A mechanistic understanding of tuberculosis progression through bacterial reporter strains

项目摘要/摘要 由于其广泛的人类渗透性，结核分枝杆菌(Mtb)仍然是一种 对艾滋病毒携带者的健康构成严重威胁。结核病疫苗开发计划受到我们的阻碍 对疾病进展的免疫机制缺乏了解。我们在这篇文章中提出的 应用是利用结核分枝杆菌报告菌株提供微生物适合性的功能读数和 复制以使我们能够识别和表征那些限制细菌生长的吞噬细胞(控制者) 与那些吞噬细胞相比，促进细菌生长(允许)以了解疾病的基础 人类结核病的进展。 我们的假设是结核分枝杆菌报告菌株代表了一种鉴定吞噬细胞的新途径。 最好地限制或促进细菌复制的种群，以及定义这些细胞种群将提供 理解结核病免疫控制的合理框架。 目的1：在小鼠系统中开发和验证结核分枝杆菌报告菌株和挑战模型。 我们将(A)利用结核分枝杆菌报告菌株来识别允许和控制吞噬细胞的种群；(B)优化 一种体外感染方案，用于将细胞招募到结核杆菌感染的小鼠肺，用于NHP肉芽肿，以及 人类呼吸道和肉芽肿吞噬细胞；以及(C)在不同的吞噬细胞上执行RNAseq分析 由Mtb报告菌株定义的群体用于表型分析并产生一组候选细胞 NHP和人类体外挑战研究的表面标志物。 目的2.NHP肺肉芽肿吞噬细胞亚群的功能特征。我们将表演 报告菌株分离后NHP吞噬细胞群体的平行分析 感染猴肺的单个肉芽肿。我们提出了类似的表型特征，使用 NHP中功能识别允许和控制吞噬细胞群的有偏和无偏方法 结核病在体内感染和体外挑战实验。 特异性目标3：结核分枝杆菌体外攻击测定人吞噬细胞表型 记者菌株。此外，与Henry Mwandumba博士(马拉维)和Alasdair Leslie博士(南部)合作 我们将确定人类呼吸道和人类结核肉芽肿吞噬细胞的功能表型 用Mtb荧光报告菌株在体外探测细胞亚群。

Project Summary / Abstract Due to its extensive penetrance of the human population, Mycobacterium tuberculosis (Mtb) remains a serious health risk to those individuals living with HIV. TB vaccine development programs are hampered by our poor understanding of the immune mechanisms underpinning disease progression. What we propose in this application is the utilization of Mtb reporter strains to provide a functional readout of microbial fitness and replication to enable us to identify and characterize those phagocytes that restrict bacterial growth (controllers) versus those phagocytes the promote bacterial growth (permissive) to understand the basis of disease progression in human tuberculosis. Our hypothesis is that Mtb reporter strains represent a novel route to the identification of the phagocyte populations that best restrict or promote bacterial replication, and that defining these cell populations will provide a rational framework for understanding immune control of tuberculosis. Aim 1: Development and validation of Mtb reporter strains and challenge models in the murine system. We will (a) exploit Mtb reporter strains to identify permissive and controller phagocyte populations; (b) optimize an ex vivo infection protocol for cells recruited to Mtb-infected mouse lung, to be used on NHP granulomas, and human airway and granuloma phagocytes; and (c) perform RNASeq profiling on the different phagocyte populations defined by the Mtb reporter strains for phenotypic analysis and to generate a panel of candidate cell surface markers for NHP and human ex vivo challenge studies. Aim 2. Functional characterization of phagocyte subsets in NHP pulmonary granulomas. We will perform parallel analysis of NHP phagocyte populations infected with reporter bacterial strains following the isolation of individual granulomas from infected monkey lungs. We propose comparable phenotypic characterization using biased and unbiased methods to functionally identify permissive and controller phagocyte populations in NHP tuberculosis in in vivo infections and ex vivo challenge experiments. Specific Aim 3: Determination of human phagocyte phenotypes through ex vivo challenge with Mtb reporter strains. In addition, in collaboration with Drs. Henry Mwandumba (Malawi) and Alasdair Leslie (South Africa) we will determine the functional phenotypes of human airway and human TB granuloma phagocyte subsets by probing the cells ex vivo with the fluorescent Mtb reporter strains.