Identification of M. tuberculosis Protein Secreted During Growth in Macrophages

描述(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生存和生长的细胞内病原体。结核分枝杆菌分泌或定位于细菌细胞表面的蛋白质可与宿主细胞相互作用，参与致病和免疫应答。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，有一类重要的分泌性和表面蛋白--那些在结核杆菌处于细胞内时专门输出的蛋白--是目前检测方法所遗漏的。我们最近建立了一个遗传报告系统，在该系统中，β-内酰胺酶用于蛋白质融合，以直接报告结核分枝杆菌对β-内酰胺敏感的突变体的蛋白质输出。因为β-内酰胺类抗生素针对的是细胞壁合成酶，所以必须将β-内酰胺酶从细胞质中排出，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，可以在β-内酰胺处理的巨噬细胞中识别结核分枝杆菌在细胞内生长过程中分泌的蛋白质。在这个R03应用中，我们建议在基因选择方案中使用该报告来识别结核分枝杆菌在宿主环境中生长期间输出的蛋白质。已确定的蛋白质的一个子集将进一步研究在巨噬细胞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，应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.