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.

摘要 治疗耐药结核病的负担和出现本质上耐药的新菌株 对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。一次这样的治疗 方式是使用β-内酰胺类抗生素，其靶向是细菌的多肽交联途径 肽聚糖(PG)。这些抗生素不被用来治疗结核分枝杆菌感染，因为固有的 细菌对BLACβ-内酰胺酶的耐药性。然而，结核分枝杆菌可以通过这些方法致死。 抗生素与β-内酰胺酶抑制剂克拉维酸联合使用。这一观察，由我们和 其他团体对利用β-内酰胺类抗生素作为治疗结核病感染的一种方法重新产生了兴趣。然而， 有关分枝杆菌中PG生物合成的知识一直落后于其他细菌。 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 肽聚糖(PG)。在这项提议中，我们感兴趣的是新的3-3肽的生物学意义 PG内部的交联链。几十年前在结核分枝杆菌中首次描述的这些联系，在许多 其他细菌，但对它们在细胞壁生物学中的意义知之甚少。3-3交联剂不同于 经典的4-3交联物，也存在于分枝杆菌中，因为3-3交联物是由一种新途径催化的 这与4-3交联途径不同。而4-3交联物是由青霉素敏感的DD- 转肽酶(也称为青霉素结合蛋白或PBPS)，3-3个交联物由独特的， 青霉素不敏感LD-转肽酶(LDTS)。我们之前的工作已经证明，分枝杆菌 LDT酶可以根据序列同一性被分成特定的类别。使用污垢分枝杆菌作为一种 替代生物体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才会产生突变表型。是所有突变体的中心 一个重要的表型是5类ldtC基因的缺失，这表明ldtC是这一过程中的关键酶 路径。本申请旨在进一步研究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-羧基肽酶在3-3交联键中的作用 路径。