Characterization of gene cluster for glycophosphoinositides of mycobacteria

描述(申请人提供)：结核分枝杆菌多药耐药(MDR)菌株的出现导致了对新的结核病治疗药物的迫切需求。结核分枝杆菌含有多种糖基磷脂酰肌醇甘露糖苷(PIMs)、脂甘露聚糖(LM)和脂阿拉伯甘露聚糖(LAM)，在分枝杆菌感染的生理和免疫发病机制中发挥重要作用。在描述PIM的结构和生物合成方面已经取得了巨大的进展，但直到最近，导致LM/LAM生物合成的酶和步骤还不清楚。阐明参与合成这些分子的催化步骤将有助于确定分枝杆菌最复杂的生物合成途径之一，并为新型抗分枝杆菌药物的鉴定提供新的靶点。合成有缺陷的突变体也应该能够使这些分子在结核病的免疫发病机制中做出精确的贡献。我们最近发现了两个编码甘露糖转移酶的基因，它们负责LM/LAM生物合成的末端步骤。对这些基因的遗传位点的电子分析表明，结核分枝杆菌基因组中可能存在PIM/LM/LAM生物合成基因簇。为了确定分枝杆菌的PIM/LM/LAM生物合成途径，我们已经开始对该簇的基因进行表征。因此，这项建议的具体目标是：1.通过遗传和生化方法表征酰基转移酶；2.潜在参与PIMs跨质膜运输的蛋白质的功能表征。公共卫生相关性：分枝杆菌糖基磷脂酰肌醇基因簇的鉴定和特征。对分枝杆菌糖基磷脂酰肌醇(GPI)途径的剖析将导致一类新的结核分枝杆菌必需酶的发现，这些酶可能是治疗干预的新靶点。此外，在GPI合成的某些方面有缺陷的突变体将有助于确定这些分子在免疫发病机制中的确切作用。

DESCRIPTION (provided by applicant): The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis has led to a critical need for new therapeutic agents against tuberculosis. M. tuberculosis is endowed with a diverse range of glycosylphosphatidylinositides such as phosphatidylinositol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM), which play an important role in the physiology as well as the immunopathogenesis of mycobacterial infection. Enormous progress has been made in delineating the structure and biosynthesis of PIMs, but until recently the enzymes and steps leading to LM/LAM biosynthesis were not known. The elucidation of the catalytic steps involved in the synthesis of these molecules will help define one of the most complex biosynthetic pathways of mycobacteria in addition to providing new targets for the identification of novel anti-mycobacterial agents. Mutants defective in their synthesis should also enable a precise contribution of these molecules to the immunopathogenesis of tuberculosis. We have recently identified two genes encoding mannosyltransferases responsible for the terminal steps of LM/LAM biosynthesis. In silico analysis of the genetic locale of these genes revealed a possible PIM/LM/LAM biosynthetic gene cluster in the genome of M. tuberculosis. In order to define the PIM/LM/LAM biosynthetic pathways of mycobacteria, we have begun the characterization of genes from this cluster. Thus, the specific aims of this proposal are: 1. Characterization of an acyltransferase by genetic and biochemical approaches; 2. Functional characterization of a protein potentially involved in the transport of PIMs across the plasma membrane. PUBLIC HEALTH RELEVANCE: Identification and characterization of a gene cluster for glycosylphosphatidylinositides of mycobacteria. Dissection of the glycosylphosphatidylinositide (GPI) pathway in mycobacteria will lead to the discovery of a novel class of essential enzymes of M. tuberculosis that may represent new targets for therapeutic intervention. In addition, the mutants defective in some aspects of GPI synthesis will help define the precise role of these molecules in immunopathogenesis.