Dissecting the role of Escherichia coli peptidoglycan synthase activators

 描述(申请人提供)：肽多聚糖层(PG)是细菌细胞膜的基本成分，保护细胞免受渗透溶解。这种结构是细菌界独有的，使其成为许多抗生素的宝贵靶标。青霉素结合蛋白1a(PBP1a)和青霉素结合蛋白1b(PBP1b)被认为是负责PG合成的主要酶，因为它们同时失活导致裂解。11这两种酶通过聚合糖链并将这些链交联到现有的PG层来合成PG层。伯恩哈特实验室目前的工作表明，某些被称为裂解转糖基酶(LTS)的酶可能与青霉素结合蛋白(PBPs)一起作用，但这种相互作用的细节尚未阐明。有趣的是，LTS裂解多聚糖链，所以我们预测细胞必须拥有一种方法来协调PBPs的活性和LTS的拮抗活性。最近，大肠杆菌外膜脂蛋白LPOA和LpoB分别被发现是PBP1a和PBP1b体内活性所必需的。11、12然而，具有构成过程的激活剂的目的尚不清楚。这个项目的目的是进一步了解 通过确定LPOA和LpoB的生理作用来调节PG的合成。为了实现这一目标，我将确定LPOA和LpoB的额外调控靶点，寻找克服含有结构性活性PBP1b等位基因的菌株细胞壁缺陷的抑制因子，并确定LPOA的生理功能及其激活PBP1a的机制。拟议的实验将建立在伯恩哈特实验室在细菌细胞膜生物学方面的优势的基础上，并有可能揭示与未来抗生素开发相关的新的生物学机制。

 DESCRIPTION (provided by applicant): The peptidoglycan layer (PG) is an essential component of the bacterial cell envelope that protects the cell from osmotic lysis. This structure is unique to the bacterial kingdom, making it a valuable target of many antibiotics. Penicillin binding protein 1a (PBP1a) and penicillin binding protein 1b (PBP1b) are thought to be the primary enzymes responsible for PG synthesis, because their simultaneous inactivation leads to lysis.11 These two enzymes synthesize the PG layer by polymerizing glycan strands and then cross-linking these strands into the existing PG layer. Current work in the Bernhardt lab suggests that certain enzymes known as lytic transglycosylases (LTs) may act in conjunction with the penicillin binding proteins (PBPs), however the specifics of this interaction are not yet elucidated. Interestingly, LTs cleave glycan strands, so we predict that cells must possess a way to coordinate the activity of PBPs with the antagonistic activity of LTs. Recently, the Escherichia coli outer membrane lipoproteins LpoA and LpoB were discovered to be required for the in vivo activity of PBP1a and PBP1b, respectively.11, 12 However, the purpose of having activators of a constitutive process is unclear. This project aims to gain further understanding of the regulation of PG synthesis by determining the physiological role of LpoA and LpoB. To accomplish this, I will identify additional regulatory targets of LpoA and LpoB, identify suppressors that overcome the cell wall defects of strains containing a constitutively active PBP1b allele, and determine the physiological function of LpoA and the mechanism of its activation of PBP1a. The proposed experiments will build on the strengths of the Bernhardt lab in bacterial cell envelope biology and have the potential to reveal novel biological mechanisms relevant to future antibiotic development.