A mechanistic understanding of tuberculosis progression through bacterial reporter strains

项目总结/摘要 由于结核分枝杆菌（Mtb）在人群中的广泛分布，它仍然是一种常见的结核病。 对艾滋病毒感染者的健康构成严重威胁。结核病疫苗开发计划受到我们 对疾病进展的免疫机制缺乏了解。我们的建议是， 应用是利用Mtb报告菌株提供微生物适合度的功能读数， 复制，使我们能够识别和表征那些限制细菌生长的吞噬细胞（控制器） 与那些促进细菌生长（允许）的吞噬细胞相比， 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新的途径，以确定吞噬细胞 最好地限制或促进细菌复制的细胞群，并且定义这些细胞群将提供 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Mtb报告菌株和攻击模型。 我们将（a）利用Mt B报告菌株来鉴定容许和控制吞噬细胞群体;（B）优化 - 用于募集至Mtb感染的小鼠肺的细胞的离体感染方案，用于NHP肉芽肿，和 人气道和肉芽肿吞噬细胞;和（c）对不同的吞噬细胞进行RNASeq分析 用于表型分析的Mtb报告菌株定义的群体，并产生一组候选细胞 用于NHP和人离体激发研究的表面标志物。 目标2. NHP肺肉芽肿中吞噬细胞亚群的功能特征我们将执行 在分离出NHP噬菌体后，用报告菌株感染的NHP吞噬细胞群的平行分析 感染猴肺的单个肉芽肿。我们提出了可比较的表型表征， 有偏和无偏的方法，以功能性地鉴定NHP中的容许和控制吞噬细胞群体 结核病在体内感染和离体攻击实验中的应用。 具体目标3：通过Mtb体外激发测定人吞噬细胞表型 报道菌株。此外，还与亨利·姆万敦巴博士（马拉维）和阿拉斯代尔·莱斯利博士（南非）合作， 非洲），我们将确定人气道和人结核肉芽肿吞噬细胞的功能表型 通过用荧光Mtb报告菌株离体探测细胞来检测亚群。

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.