GENES FOR METHY1-BRANCHED WALL LIPIDS AND TUBERCULOSIS

结核病和其他与艾滋病相关的分枝杆菌感染的死灰复燃威胁着世界范围内的人类健康。耐药结核病的出现使发现新的药物靶点变得至关重要。结核分枝杆菌的细胞壁含有多种不寻常的脂类，其中含有致病分枝杆菌所特有的多种甲基支链脂肪酸。这些脂质被认为在病原体抵抗抗菌剂和逃避宿主防御反应的能力中发挥重要作用。这些含有多个甲基支链脂肪酸的独特脂质的生物合成可能为新的抗分枝杆菌药物提供理想的靶点。基因组测序显示，分枝杆菌基因组的一个非常独特的特征是，它包含了异常大量的涉及脂类代谢的基因。基于与MAS的同源性，我们已经鉴定了两类聚酮合成酶(PKS)样基因，它们包含完整的活性部位结构域，参与催化合成多甲基支链脂肪酸所需的所有步骤：第一类MAS样基因(msl1、msl2和msl3)，与Mas高度同源；第二类(msl4mmsl5、msl6和msl7)，其同源性较低。这些开放阅读框架(ORF)最有可能编码在结核分枝杆菌中发现的八种以上的甲基分支脂肪酸。为了阐明这些基因的功能并研究它们在宿主-病原体相互作用中的可能作用，我们提出了以下具体目标：1)通过鉴定结核分枝杆菌表达的ms1、ms1、msl2和msl3类基因的产物，以及通过确定它们被干扰的生化和功能后果，来确定结核分枝杆菌类ms1基因的功能。2)确定结核分枝杆菌基因组中2类(msl4、msl5、msl6和msl7)Mas样基因的功能，方法是对其在耻垢分枝杆菌中表达的产物进行鉴定，并确定其干扰的生化和功能后果。3)确定上述基因突变导致的特定脂质的缺乏是否影响宿主与病原菌的相互作用和毒力。这些结果将有助于识别对发病机制至关重要的细胞壁脂质，这些脂质可能是新的抗分枝杆菌药物的合适靶点。

Resurgence of tuberculosis (TB) and other mycobacterial infections associated with AIDS threaten human health world-wide. Emergence of drug resistant TB makes it critical to discover new drug targets. Cell walls of Mycobacterium tuberculosis contain a variety of unusual lipids with many types of multiple methyl-branched fatty acids that are unique to pathogenic mycobacteria. These lipids are thought to play important roles in the ability of the pathogens to resist antimicrobial agents and evade the defense reactions of the host. Biosynthesis of these unique lipids containing multiple methyl-branched fatty acids could offer ideal targets for new anti mycobacterial drugs. Genomic sequencing revealed that a remarkably unique feature of the mycobacterial genome is that it contains an unusually large number of genes involved in lipid metabolism. Based on the homology to mycocerosic acid synthase (mas) we have identified two classes of polyketide synthase (pks)-like genes which contain a full complement of active site domains that should be involved in the catalysis of all of the steps required for the synthesis of multimethyl- branched fatty acids: Class 1 mas-like genes mas-like genes (msl1, msl2 and msl3) that are highly homologous to mas and Class 2 (msl4m msl5, msl6 and msl7) which show a lesser degree of homology to mas. These open reading frames (ORFs) most probably encode the more than eight classes of methyl-branched fatty acids found in M. tuberculosis. To elucidate the function of these genes and to examine their possible role in the host-pathogen interaction, we propose to pursue the following specific aims: 1) Determine the functions of Class 1 mas-like genes, ms1, msl2 and msl3 of M. tuberculosis by characterization of their products expressed in M. smegmatis, and by determination of the biochemical and functional consequences of their disruption. 2) Determine the functions of Class 2 (msl4, msl5, msl6 and msl7) mas-like genes in M. tuberculosis genome, by characterization of their products expressed in M. smegmatis, and by determination of the biochemical and functional consequences of their disruption. 3) Determine whether lack of specific lipids caused by the above gene-disruptions affects host-pathogen interaction and virulence. The results will help identify cell wall lipids critical for pathogenesis that might be suitable targets for new anti-mycobacterial drugs.