Altered Mycobacterium tuberculosis Mannosylation and the Macrophage

描述(申请人提供)：结核病继续在全世界人口中造成巨大的发病率和死亡率。在人类宿主内建立结核分枝杆菌(M.tb)感染的关键是进入巨噬细胞并存活。巨噬细胞甘露糖受体(MR)参与结核分枝杆菌强毒株的吞噬作用。结核分枝杆菌细胞壁的成分作为宿主细胞受体的配基，可以调节宿主的杀菌和炎症反应。结核分枝杆菌细胞被大量甘露糖化，含有脂多糖，如阿拉伯甘露聚糖(LAM)，它是结核分枝杆菌的配体。我们推测，结核分枝杆菌表面甘露糖化的性质对结核分枝杆菌与MR相互作用的能力以及调节巨噬细胞功能从而调节宿主反应的能力有重大影响，从而能够建立感染。PimB是新近发现的一种肌醇磷脂转移酶(PimB)。我们利用等位基因交换技术灭活了结核分枝杆菌Erdman株中的pimB。巨噬细胞在感染野生型结核分枝杆菌后表现出明显的细胞黏附。相比之下，感染pimB突变体的巨噬细胞显示出最小的细胞黏附和显著增加的巨噬细胞死亡率。我们已经开发了一种分析方法，其中沙门氏菌甘露糖特异性结合菌毛凝集结核分枝杆菌LAM包被微球，我们将开发该微球作为对结核分枝杆菌表面甘露糖化变化的筛查。我们建议进一步研究pimB和其他可能参与结核分枝杆菌表面分子甘露糖化的酶在结核分枝杆菌-宿主相互作用生物学中的作用，开发一种新的筛选表面甘露糖化改变的结核分枝杆菌克隆的策略，并评估这些细菌克隆是否存在异常的宿主细胞相互作用。我们的具体目标是：1.确定感染结核分枝杆菌pimB突变株后同型黏附减少和巨噬细胞死亡率增加的机制。确定pimB突变的生化性质。分析来自野生型、pimB突变体和pimB高产结核分枝杆菌菌株的LAM和其他甘露糖化细胞壁糖结合物的结构。2.对编码LAM和甘露糖共轭化合物的生物合成酶的基因进行转录和遗传学研究：2a。使用ABI 7700(TaqMan)“实时”定量聚合酶链式反应系统，量化在肉汤、固体培养基和人巨噬细胞内生长的结核分枝杆菌中pimB及其同系物的转录水平；构建和分析具有甘露糖生物合成基因改变的遗传定义的结核分枝杆菌菌株。3)。利用沙门氏菌甘露糖结合(1型)菌毛从M.tb转座子文库和带有M.tb基因的耻垢分枝杆菌文库中分别筛选表面甘露糖化改变的突变体和克隆，以表征细菌甘露糖化改变对巨噬细胞相互作用的影响。聚集的研究人员将结合遗传学、生物化学和细胞生物学的技术来实现这项提案的目标。

DESCRIPTION (provided by applicant): Tuberculosis continues to cause tremendous morbidity and mortality throughout the world's population. Critical in establishment of a M. tuberculosis (M.tb) infection within its human host are entry and survival in the macrophage. The macrophage mannose receptor (MR) participates in the phagocytosis of virulent strains of M.tb. Components of the M.tb cell wall serve as ligands for host cell receptors and can modulate host microbicidal and inflammatory responses. The M.tb cell envelope is heavily mannosylated containing lipoglycans such as lipoarabinomannan (LAM) which serves as a ligand for the MR. We hypothesize that the nature of surface mannosylation of M.tb has a major impact on the ability of M.tb to interact with the MR as well as to modulate macrophage function and consequently host responses, enabling the establishment of infection. PimB was recently described as M.tb phosphatidyl myo-inositol monomannoside transferase (pimB). We have used allelic-exchange to inactivate pimB in M.tb strain Erdman. Macrophages display marked cellular adhesion following infection with wild-type M.tb. In contrast, macrophages infected with the pimB mutant display minimal cellular adhesion and a significant increase in the rate of macrophage death. We have developed an assay in which Salmonella mannose-specific binding pili agglutinate M.tb LAM coated microspheres that we will develop as a screen for alterations in M.tb surface mannosylation. We propose to further characterize the role of pimB and other selected enzymes potentially involved in mannosylation of M.tb surface molecules in the biology of the M.tb-host interaction, to develop a novel screening strategy for M.tb clones altered in surface mannosylation, and to evaluate these bacterial clones for anomalous host cell interaction. Our specific aims are to: 1A. Determine the mechanism for reduced homotypic adhesion and increased rate of macrophage death following infection with the pimB mutant of M.tb: 1 B. Determine the biochemical nature of the pimB mutation. Analyze the structure of LAM and other mannosylated cell wall glycoconjugates from wild type, the pimB mutant, and pimB overproducing M.tb strains. 2. Perform transcription and genetic studies of genes encoding the biosynthetic enzymes of LAM and mannose glycoconjugates: 2A. Quantify the level of transcription of pimB and its homologues in M.tb grown in broth, solid medium and within human macrophages using the AbI 7700 (TaqMan) "real-time" quantitative PCR system; 2B. Construct and analyze genetically defined M.tb strains with alterations in the mannose biosynthetic genes. 3). Utilize Salmonella mannose-binding (type 1) pili to screen for M.tb mutants and clones respectively altered in surface mannosylation from an M.tb transposon library and M. smegmatis library complemented with M.tb genes to characterize the effect of alterations in bacterial mannosylation on macrophage interaction. The assembled investigators will combine techniques in genetics, biochemistry, and cell biology to accomplish the goals of this proposal.