Genetics of Mycobacterium tuberculosis adaptation to the macrophage phagosome

描述（由申请人提供）：结核分枝杆菌（Mtb）是一种细胞内病原体，其成功的关键是能够在巨噬细胞吞噬体中存活。对于许多微生物，由于转移到溶酶体或暴露于氧化或亚硝化应激，巨噬细胞的吞噬作用杀死。结核分枝杆菌通过阻止吞噬体成熟并驻留在与早期内体相似的相对安全的空泡中来避免这些杀伤机制。本申请的目的是使用功能基因组技术探索Mtb适应巨噬细胞吞噬体的机制，包括：巨噬细胞内生长的Mtb的转录谱分析和用于遗传筛选的新型转座子诱变方法，以发现在适应吞噬体方面有缺陷的突变体。本申请的具体目的是：1）开发在体外和体内响应特定环境线索的荧光Mtb报告菌株。为此，将通过基于微阵列的细胞内和培养物中生长的结核分枝杆菌的转录谱来鉴定由吞噬体刺激物调控的基因。来自受体外和体内特异性刺激调节的基因的启动子将用于在Mtb中表达绿色荧光蛋白。2)使用基于流式细胞术的遗传筛选鉴定在其对吞噬体环境的响应中改变的Mtb突变体。在该筛选中，Mtb报告菌株将被转座子诱变并暴露于感兴趣的刺激。与野生型菌株相比具有更少荧光的突变体将通过基于流式细胞术的分选富集。这些功能丧失的突变体可能在与感知或调节对刺激的反应相关的基因中具有损伤。这项工作将确定Mtb用于适应巨噬细胞吞噬体的基因。其中一些基因将成为新药或疫苗策略的靶点。此外，细胞内结核分枝杆菌基因表达的转录谱将是不同研究领域的微生物学家的宝贵资源。公共卫生相关性：结核分枝杆菌是人类结核病的病原体，是一种需要深入研究的微生物。在世界范围内，它是传染病导致死亡的主要原因，没有针对成人的有效疫苗，并且由于对长期抗生素治疗的低依从性，目前的药物治疗正在导致多药耐药菌株的进化。结核病是一种高度传染性的空气传播疾病，鉴于全球旅行相对容易，世界任何地方的结核病高发病率都对所有美国人的健康构成严重威胁。如果我们要发现和实施新的疗法或疫苗，就必须更全面地了解结核病生物学。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb) is an intracellular pathogen and a key to its success is the ability to survive in the macrophage phagosome. For many microbes, phagocytosis by a macrophage kills, due to transfer to the lysosome or exposure to oxidative or nitrosative stresses. Mtb avoids these killing mechanisms by arresting phagosome maturation and residing in a relatively safe vacuole that bears similarities to the early endosome. The goal of this application is to explore the mechanisms of Mtb adaptation to the macrophage phagosome using functional genomic techniques including: transcriptional profiling of Mtb growing inside macrophages and novel transposon mutagenesis approaches for genetic screens to discover mutants defective in adaptation to the phagosome. The specific aims of this application are: 1) the development of fluorescent Mtb reporter strains that respond to specific environmental cues in vitro and in vivo. For this aim, genes regulated by phagosomal stimuli will be identified by microarray-based transcriptional profiling of Mtb growing intracellularly and in culture. Promoters from genes regulated by a specific stimulus both in vitro and in vivo will be used to express green fluorescent protein in Mtb. 2) Identification of Mtb mutants that are altered in their response to the phagosomal environment using a flow cytometry-based genetic screen. In this screen, the Mtb reporter strains will be transposon mutagenized and exposed to a stimulus of interest. Mutants with less fluorescence as compared to the wild type strain will be enriched by flow cytometry-based sorting. These loss-of-function mutants may have lesions in genes associated with sensing or modulating a response to the stimulus. This work will identify genes that Mtb uses to adapt to the macrophage phagosome. Some of these genes will represent targets for novel drugs or vaccine strategies. Additionally, the transcriptional profiles of intracellular Mtb gene expression will be a valuable resource for microbiologists in diverse fields of study. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is a microbe that requires intensive study. Worldwide, it is a leading cause of death by an infectious disease, there is no effective vaccine for adults, and current drug therapies are leading to the evolution of multidrug-resistant strains because of low compliance to prolonged antibiotic treatments. Tuberculosis is a highly infectious, airborne disease and given the relative ease of global travel, high rates of tuberculosis anywhere in the world represents a serious threat to the health of all Americans. A more complete understanding of tuberculosis biology is essential if we are to discover and implement novel therapies or vaccines.