Novel Mechanisms of Beta-lactam Resistance in Staph Aureus

摘要 我们已经在金黄色葡萄球菌中发现了一种新的高水平、广谱的β-内酰胺类耐药模式，而不是 由导致甲氧西林耐药的青霉素结合蛋白(PBP)PBP2a介导。PBP4，一种非 Essential PBP和GdpP，GdpP是已知的唯一一种介导环二腺苷单核苷酸的磷酸二酯酶。 磷酸盐(CDA)的降解，在这类抗性中起着关键作用。PBP4基因突变增强与S 产生高度交联细菌细胞壁的能力和GdpP功能丧失突变是基因 对这种非规范抵抗负有责任的基础。高度交联的细胞壁的形成是由 独立地或协同地由PBP4的两个不同的生化特征：a)其蛋白质的结构变化 由于错义突变和b)由于其启动子区域的突变而过度表达。 GdpP功能缺失突变导致细菌细胞中CDA浓度升高。CDA 是细菌中一种新发现的细胞信号第二信使，它通过 与其效应器(蛋白质和RNA)结合。CDA广泛影响基因表达并控制GdpP相关 β-内酰胺类耐药表型呈浓度依赖关系，提示它是确定性的 阻力因素。然而，CDA在介导β-内酰胺类耐药以及其他方面的确切作用(S) 金黄色葡萄球菌的生命过程目前尚不清楚。 PBP4和GdpP的这些功能变化可能是以细菌毒力为代价的，这是由于 细胞壁相关蛋白的耗尽和细胞溶血素的产生分别减弱。这表明一个 金黄色葡萄球菌两个关键致病因子β-内酰胺类耐药与阴阳关系的研究 致命性。我们将研究导致PBP4和GdpP功能变化的基本基础 耐药性及其对细菌毒力的影响。目的1：确定PBP4介导的信号转导机制 β-内酰胺类抗生素耐药性及其在细胞壁组成中的作用。PBP4的相对贡献 对细胞壁合成的错义和启动子突变将进行生化和结构评估。这个 控制pbp4表达的机制(S)将被调查，以确定调控因子(S)，并确定如何 它们使PbP4介导的β-内酰胺类耐药。PBP4‘S在细菌细胞表面相关毒力中的作用 将确定因子表达式。目的2：确定环二腺苷一磷酸的作用 (CDA)信号在金黄色葡萄球菌。将采用遗传和化学蛋白质组学方法来鉴定CDA 介体/S在导致β-内酰胺耐药和毒力缺陷的细菌中的作用。最后，我们的 初步数据表明，除GdpP外，金黄色葡萄球菌中还存在一种新的CDA特异性磷酸二酯酶。 我们将对这种新的磷酸二酯酶进行鉴定。这项拟议的研究将促进对基础细胞的了解 金黄色葡萄球菌的过程。

Abstract We have identified a novel mode of high-level, broad-spectrum β-lactam resistance in S. aureus that is not mediated by PBP2a, the penicillin-binding protein (PBP) that confers methicillin resistance. PBP4, a non- essential PBP, and GdpP, the only known phosphodiesterase (PDE) that mediates cyclic-di-adenosine-mono- phosphate (CDA) degradation, have critical roles in this type of resistance. Mutations that enhance PBP4's ability to make a highly cross-linked bacterial cell wall and loss-of-function mutations in GdpP are the genetic basis responsible for this uncanonical resistance. The highly cross-linked cell wall formation is driven either independently or cooperatively by two distinct biochemical features of PBP4, a) structural changes in its protein due to missense mutations and b) its overexpression due to mutations in its promoter region. The loss-of-function mutations in GdpP result in elevated concentrations of CDA in bacterial cells. CDA is a newly discovered cell-signaling second messenger in bacteria which acts as an allosteric regulator by binding to its effectors (proteins and RNAs). CDA broadly affects gene expression and controls GdpP related β-lactam resistant phenotypes in a concentration dependent manner, suggesting that it is the deterministic factor in resistance. However, the precise role(s) of CDA in mediating β-lactam resistance as well as other vital processes of S. aureus is currently unknown. These functional alterations of PBP4 and GdpP likely come at the cost of bacterial virulence due to depletion of cell wall associated proteins and attenuated production of cytolysins, respectively. This indicates a unique yin-yang relationship between two key pathogenic factors of S. aureus, β-lactam resistance and virulence. We will investigate the fundamental basis of the functional changes in PBP4 and GdpP that lead to resistance and their impact on bacterial virulence. Aim 1: To determine the mechanism of PBP4-mediated β-lactam resistance and the role of PBP4 in cell wall composition. The relative contribution of PBP4 missense and promoter mutations on cell wall synthesis will be evaluated biochemically and structurally. The mechanism(s) that control pbp4 expression will be investigated to identify regulator(s) and to determine how they confer PBP4-mediated β-lactam resistance. PBP4's role on bacterial cell surface associated virulence factor expression will be determined. Aim 2: To define the role of cyclic-di-adenosine-mono-phosphate (CDA) signaling in S. aureus. Genetic and chemical proteomic approaches will be taken to identify CDA mediator/s in the bacteria that are responsible for β-lactam resistance and virulence defect. Finally, our preliminary data suggest the presence of a novel CDA specific phosphodiesterase in S. aureus besides GdpP. We will identify this novel phosphodiesterase. The proposed research will advance knowledge of basic cellular processes in S. aureus.