Molecular dissection of vesiculogenesis in Mycobacterium tuberculosis

 描述（由申请人提供）：结核病（TB）仍然是全球发病率和死亡率的主要原因。结核分枝杆菌（M.tb）是结核病的病原体，能够建立原发性感染，这些感染通常是潜伏性的，即使免疫系统完好，也会持续存在。众所周知，结核分枝杆菌具有多种高度进化的机制来操纵宿主细胞机器并逃避宿主免疫系统。我们通过发现基于膜囊泡（MV）的替代抗原释放机制，为扩大结核分枝杆菌生理学知识做出了贡献。MV的成分分析显示，极性脂质和脂蛋白的富集。MV释放事件在体外和体内感染过程中可视化。MV刺激巨噬细胞和小鼠引起依赖于Toll样受体2（TLR-2）的炎症反应。MV刺激与雾化感染在小鼠中的组合产生了促进肺和脾中分枝杆菌生长的“Koch现象”，表明MV与结核分枝杆菌发病机制之间存在联系。然而，结核分枝杆菌中囊泡形成和毒力之间更强的联系仍有待确定。我们最近致力于表征Rv 0431结核分枝杆菌突变体的表型，其由于MV的过度产生而在巨噬细胞中表现出高度炎症反应。这一发现表明，MV生产是一个受调控的过程，鉴于该过程的内在复杂性，可能涉及其他基因。本申请提出了结核分枝杆菌水疱发生的分子解剖。首先，我们将确定 通过筛选结核分枝杆菌转座子插入突变体库，使用灵敏的高通量斑点印迹系统检测低容量培养物中的MV，从而确定负责囊泡产生的基因。筛选试验使用能够识别囊泡的抗血清，其特异性由用于制备免疫原的物理分离技术赋予。MV产量增加的突变体、MV产量低的突变体和不产生MV的突变体将通过显微镜方法进行表征，以深入了解超微结构变化，并且将采用基于生物化学和质谱的技术来鉴定可能改变的MV组成。此外，我们将研究Rv 0431在水疱发生中的作用。这里提出的方法将允许识别在囊泡产生中受影响的其他遗传变异，这对于研究它们在分枝杆菌感染的发病机制中的作用至关重要。

 DESCRIPTION (provided by applicant): Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide. Mycobacterium tuberculosis (M.tb), the causative agent of TB, is capable of establishing primary infections that often become latent and can persist despite an intact immune system. It is well known that M.tb possesses multiple highly evolved mechanisms to manipulate host cellular machinery and evade the host immune system. We have contributed to expanding the knowledge of M.tb physiology through the discovery of an alternative antigen release mechanism based on membrane vesicles (MV). Compositional analysis of MV revealed enrichment in polar lipids and lipoproteins. MV release events were visualized during in vitro and in vivo infections. MV stimulation of macrophages and mouse elicited inflammatory response dependent on Toll-like receptor 2 (TLR-2). The combination of MV stimulation with an aerosolized infection in mice produced a `Koch phenomenon' promoting the growth of mycobacteria in lungs and spleen, suggesting a link between MV and M.tb pathogenesis. Nevertheless a stronger association between vesiculation and virulence in M.tb remains to be established. We recently contributed to characterizing the phenotype of the Rv0431 M.tb mutant, which manifested a hyper- inflammatory response in macrophages due to overproduction of MV. This finding suggests that MV production is a regulated process and given the intrinsic complexity of the process, additional genes are likely to be involved. This application proposes a molecular dissection of vesiculogenesis in M.tb. Initially, we will identify genes responsible for vesicle production by screening a library of M.tb transposon insertion mutants using a sensitive, high-throughput dot blot system to detect MV in low-volume cultures. The screening assay uses antiserum capable of recognizing vesicles whose specificity is imparted by the physical separation techniques used to prepare the immunogen. Mutants with increased MV production, low MV production and non-producing mutants will be characterized by microscopic methods to gain insight into ultrastructural changes, and biochemical and mass spectrometry based-techniques will be employed to identify potentially altered MV composition. Further, we will investigate the role of Rv0431 in vesiculogenesis. The approach proposed here will allow identification of additional genetic variants affected in vesicle production, which willbe essential for studying their role in the pathogenesis of mycobacterial infection.