Identifying and validating new antibiotic targets in cell wall synthesis pathways

描述（由申请人提供）：抗生素耐药性对我们的医疗保健系统构成了重大威胁。六种问题病原体，即所谓的ESKAPE细菌，是医院中大多数耐药感染的罪魁祸首。迫切需要治疗这些感染的新策略。针对肽聚糖(PG)/细胞壁生物发生的抗生素是治疗细菌感染最有效的药物之一，但目前临床使用的所有抗生素都出现了耐药性。这项提议的工作源于最近使用¿-内酰胺作为PG生物合成的化学探针的发现。它旨在鉴定和验证耐甲氧西林金黄色葡萄球菌（MRSA）和大肠杆菌（E. coli）细胞壁组装途径中的新靶点。MRSA是ESKAPE病原菌中毒性最强的，而大肠杆菌本身就是一种重要的病原体，是所有致病性革兰氏阴性棒中PG生物发生的模式系统。我们的前两个目标是验证一种新的抑制剂靶点，使MRSA对内酰胺重新敏感。MRSA获得了一种名为PBP2A的PG转肽酶，可以促进内酰胺耐药性。我们发现PBP2A的功能依赖于糖基转移酶（TarS）的活性，该酶将glcnac残基附着在壁壁壁酸（WTAs）上，这是金黄色葡萄球菌细胞分裂的一种额外的细胞壁聚合物。这表明PG和WTA的合成途径在某种程度上相互关联。我们将使用遗传和化学方法的结合来揭示这些连接的机制基础，以便我们可以利用它们作为对抗MRSA的内酰胺耐药性的目标。我们还将通过监测小分子内酰胺增强剂对其活性的影响，并在有和没有结合抑制剂的情况下解决其结构问题，来探索TarS本身作为药物靶点。我们的第二组目标集中在了解PG合成机器的功能并验证它们作为抗生素靶点。鉴于它们作为潜在药物靶点的重要性，令人惊讶的是，人们对这些机器组装PG的机制知之甚少。这主要是由于遗传分析的可用性有限，以解剖其功能。利用大肠杆菌系统的遗传易感性，我们开发了第一个阳性

DESCRIPTION (provided by applicant): Antibiotic resistance poses a major threat to our healthcare system. Six problem pathogens, the so-called ESKAPE bacteria, are responsible for the majority of drug resistant infections in hospitals. New strategies to treat these infections ar sorely needed. Antibiotics that target peptidoglycan (PG)/cell wall biogenesis are among the most effective drugs for treating bacterial infections, but resistance has emerged to all those currently in clinical use. The proposed work grew out of recent discoveries made using ¿-lactams as chemical probes of PG biosynthesis. It is aimed at identifying and validating new targets in the pathways for cell wall assembly in methicillin resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli). MRSA is the most virulent of the ESKAPE pathogens, and E. coli, an important pathogen in its own right, is the model system for PG biogenesis in all pathogenic Gram-negative rods. Our first two aims are focused on validating a new target for inhibitors that resensitize MRSA to ¿-lactams. MRSA have acquired a PG transpeptidase called PBP2A that promotes ¿-lactam resistance. We discovered that PBP2A function is dependent on the activity of a glycosyltransferase, TarS, that attaches ¿-O-GlcNAc residues to wall teichoic acids (WTAs), an additional cell wall polymer important for cell division in S. aureus. This suggests that the pathways of PG and WTA synthesis are somehow interconnected. We will use a combination of genetic and chemical approaches to uncover the mechanistic basis for these connections so that we can exploit them as targets to combat ¿-lactam resistance in MRSA. We will also explore TarS itself as a drug target by monitoring the effect of small molecule ¿-lactam potentiators on its activity and solving its structure with and without bound inhibitors. Our second set of aims focus on understanding the function of PG synthesizing machines and validating them as antibiotic targets. Given their importance as potential drug targets, surprisingly little is known about the mechanism of PG assembly by these machines. This has primarily been due to a limited availability of genetic assays to dissect their function. Taking advantage of the genetic tractability of the E. coli system, we developed the first positive selection against the activity of a PG assembly machine, the highly conserved Rod system needed for cell elongation. We used this selection to identify small molecule antagonists of Rod function and propose to determine their specific targets and mode of action. We will also use our selection to genetically interrogate the structure of the multi-protein Rod complex and identify amino acid residues critical for the function of each component. The combined chemical genetic analysis will help us identify and validate aspects of Rod system function amenable to targeting by novel therapeutics. Because the PG and WTA synthesis machineries we will study are highly conserved, our findings in MRSA and E. coli will be broadly relevant to our understanding of cell wall polymer biogenesis in other microorganisms and should significantly impact and inform efforts to generate therapies against MRSA and Gram-negative ESKAPE pathogens.