Mycobacteria Invasion and Persistence

主要评估者/PD_riraemct（oLarst，first，middle）：BFO_I_ EI_iC J.描述：说明申请的广泛、长期目标和具体目标，参考项目的健康相关性。简明扼要地描述研究设计和实现这些目标的方法-避免总结过去的成就和使用第一人称。本摘要旨在作为与申请分开时艾德工作的简洁准确描述，如果申请获得资助，则本描述将成为公开信息，因此，不包括专有/机密信息。请勿占用所提供的空间。分枝杆菌感染仍然是人类健康的主要问题。结核分枝杆菌感染了大约1/3的世界人口;麻风分枝杆菌（ML）是世界许多地区的地方性感染。M.海洋结核菌（Marinum，Mm）是鱼类的一种天然病原体，可引起青蛙的慢性疾病，具有结核病的许多特征。由于其在体外快速生长，适合正向遗传学，对实验室工作者的致病性最小，以及其在体外哺乳动物巨噬细胞中生长的能力，Mm可以成为发现参与分枝杆菌感染发病机制的基因的简单模型。我们已经开发了一种转座子诱变质粒用于Mm已被证明提供了一个很好的随机突变库。从该随机插入文库中，我们筛选了>1000个突变体在巨噬细胞中生长的能力，并表征了>25个不能正常生长的突变体。对于每个突变体，我们已经测序的转座子插入位点，并利用这些信息，我们已经发现了几个基因所需的细胞内生长的MM。我们建议专注于一个更详细的表征两个最有趣的突变体，以了解宿主细胞入侵的靶基因的作用，和胞内生长，分枝杆菌。在每种情况下，我们已经表明，Mtb的同源物的目标Mm基因将补充突变体中发现的表型缺陷。这允许快速和彻底的调查，这些Mtb基因的作用，在一个模型中，是能够的细胞内生长，并最终扩展到体内感染模型。目前的建议的目的是使用这些突变体，以发展一个更好地了解分枝杆菌进入和生存在巨噬细胞，巨噬细胞对分枝杆菌感染的反应，在疾病的发病机制中的关键事件。我们的具体目标是：1.表征Mm在巨噬细胞中宿主细胞侵袭和细胞内存活所需的mip基因座2。确定GDP-甘露糖合成操纵子如何调节巨噬细胞活化和细胞内存活3.对巨噬细胞细胞内生长所需的Mm基因进行遗传筛选。性能现场=

PrincipInavl estk3ator/PD_riraemct(oLarst, first, middte): BFO_'I_ EI_iC J. DESCRIPTION: State the application's broad, long-term objectives and specific aims, making reference to the health relatedness of the project. Describe concisely the research de-sign and methods for achieving these goals_ Avoid summaries of past accomplishments and the use of the first person. This abstract is meant tQ serve as a succinct and accurate description of the prep_ed work when separated from the application, If the applicatiori is funded, this description, as is, will become public information, Therefore, do not include proprietary/confidential information. DO NOT _CEED THE SPACE PROVIDED. Mycobacteria infections remain a major problem in human health. Mycobacterium tuberculosis infects approximately 1/3 of the world's population; Mycobacterium leprae (ML) is an endemic infection in many parts of the world. M. marinum (Mm) is a natural pathogen of fish and can cause chronic disease in frogs with many features of tuberculosis. Because of this, its rapid growth in vitro, its suitability for forward genetics, its minimal pathogenicity for laboratory" workers, and its ability to grow in mammalian macrophages in vitro, Mm can be a facile model to discover genes involved in pathogenesis of Mycobacteria infections. We have developed a transposon mutagenesis plasmid for use in Mm that has proven to provide an excellent library of random mutations. From that random insertion library, we have screened >1000 mutants for ability to grow in macrophages and have characterized >25 mutants that fail to grow normally. For each mutant, we have sequenced the transposon insertion site, and, using this information, we have discovered several genes required for intracellular growth of Mm. We propose to focus on a more detailed characterization of two most interesting mutants to understand the roles of the targeted genes in host cell invasion by, and intracettular growth of, Mycobacteria. In each case, we have shown that the Mtb homologue of the targeted Mm genes will complement the phenotypic defects discovered in the mutant. This allows a rapid and thorough investigation of the roles of these Mtb genes in a model that is capable of intracettular growth and ultimately of extension to in vivo models of infection. The purpose of the current proposal is to use these mutants to develop a better understanding of Mycobacteria entry into and survival in macrophages, and of macrophage response to Mycobacteria infection, cdtical events in the pathogenesis of disease. Our specific aims are: 1. Characterize the mip locus, required by Mm for host cell invasion and intracellular survival in macrophages 2. Determine how the GDP-mannose synthesis operon regulates macrophage activation and intracellular survival 3. Carry out a genetic screen for Mm genes specifically required for intracellular growth in macrophages. PERFORMANCE SITE ========================================Section End===========================================