源于确定性的、不对称的生长 和分工模式。这种独特的生长模式在细胞大小、生长速度和分区方面产生了变化 蜂窝组件。分枝杆菌细胞大小提供了对细胞生理学的关键洞察，因为细胞大小 与抗生素敏感性密切相关。结核分枝杆菌在不同环境下细胞大小分布的变化 在宿主体内遇到的压力源。尽管应激在致病菌的相互作用中起着关键作用 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、非结核分枝杆菌中进行了研究。匮乏 了解特定环境控制结核分枝杆菌细胞大小和细胞数量所涉及的细节 结构是一个关键的实验鸿沟，必须弥合才能进入设计结核病治疗的新阶段 以耐药亚群为目标。为了弥补这一差距，我们试图了解结核分枝杆菌细胞如何生长和 复制过程在宿主组织中遇到的各种环境条件中被介导，以及如何 这些特性决定了抗生素的敏感性。系统的、量化的方法是关键 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出更有效的 结核病治疗方案。在这个项目中，我们将研究Mtb使信元大小控制适应不同的过程 在宿主感染过程中遇到的生长环境，并详细定量地描述了不同的 耐药结核分枝杆菌亚群。我们将使用活细胞显微镜、荧光标记和 图像分析。我们将把这些定量分析集成到数学模型中，以严格测试细胞 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 寄主在不同元素胁迫条件下的大小和细胞周期进程与药物敏感性的关系 环境。为了将结核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 将重点放在治愈或复发患者的临床分离株上。我们预计这些模式将形成 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.