Identification of M. tuberculosis Protein Secreted During Growth in Macrophages

描述（由申请人提供）：结核分枝杆菌是导致结核病的细菌，是一种在巨噬细胞内存活和生长的细胞内病原体。由M分泌或定位于细菌细胞表面的蛋白质。结核病可以与宿主细胞相互作用，并且它们有助于发病机制和免疫应答。在这一亚细胞类别中的许多蛋白质仍有待鉴定或研究。此外，我们假设有一类重要的分泌和表面蛋白-那些专门出口，而M。结核病是细胞内的--目前的检测方法错过了这一点。我们最近建立了一个基因报告系统，其中β-内酰胺酶用于蛋白融合，以直接报告β-内酰胺敏感的M突变体中的蛋白输出。结核因为β-内酰胺抗生素靶向细胞壁合成酶，所以β-内酰胺酶必须从细胞质中输出以保护细菌。我们相信，我们的β-内酰胺酶报告系统具有一种新的能力，确定分泌蛋白质在细胞内生长的M。β-内酰胺处理的巨噬细胞中的结核病。在此RO 3应用中，我们建议在遗传选择方案中使用该报告基因来鉴定由M.结核病在宿主环境中生长。将进一步研究鉴定的蛋白质的子集在巨噬细胞的细胞内生长中的作用。这项研究将补充现有的基因组方法，并发现新的毒力因子和候选抗原，有助于开发新的疾病干预和诊断策略。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis, the bacterium responsible for tuberculosis disease, is an intracellular pathogen that survives and grows within macrophages. Proteins secreted or localized to the bacterial cell surface by M. tuberculosis can interact with host cells, and they contribute to pathogenesis and the immune response. Many of the proteins in this subcellular category remain to be identified or studied. Further, we hypothesize that there is an important class of secreted and surface proteins - those exclusively exported while M. tuberculosis is intracellular - which are missed by current detection methods. We recently established a genetic reporter system in which beta-lactamase enzymes are used in protein fusions to report on protein export directly in a beta-lactam sensitive mutant of M. tuberculosis. Because beta-lactam antibiotics target cell wall synthesis enzymes, beta-lactamases must be exported out of the cytoplasm to protect the bacterium. We believe our beta-lactamase reporter system has a novel capability of identifying proteins secreted during intracellular growth of M. tuberculosis in beta-lactam treated macrophages. In this RO3 application we propose to use this reporter in a genetic selection scheme to identify proteins exported by M. tuberculosis during growth in the host environment. A subset of the proteins identified will be further studied for roles in intracellular growth in macrophages. This research will complement existing genomic approaches and uncover new virulence factors and candidate antigens that should aid development of novel disease intervention and diagnostic strategies.