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

描述(申请人提供)：比其他任何人类病原体都多，肠球菌。由于对常用抗菌药，特别是对β-内酰胺类抗生素的耐药性，粪便杆菌的重要性日益增长。高层？粪肠球菌对内酰胺类抗生素的耐药不仅影响了细菌感染的治疗，而且促进了肠道定植和耐药菌株的进一步传播。粪肠球菌对β-内酰胺类抗生素高度耐药的特点是表达低亲和力的B类青霉素结合蛋白Pbp5。Pbp5作为一种只具有转肽酶活性的B类PBP，必须与糖基转移酶的活性相协调才能合成成熟的肽聚糖。B类PbP最常见的配位方式是双功能(同时具有糖基转移酶和转肽酶活性)A类PbP，在粪肠球菌中为PbpF、PbpZ和PONA。在这项资助的前一阶段进行的工作中，我们描述了粪肠球菌表达对β-内酰胺类抗生素耐药性的辅助和平行机制。我们已经删除了每个组合中的粪肠球菌A类PBPs，并发现PbpF和PONA的缺失导致了对头孢曲松的异质性敏感性(提示Pbp5与这两个A类PBPs中的任何一个配位使其对头孢曲松产生耐药性)，从中可以通过在头孢曲松上生长(自发突变体的选择)或通过接触青霉素来选择同质耐药性。PbpF的丢失也与粪肠球菌的自溶表型有关。我们还鉴定了一种L，D-转肽酶(LDTfm)，它能够在缺乏Pbp5的情况下与D，D-羧基肽酶的活性相结合地产生β-内酰胺类和万古霉素抗性。本提案将以下列方式继续这项重要的工作：1)我们将通过鉴定粪肠球菌肽多聚糖合成复合体来研究A类PBP缺失表型的机制，针对通过基因组搜索确定的可能的替代糖基转移酶，并利用微阵列分析在ponApbpF双突变体中青霉素诱导的头孢曲松耐药的调控框架2)我们将通过对PbpF的定点突变和对粪肠球菌D344R的功能研究来研究PbpF与头孢曲松-莫诺霉素协同作用的机制以及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β-内酰胺类药物抑制酶的惊人模式4)我们将使用蛋白质组学的方法来鉴定Ldtfm在多肽聚合中的合作伙伴的生理方面的特征。我们还将使用转录组分析来确定差异表达的基因，并进行全基因组测序，以确定所有导致通过Ldtfm途径表达高水平氨苄西林和万古霉素耐药性的突变。这些研究将确定和表征一种细菌的关键和多余的β-内酰胺耐药机制，在这种细菌中，β-内酰胺耐药对其在医院环境中的传播是绝对必要的。它们还将加深我们对革兰氏阳性球菌细胞壁合成机制的理解，并揭示抗菌治疗的新靶点。

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.