CHARACTERIZATION OF MYCOBACTERIAL AUTOLYSINS

描述(改编自申请者摘要)：结核分枝杆菌 仍然是全球范围内细菌死亡的主要原因，并对多种药物产生耐药性 菌株已成为发达国家和发展中国家面临的新问题。 国家。结核分枝杆菌的细胞壁具有独特、复杂的结构和 非常厚，坚硬，疏水性。正因为如此 特点，对普通抗菌剂具有高度的防渗性。 细菌自溶素是一种能够分解细菌的酶 细胞壁，与正常的细菌细胞分裂、生长和 自溶。人们对分枝杆菌的自溶素知之甚少。中环 这一提议的假设是，分枝杆菌细胞壁自溶蛋白是 对生物的生长和生存是必不可少的，可以被开发为 抗分枝杆菌药物的新靶点。 在初步研究中，研究人员(I)准备了一种分枝杆菌 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增加了细胞壁的水解率；以及 在结核分枝杆菌基因组中发现了一个开放阅读框架(ORF)，它是 与枯草芽孢杆菌细胞壁水解酶高度同源，即 N-乙酰-L-丙氨酸酰胺酶。调查人员现在提出 以下为：AIM1.上面提到的推测的结核分枝杆菌酰胺酶基因将是 经聚合酶链式反应扩增、克隆、表达。如果该蛋白质被确认含有 预测酶的活性，然后预测其功能和生化 将确定该基因的特征和表达特性 将会被分析。 目的2.(A)建立快速、灵敏的原位表型分析方法并应用 这些用于筛选在大肠杆菌中表达的克隆的分枝杆菌自溶素。 (B)开发改进的酶谱分析和无细胞酶分析，并应用这些方法 对可能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 由MALDI/TOF质谱学测定的蛋白质质量)，将使用 鉴定已发表的结核分枝杆菌基因组中的编码基因 序列。

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.