Analysis of a novel peptidoglycan assembly pathway in mycobacteria

总结 治疗耐药结核病的负担和基本上耐药的新菌株的出现 对所有结核病药物的使用促使人们重新评估治疗M.结核病。一种这样的处理 形式是使用β-内酰胺类抗生素，其靶向细菌的肽交联途径， 肽聚糖（PG）。这些抗生素不用于治疗M。结核病感染由于固有的 细菌通过BlaC β-内酰胺酶产生耐药性。然而，M。结核病可以通过这些 抗生素联合β-内酰胺酶抑制剂克拉维酸。这一观察，由我们和 其他研究小组重新对开发β-内酰胺抗生素作为治疗结核病感染的方法产生了兴趣。然而，在这方面， 分枝杆菌中PG生物合成的知识落后于其它细菌。 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）。在本研究中，我们对新的3-3肽的生物学意义感兴趣 PG内的交联。这些连接，首先在M.几十年前的结核病， 其他细菌，但它们在细胞壁生物学中的重要性知之甚少。3-3交联不同于 经典的4-3交联，也在分枝杆菌中发现，因为3-3交联由一种新的途径催化 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PBPs），3-3交联是由独特的， 青霉素不敏感LD-转肽酶（Ldts）。我们以前的工作已经证明， Ldt酶可以根据序列同一性分为特定的类别。利用M.作为一个 我们构建了每个LDT单个或多个缺失的突变体 基因，并表明只有某些突变组合产生突变表型。所有变种人的中心 具有实质性表型的是5类ldtC基因的缺失，这表明LdtC是这一过程中的关键酶。 通路本申请旨在进一步研究Ldt酶在M.结核病细胞壁生物学， 特别关注LdtC，并表征DD-羧肽酶在3-3交联中的作用， 通路

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.