Altered Mycobacterium tuberculosis Mannosylation and the Macrophage

描述（由申请人提供）：结核病继续在全世界人口中造成巨大的发病率和死亡率。巨噬细胞的进入和存活是结核分枝杆菌在人类宿主内感染的关键。巨噬细胞甘露糖受体（MR）参与结核分枝杆菌毒力菌株的吞噬作用。结核分枝杆菌细胞壁的成分作为宿主细胞受体的配体，可以调节宿主的杀微生物和炎症反应。结核分枝杆菌包膜含有大量甘露糖基化的脂聚糖，如脂arabinman聚糖（LAM），作为MR的配体。我们假设结核分枝杆菌表面甘露糖基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和宿主反应的能力有重大影响，从而使感染得以建立。PimB最近被描述为M.tb磷脂酰肌醇单甘露糖转移酶（PimB）。我们用等位基因交换法灭活了M.tb菌株Erdman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粘附。相比之下，感染了pimB突变体的巨噬细胞表现出最小的细胞粘附，巨噬细胞死亡率显著增加。我们已经开发了一种检测方法，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，我们将开发作为结核分枝杆菌表面甘露糖基化变化的筛选。我们建议进一步表征pimB和其他可能参与结核分枝杆菌表面分子甘露糖基化的酶在结核分枝杆菌与宿主相互作用的生物学过程中的作用，开发一种新的筛选表面甘露糖基化改变的结核分枝杆菌克隆的策略，并评估这些细菌克隆对异常宿主细胞相互作用的影响。我们的具体目标是：确定感染M.tb: 1 b的pimB突变体后，巨噬细胞同型粘附减少和死亡率增加的机制。确定pimB突变的生化性质分析野生型、pimB突变株和pimB高产株的LAM和其他甘露糖基细胞壁糖缀合物的结构。2. 对编码LAM和甘露糖缀合物生物合成酶的基因进行转录和遗传研究：2A。利用AbI 7700 （TaqMan）“实时”定量PCR系统，定量在肉汤、固体培养基和人巨噬细胞中培养的结核分枝杆菌中pimB及其同源物的转录水平；2 b。构建和分析具有甘露糖生物合成基因改变的遗传定义结核分枝杆菌菌株。3）. 利用沙门氏菌甘露糖结合（1型）菌毛从M.tb转座子文库和与M.tb基因互补的耻毛分枝杆菌文库中分别筛选表面甘露糖基化改变的M.tb突变体和克隆，以表征细菌甘露糖基化改变对巨噬细胞相互作用的影响。与会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完成本提案的目标。

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.