MOLECULAR SEARCH FOR BIOSYNTHETIC GENES OF MYCOBACTERIUM TUBERCULOSIS CORD FACTOR

结核分枝杆菌索因子生物合成基因的分子搜索

• 批准号: 6161563
• 负责人: X QIN
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6161563
• 关键词: Mycobacterium tuberculosis; bacterial genetics; cell wall; gene expression; genetic strain; lipid biosynthesis; polymerase chain reaction; virulence

The principal focus of this work has been to search for biosynthetic genes of cord factor unique to Mycobacterium tuberculosis (Mtb). Since the virulence factor trehalose dimycolate (TDM), the molecular element responsible for "cord" formation in virulent Mtb (cord factor), is found exclusively in the lipid constituents of the cell walls of pathogenic Mtb, study of the genes encoding the synthesizing enzymes will increase our knowledge of mycobacterial cell-wall biosynthesis and function, hence permitting the design of a tailor-made drug(s) to inhibit key catalytic enzymes. Furthermore, since the cell wall mycolic glycoconjugates are large and complex molecules, the biosynthetic enzymes are believed to be membrane-bound proteins that carry out some of the major condensation steps outside of the cytoplasm to avoid employing complex transport systems. Therefore, these membrane-bound proteins could, in theory, be used for vaccine targets. Though biosynthetic pathway(s) of Mtb wall lipid constituents are not known, experience in well-studied cell envelope features of gram-positive and gram-negative bacteria enables one to speculate on some principal catalytic steps involved in the formation of distinctive molecules such as TDM. The biosynthesis of Escherichia coli lipid A has been elucidated and several of the relevant genes are known. Among the known genes are lpxA and lpxD, which encode UDP-GlcNAc O-acyltransferases. Based on the primary sequence alignment of a number of lpxA/lpxD genes from various organisms (E. coli, Salmonella typhimurium, Yersinia enterocolitica, and Rickettsia rickettsii), degenerate oligonucleotide primers were designed to amplify the analogs in Mtb. A polymerase chain reaction (PCR) procedure amplified a lKb fragment from the chromosomal DNA of a virulent Mtb reference strain, H37Rv, while no comparable amplification product was observed from DNA controls of Bacillus subtilis or M. smegmatis. The length of the possible interdomain sequence amplified appeared to be about 2 times longer than those in the other species. A large open-reading frame in the sequence was deduced to contain hexad repeats, similar to those observed in LpxA/LpxD protein sequences. A Southern blot of chromosomal materials from H37Rv, H37Ra, M. smegmatis, and B. subtilis probed with the lKb fragment suggested that this PCR product was Mtb-specific - not present in any other species tested. The DNA sequence obtained from the lKb PCR product produced a perfect match to a Mtb gene proposed to encode a potential membrane-associated protein with an ATPase motif. Further investigations of the location and the biological function of this gene will be carried out in the next 12 months.

这项工作的主要重点是寻找生物合成基因 结核分枝杆菌（Mtb）特有的脐带因子。以来 毒力因子海藻糖二霉菌酸酯（TDM），分子元件 在毒性Mtb中负责“索”形成（索因子）， 仅存在于致病性Mtb细胞壁的脂质成分中， 对编码合成酶的基因的研究将增加我们的 分枝杆菌细胞壁生物合成和功能的知识，因此 允许设计定制的药物来抑制关键的催化活性， 内切酶此外，由于细胞壁分枝菌糖缀合物是 大而复杂的分子，生物合成酶被认为是 膜结合蛋白质，进行一些主要的缩合， 在细胞质外的步骤，以避免采用复杂的运输 系统.因此，理论上，这些膜结合蛋白质可能是 用于疫苗靶点。 虽然Mtb壁脂质成分的生物合成途径不是 已知的，在充分研究的革兰氏阳性细胞的细胞包膜特征的经验， 和革兰氏阴性菌的研究使人们能够推测出 参与形成独特分子的催化步骤， TDM。大肠杆菌脂质A的生物合成已被阐明， 几个相关基因是已知的。在已知的基因中有lpxA 和lpxD，其编码UDP-GlcNAc O-酰基转移酶。基于主要 来自各种生物体的许多lpxA/lpxD基因的序列比对 （E.大肠杆菌，鼠伤寒沙门氏菌，小肠结肠炎耶尔森氏菌，和 立克次体），设计简并寡核苷酸引物 以扩增Mtb中的类似物。聚合酶链反应（PCR）程序 从Mtb强毒株的染色体DNA中扩增出1 Kb片段， 参考菌株H37 Rv，而没有可比较的扩增产物。 从枯草芽孢杆菌或M.恶臭的 扩增的可能的结构域间序列的长度似乎约为 是其他物种的2倍。一个大的开放阅读框架 在序列中被推断含有六联体重复，类似于那些 在LpxA/LpxD蛋白质序列中观察到。染色体的Southern印迹 材料来自H37 Rv、H37 Ra、M.斯麦葛碱和B.枯草杆菌探测与 1 Kb片段表明该PCR产物为Mtb特异性的，而非Mtb特异性的。 存在于测试的任何其他物种中。DNA序列从 1 Kb PCR产物与Mtb基因完全匹配，该基因被认为编码 具有ATP酶基序的潜在膜相关蛋白。进一步 对该基因的定位和生物学功能的研究 将在未来12个月内进行。