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

描述（由申请人提供）：比任何其他人类病原体，肠球菌。由于粪菌对常用的抗菌剂，特别是-内酰胺类抗菌剂具有耐药性，其重要性日益提高。高层?粪肠杆菌表达的内酰胺耐药不仅影响细菌感染的治疗，还促进胃肠道定植和耐药菌株的进一步传播。高水平的标志是什么？粪肠杆菌的-内酰胺耐药是低亲和力B类青霉素结合蛋白Pbp5的表达。作为仅具有转肽酶活性的B类Pbp， Pbp5必须与糖基转移酶的活性协调才能合成成熟的肽聚糖。B类Pbps通常与双功能（同时具有糖基转移酶和转肽酶活性）A类Pbps配合，A类Pbps在粪肠杆菌中是PbpF、PbpZ和PonA。在此资助的前一阶段进行的工作中，我们描述了粪肠杆菌表达对？内酰胺抗生素。我们删除了每个组合中的屎肠杆菌A类pbps，并发现PbpF和PonA的删除导致对头孢曲松的异质易感（这表明Pbp5与这些A类pbps中的任何一种协同产生对头孢曲松的抗性），从中可以通过在头孢曲松上生长（选择自发突变体）或暴露于青霉素诱导产生同质抗性。PbpF的缺失也与粪肠杆菌的自溶表型有关。我们还鉴定并鉴定了一种L， d -转肽酶（Ldtfm），它能够赋予？-内酰胺和万古霉素耐药在缺乏Pbp5的情况下，与D，D-羧肽酶的活性一致。本提案将以下列方式继续这项重要的工作：1)我们将通过表征粪肠菌肽聚糖合成复合物来研究A类Pbp缺失表型的机制。我们将通过PbpF的定点诱变和E. faecium自溶酶的功能研究来研究PbpF对头孢曲松的协同作用机制，以及PbpF对E. faecium自溶表型的影响Ldtfm底物特异性的分子基础，以了解酶的抑制令人惊讶的模式？4)我们将利用蛋白质组学方法来识别肽聚糖聚合复合物中Ldtfm的伙伴，表征L， d -转肽化途径的生理方面。我们还将使用转录组分析来鉴定差异表达基因，并进行全基因组测序，以鉴定通过Ldtfm途径导致氨苄西林和万古霉素高水平耐药表达的所有突变。这些调查将确定关键和冗余的特征。-内酰胺耐药机制的细菌种类？-内酰胺耐药性对其在医院环境中的传播是绝对必要的。它们也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。

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.