Analysis of a novel peptidoglycan assembly pathway in mycobacteria

分枝杆菌中新型肽聚糖组装途径的分析

基本信息

批准号：
10431963
负责人：
Martin S. Pavelka
金额：
$ 44.15万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10431963
关键词：
Aerosols Alleles Anabolism Antibiotics Attenuated Bacteria Biological Biology Carbapenems Carboxypeptidase Cell Wall Clavulanic Acids Complement Disease Drug resistance in tuberculosis Enzymes Genes Genus Mycobacterium Goals Gram-Negative Bacteria Growth Knowledge Learning Maintenance Measures Modality Monobactams Mus Mutation Mycobacterium smegmatis Mycobacterium tuberculosis Names Nomenclature Organism Pathway interactions Penicillin-Binding Proteins Penicillins Peptides Peptidoglycan Peptidyltransferase Pharmaceutical Preparations Phenotype Phosphorylation Physiology Proteins Publishing Recombinants Research Research Personnel Resistance Role Salvelinus Temperature Testing Time Tuberculosis Work bacterial resistance base beta-Lactamase beta-Lactams carbapenem resistance cell envelope crosslink fitness in vivo inhibitor interest mouse model mutant mycobacterial novel rational design therapy development tuberculosis drugs

项目摘要

Summary The burden of treating drug resistant TB and the emergence of new strains that are essentially resistant to all TB drugs has prompted the re-assessment of ways to treat M. tuberculosis disease. One such treatment modality is the use of the β-lactam class of antibiotics that target the peptide crosslinking pathways of bacterial peptidoglycan (PG). These antibiotics are not used to treat M. tuberculosis infections due to the inherent resistance of the bacteria via the BlaC β-lactamase. Nevertheless, M. tuberculosis can be killed by these antibiotics in combination with the β-lactamase inhibitor clavulanic acid. This observation, made by us and other groups, has renewed interest in exploiting β-lactam antibiotics as a way to treat TB infections. However, knowledge of PG biosynthesis in mycobacteria has lagged behind that of other bacteria. Our overall research goals are to learn more about the assembly and maintenance of mycobacterial peptidoglycan (PG). In this proposal, we are interested in the biological significance of novel 3-3 peptide crosslinks within the PG. These linkages, first described in M. tuberculosis decades ago, are found in many other bacteria but their significance in cell wall biology is poorly understood. The 3-3 crosslinks differ from classical 4-3 crosslinks, also found in mycobacteria, in that 3-3 crosslinks are catalyzed by a novel pathway that is distinct from the 4-3 crosslink pathway. While the 4-3 crosslinks are catalyzed by penicillin sensitive DD- transpeptidases (also called penicillin-binding proteins or PBPs), the 3-3 crosslinks are made by unique, penicillin insensitive LD-transpeptidases (Ldts). Our previous work has demonstrated that the mycobacterial Ldt enzymes can be grouped into specific classes based upon sequence identity. Using M. smegmatis as a surrogate organism we have constructed mutants with either single or multiple deletions of each of the ldt genes and showed that only certain combinations of mutations yield mutant phenotypes. Central to all mutants with a substantial phenotype is loss of the Class 5 ldtC gene, suggesting that LdtC is a key enzyme in this pathway. This application aims to further examine the role of Ldt enzymes in M. tuberculosis cell wall biology, with a particular focus on LdtC, and to characterize the role of DD-carboxypeptidases in the 3-3 crosslink pathway.

概括治疗耐药结核病的负担和本质上具有耐药性的新菌株的出现所有结核病药物的使用促使人们重新评估治疗结核分枝杆菌疾病的方法之一。方式是使用β-内酰胺类抗生素，针对细菌的肽交联途径由于其固有的性质，这些抗生素不用于治疗结核分枝杆菌感染。然而，结核分枝杆菌可以被这些细菌杀死。抗生素与β-内酰胺酶抑制剂克拉维酸联合使用这一观察结果是由我们和其他团体对利用 β-内酰胺抗生素作为治疗结核感染的方法重新产生了兴趣。对分枝杆菌中 PG 生物合成的了解落后于其他细菌。我们的总体研究目标是更多地了解分枝杆菌的组装和维护在本提案中，我们对新型 3-3 肽的生物学意义感兴趣。 PG 内的交联在几十年前首次在结核分枝杆菌中被描述，现已在许多疾病中发现。与其他细菌不同，但它们在细胞壁生物学中的意义知之甚少。3-3 个交联与其他细菌不同。经典的 4-3 交联，也存在于分枝杆菌中，其中 3-3 交联是由新途径催化的这与 4-3 交联途径不同，而 4-3 交联是由青霉素敏感的 DD- 催化的。转肽酶（也称为青霉素结合蛋白或 PBP），3-3 个交联是由独特的、我们之前的工作已证明分枝杆菌对青霉素不敏感 LD 转肽酶 (Ldts)。 Ldt 酶可根据序列同一性分为特定类别。我们构建了替代生物体，其中每个 ldt 具有单个或多个缺失基因并表明只有某些突变组合才会产生对所有突变体至关重要的突变表型。具有显着表型的是 5 类 ldtC 基因的丢失，表明 LdtC 是该过程中的关键酶该应用旨在进一步研究 Ldt 酶在结核分枝杆菌细胞壁生物学中的作用，特别关注 LdtC，并表征 DD-羧肽酶在 3-3 交联中的作用途径。