Cell wall synthesis enzymes and beta-lactam resistance in Enterococcus faecium

屎肠球菌细胞壁合成酶和β-内酰胺耐药性

• 批准号: 8080880
• 负责人: Louis B. Rice
• 金额: $ 33.37万
• 依托单位: RHODE ISLAND HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8080880
• 关键词: Cell; wall; synthesis; enzymes; beta

DESCRIPTION (provided by applicant): More than any other human pathogen, Enterococcus. faecium has grown in importance as a result of its resistance to commonly used antimicrobial agents, in particular to the ?-lactams. High-level ? lactam resistance expressed by E. faecium not only compromises therapy of bacterial infections, it promotes gastrointestinal colonization and further dissemination of resistant strains. The hallmark of high-level ?-lactam resistance in E. faecium is the expression of low affinity class B penicillin-binding protein Pbp5. As a class B Pbp with only transpeptidase activity, Pbp5 must coordinate its activities with that of glycosyltransferases to synthesize mature peptidoglycan. Class B Pbps most commonly coordinate with bifunctional (possessing both glycosyltransferase and transpeptidase activities) class A Pbps, which in E. faecium are PbpF, PbpZ and PonA. In work performed during the previous period of this grant, we described auxiliary and parallel mechanisms by which E. faecium expresses resistance to ?-lactam antibiotics. We have deleted the E. faecium class A pbps in every combination and have discovered that deletion of PbpF and PonA results in a heterogeneous susceptibility to ceftriaxone (suggesting that Pbp5 coordinated with either of these class A Pbps to confer resistance to ceftriaxone), from which homogeneous resistance can be selected by growth on ceftriaxone (selection of spontaneous mutants) or induced by exposure to penicillin. Loss of PbpF is also associated with the autolytic phenotype of E. faecium. We have also identified and characterized an L,D-transpeptidase (Ldtfm) that is able to confer ?-lactam and vancomycin resistance in the absence of Pbp5, in concert with the activity of a D,D-carboxypeptidase. The present proposal will continue with this important work in the following manner: 1) We will investigate the mechanisms underlying the class A Pbp deletion phenotypes by characterizing the E. faecium peptidoglycan synthesis complex, targeting a likely alternative glycosyltransferases identified through a genome search and using microarray analysis to analyze the regulatory framework of penicillin-inducible ceftriaxone resistance in the ponApbpF double mutant 2) We will investigate the mechanisms underlying PbpF ceftriaxone-moenomycin synergism vs. E. faecium D344R and the impact of PbpF on the autolytic phenotype through site-directed mutagenesis of PbpF and functional studies of E. faecium autolysins 3) We will explore the molecular basis for the substrate specificity of Ldtfm to understand the surprising pattern of inhibition of the enzyme by ?-lactams 4) We will characterize the physiological aspects of the L,D-transpeptidation pathway using a proteomic approach to identifying partners of Ldtfm in the peptidoglycan polymerization complexes. We will also use transcriptome analysis to identify differentially expressed genes and perform whole genome sequencing to identify all of the mutations leading to expression of high levels of ampicillin and vancomycin resistance through the Ldtfm pathway. These investigations will identify and characterize critical and redundant ?-lactam resistance mechanisms in a bacterial species in which ?-lactam resistance is absolutely essential to its propagation in the hospital setting. They will also enhance our understanding of cell wall synthesis mechanisms in Gram-positive cocci and reveal promising new targets for antibacterial therapy. PUBLIC HEALTH RELEVANCE: Enterococcus faecium is a common and increasingly important cause of nosocomial infections. Its characteristic high level of resistance to ?-lactam antibiotics limits treatment and promotes dissemination. This project aims to identify and characterize the complex and redundant mechanisms of ?-lactam resistance in an effort to better understand enterococcal physiology and promote identification of novel therapeutic targets.

描述（由申请人提供）：肠球菌比任何其他人类病原体都多。由于其对常用抗菌剂的耐药性，特别是对？内酰胺。高级？内酰胺类耐药表现为E.屎肠菌不仅损害细菌感染的治疗，而且促进胃肠道定植和耐药菌株的进一步传播。高水平的标志？内酰胺类耐药大肠杆菌。屎肠是低亲和力B类青霉素结合蛋白Pbp 5的表达。Pbp 5是一种仅具有转肽酶活性的B类Pbp，必须与糖基转移酶协调其活性才能合成成熟肽聚糖。B类Pbp最常与双功能（具有糖基转移酶和转肽酶活性）的A类Pbp配位，其在E. pbpF、pbpZ和PonA。在本基金前期的工作中，我们描述了辅助和平行机制，通过这些机制E。屎肠对？内酰胺类抗生素我们删除了E。已经发现PbpF和PonA的缺失导致对头孢曲松的异质性易感性（表明Pbp 5与这些A类Pbp中的任一个协调以赋予对头孢曲松的抗性），由此可以通过在头孢曲松上生长（自发突变体的选择）或通过暴露于青霉素诱导来选择同质抗性。PbpF的缺失也与E.屎室我们还确定了L，D-转肽酶（Ldtfm），能够赋予？内酰胺和万古霉素耐药性，与D，D-羧肽酶的活性一致。本研究将以如下方式继续这一重要工作：1）我们将通过鉴定E.屎肠肽聚糖合成复合物，靶向通过基因组搜索鉴定的可能的替代糖基转移酶，并使用微阵列分析来分析ponApbpF双突变体中青霉素诱导的头孢曲松耐药性的调控框架。faecium D344 R的研究以及PbpF对自溶表型的影响。3）我们将探索Ldtfm底物特异性的分子基础，以了解？4）我们将使用蛋白质组学方法表征L，D-转肽途径的生理学方面，以鉴定肽聚糖聚合复合物中Ldtfm的配偶体。我们还将使用转录组分析来鉴定差异表达的基因，并进行全基因组测序，以鉴定通过Ldtfm途径导致高水平氨苄青霉素和万古霉素耐药表达的所有突变。这些调查将确定和描述关键和冗余的？内酰胺耐药机制的细菌物种，其中？内酰胺抗性对其在医院环境中的传播是绝对必要的。它们还将增强我们对革兰氏阳性球菌细胞壁合成机制的理解，并揭示有希望的抗菌治疗新靶点。 公共卫生相关性：屎肠球菌是医院感染的常见且日益重要的原因。它的特点是对？-内酰胺类抗生素限制了治疗并促进了传播。该项目旨在识别和表征？-内酰胺耐药性，以更好地了解肠球菌生理学并促进新治疗靶点的鉴定。