CHARACTERIZATION OF MYCOBACTERIAL AUTOLYSINS

描述（改编自申请人摘要）：结核分枝杆菌 仍然是全球死亡的主要原因， 在发达国家和发展中国家， 国家M.结核病具有独特、复杂的结构， 是非常厚的、刚性的、疏水的。因为这些 由于其独特的特性，它对普通抗菌剂具有高度的不可渗透性。 细菌自溶素是能够水解细菌自溶素的酶。 细胞壁，并与正常的细菌细胞分裂，生长， 自溶对分枝杆菌的自溶素知之甚少。中央 这一建议的假设是分枝杆菌细胞壁自溶素 对生物体的生长和生存至关重要，可以作为 抗分枝杆菌药物的新靶点。 在初步研究中，研究人员（i）制备了一种分枝杆菌 水解细胞壁多糖的细胞裂解物;（ii）观察到， 乙胺丁醇处理的分枝杆菌具有增加的细胞壁水解;和（iii） 在M.结核病基因组， 与枯草芽孢杆菌细胞壁水解酶高度同源，即，一个 N-乙酰胞壁酰-L-丙氨酸酰胺酶。调查人员现在提出， 下一篇：AIM 1假定的M。上述结核酰胺酶基因将被 PCR扩增，克隆，表达。如果蛋白质被证实具有 预测酶活性，然后其功能和生化 将确定特征，并确定基因的表达特性， 将被分析。 AIM 2. (a)开发快速、灵敏的原位表型检测方法， 这些都为筛选克隆的分枝杆菌自溶素表达在E.杆菌 (b)开发改进的酶谱和无细胞酶测定法，并应用这些方法 涉及推定的水解酶的分离和纯化。纯化的酶将 分析结构信息（例如N-末端序列、定量 通过MALDI/TOF质谱法测定的蛋白质质量），其将被使用 对已发表的M.结核病基因组 序列的

DESCRIPTION (Adapted from the Applicant's Abstract): Mycobacterium tuberculosis remains the bacterial leading cause of death worldwide and multidrug-resistant strains have emerged as a new problem in both developed and developing countries. The cell wall of M. tuberculosis has a unique, complex structure and is extraordinarily thick, rigid, and hydrophobic. Because of these characteristics, it is highly impermeable to ordinary antimicrobial agents. Bacterial autolysins are enzymes which are capable of hydrolyzing the bacterial cell wall and are associated with normal bacterial cell division, growth, and autolysis. Little is known about the autolysins of mycobacteria. The central hypothesis of this proposal is that mycobacterial cell wall autolysins are essential for the growth and survival of the organisms and can be exploited as new targets for anti-mycobacterial agents. In preliminary studies, the investigators have (i) prepared a mycobacterial cell lysate that hydrolyzed the cell wall polysaccharide; (ii) observed that ethambutol treated mycobacteria have increased cell wall hydrolysis; and (iii) identified an open reading frame (ORF) in the M. tuberculosis genome that is highly homologous to a Bacillus subtilis cell wall hydrolases, i.e., an N-acetylmuranoyl-L-alanine amidase. The investigators now propose the following: AIM1. The putative M. tuberculosis amidase gene noted above will be amplified by PCR, cloned and expressed. If the protein is confirmed to have the predicted enzymatic activity, then its functional and biochemical characteristics will be determined and the expression properties of the gene will be analyzed. AIM 2. (a) Develop rapid and sensitive in situ phenotypic assays and apply these to screening for cloned mycobacterial autolysins expressed in E. coli. (b) Develop improved zymography and cell-free enzymatic assays and apply these to the isolation and purification of putative hydrolases. Purified enzymes will be analyzed for structural information (e.g. N-terminal sequence, quantitative protein mass as determined by MALDI/TOF mass spectroscopy), which will be used to identify the encoding gene in the published M. tuberculosis genome sequences.