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重大威胁。六种问题病原体，即所谓的ESKAPE细菌，是造成医院中大多数耐药感染的原因。迫切需要新的策略来治疗这些感染。靶向肽聚糖（PG）/细胞壁生物合成的抗生素是治疗细菌感染的最有效药物之一，但目前临床使用的所有抗生素都出现了耐药性。这项工作的提出源于最近的发现，使用戊内酰胺作为PG生物合成的化学探针。其目的是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）。MRSA是ESKAPE病原体中毒性最强的，而E.大肠杆菌本身是一种重要的病原体，是所有致病性革兰氏阴性杆菌中PG生物合成的模型系统。我们的前两个目标集中在验证一个新的抑制剂靶点，使MRSA对内酰胺类药物重新敏感。MRSA获得了一种称为PBP 2A的PG转肽酶，可促进对内酰胺的耐药性。我们发现PBP 2A的功能取决于糖基转移酶TarS的活性，该转移酶将½-O-GlcNAc残基连接到壁磷壁酸（WTA）上，壁磷壁酸是一种对S细胞分裂重要的额外细胞壁聚合物。金黄色。这表明PG和WTA合成的途径在某种程度上是相互关联的。我们将使用遗传和化学方法的组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中的内酰胺耐药性的目标。我们还将探索TarS本身作为药物靶点，通过监测小分子<$-内酰胺增效剂对其活性的影响，并在有和没有结合抑制剂的情况下解决其结构。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标。考虑到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。这主要是由于有限的可用性基因检测来剖析其功能。利用E.大肠杆菌系统，我们开发了第一个阳性 针对PG组装机的活性进行选择，PG组装机是细胞伸长所需的高度保守的杆系统。我们使用这种选择来鉴定杆功能的小分子拮抗剂，并建议确定其特定的靶标和作用方式。我们还将使用我们的选择来遗传地询问多蛋白质杆复合物的结构，并鉴定对每个组分的功能至关重要的氨基酸残基。结合化学遗传学分析将帮助我们确定和验证杆系统功能的各个方面，这些方面适合于新疗法的靶向。因为我们将要研究的PG和WTA合成机制是高度保守的，我们在MRSA和E。大肠杆菌中的细胞壁聚合物生物合成将与我们对其他微生物中细胞壁聚合物生物合成的理解广泛相关，并且应该显著影响和告知产生针对MRSA和革兰氏阴性ESKAPE病原体的疗法的努力。

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.