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

• 批准号: 10203747
• 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
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10203747
• 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; design; fitness; in vivo; inhibitor/antagonist; interest; mouse model; mutant; mycobacterial; novel; therapy development; tuberculosis drugs

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 治疗抗药性结核病的燃烧和本质上具有抗性的新菌株的出现 所有结核病药物都促使对治疗结核分枝杆菌病的方法进行了重新评估。一种这样的治疗方法 模态是靶向细菌的胡椒交联途径的β-内酰胺类抗生素类 肽聚糖（PG）。这些抗生素不用于治疗因继承而导致的结核病。 细菌通过BLACβ-内酰胺酶的抗性。然而，结核分枝杆菌可以被这些杀死 抗生素与β-内酰胺酶抑制剂克拉维酸结合使用。这个观察，由我们和 其他小组对利用β-内酰胺抗生素作为治疗结核病感染的一种方式，使人们更加兴趣。然而， 对分枝杆菌中PG生物合成的知识落后于其他细菌。 我们的整体研究目标是更多地了解分枝杆菌的组装和维护 肽聚糖（PG）。在此提案中，我们对新型3-3 Pepperide的生物识别意义感兴趣 在PG内交叉链接。这些联系是几十年前在结核分枝杆菌中首先描述的，在许多 其他细菌，但其在细胞壁生物学中的意义知之甚少。 3-3的交联不同 经典的4-3个交叉链接，在分枝杆菌中也发现，3-3个交联被新型途径催化 这与4-3个交叉链接路径不同。而4-3个交联被青霉素敏感的DD-催化 3-3个交叉链接是由独特的， 青霉素不敏感的LD-转肽酶（LDTS）。我们以前的工作证明了分枝杆菌 LDT酶可以基于序列身份分为特定类。将M. smegmatis用作 替代生物我们已经构建了每个LDT的单个或多个缺失的突变体 基因并表明只有某些突变组合会产生突变表型。所有突变体的中心 具有实质性表型的是5类LDTC基因的丧失，这表明LDTC是其中的关键酶 路径。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交叉链接中的作用 路径。