Ampicillin resistance mechanisms in E. faecium

描述(由申请人提供)：多重耐药的粪肠球菌给医院感染治疗和控制带来了严重的问题。氨苄西林是对这种药物敏感的肠球菌菌株的首选治疗方法。粪肠球菌通过表达低亲和力的PBP5而对低水平的氨苄西林产生固有的耐药性。氨苄西林耐药性的提高与多溴联苯类药物中氨基酸的取代和蛋白质产量的增加有关。氨苄西林耐药性与治疗失败和感染肠球菌胃肠道定植的风险增加有关。在该项目的头三年中完成的工作已经确定了特定的氨基酸替代组合在PBP5介导的抗性中的作用。我们还提供了令人信服的证据，证明PBP5的增加是由于高抗性突变体特异性产生的扩散激活剂对双顺反子操纵子(PSR-PBP5)的转录激活所致。最后，我们已经证明，粪肠球菌可以通过激活另一条肽聚糖交联的途径获得独立于PBP5产生的氨苄西林耐药性。这种旁路机制需要增加细胞羧肽酶的活性，以提供L D-转肽酶的底物(四肽)，以取代所有的PBPs。在这次竞争性更新中，我们将在已经创建的突变体的晶体结构研究的基础上，继续我们对多溴联苯的结构-功能关系的分析。我们还将分离和鉴定与PSR上游结合的转录激活子，并更全面地探索另一个上游基因ftsWEfm参与氨苄西林耐药表达。我们将结晶和表征新型的L，D-转肽酶的结构，以破译这一新的酶类的催化机理。我们将研究两个假定的羧基肽酶在产生L，D转肽酶所需的四肽底物中的作用。最后，我们将确定在粪肠球菌基因组中鉴定的三种A型多氯联苯中的哪一种与多氯联苯合作合成成熟的肽聚糖。这些实验的成功完成将使人们更深入地了解粪肠球菌对氨苄西林产生耐药性的复杂机制，氨苄西林是一种耐药表型，对肠球菌在医院环境中的传播和重症患者的肠球菌感染的治疗具有重要意义。

DESCRIPTION (provided by applicant): Multi-resistant Enterococcus faecium cause significant problems for both nosocomial infection treatment and control. Ampicillin is the therapy of choice for enterococcal strains that are susceptible to this agent. E. faecium are intrinsically resistant to low levels of ampicillin through expression of low affinity PBP5. Increased ampicillin resistance has been associated with both amino acid substitutions within PBPS and increased production of the protein. Ampicillin resistance has been tied to therapeutic failures and to increased risk of acquiring enterococcal gastrointestinal colonization. Work accomplished during the first three years of the project has identified the role of specific combinations of amino acid substitutions in PBP5-mediated resistance. We have also provided compelling evidence that increased production of PBP5 results from transcriptional activation of a bi-cistronic operon (psr-pbp5) by a diffusible activator specifically produced by highly resistant mutants. Finally, we have shown that E. faecium can acquire ampicillin resistance independently from PBP5 production by the activation of an alternate pathway of peptidoglycan cross-linking. This by-pass mechanism requires increased cellular carboxypeptidase activity to provide the substrate (tetrapeptide) of the L,D-transpeptidase that substitute for all the PBPs. In this competitive renewal, we will continue our analysis of structure-function relationships of PBPS, based on crystal structure studies of mutants already created. We will also isolate and characterize the transcriptional activator that binds upstream of psr, and explore more fully the involvement of another upstream gene, ftsWEfm, in ampicillin resistance expression. We will crystallize and characterize the structure of the novel L,D-transpeptidase to decipher the catalytic mechanism of this novel enzyme class. We will investigate the role of two putative carboxypeptidases in creating the tetrapeptide substrate required for L,D transpeptidation. Finally, we will determine which of the three Type A PBPs identified within the E. faecium genome cooperates with PBPS in synthesizing mature peptidoglycan. Successful completion of these experiments will yield a deeper understanding of the complex mechanisms by which E. faecium become resistant to ampicillin, a resistance phenotype that has important implications for the spread of enterococci within the hospital environment and the treatment of enterococcal infections in seriously ill patients.