Ampicillin resistance mechanisms in E. faecium

屎肠球菌的氨苄西林耐药机制

• 批准号: 6869221
• 负责人: Louis B. Rice
• 金额: $ 26.11万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 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 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6869221
• 关键词: Enterococcus; X ray crystallography; ampicillin; bacterial genetics; bacterial proteins; binding proteins; carboxypeptidase; drug resistance; enzyme mechanism; enzyme structure; gene expression; gene mutation; genetic regulation; genetic transcription; peptidoglycan; protein structure function; transpeptidation

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.

描述（申请人提供）：多重耐药屎肠球菌给医院感染的治疗和控制带来了重大问题。氨苄西林是肠球菌对这种药物敏感的首选治疗方法。粪肠杆菌通过表达低亲和力的PBP5对低水平氨苄西林具有内在抗性。氨苄西林耐药性的增加与PBPS内的氨基酸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肠球菌胃肠道定植风险增加有关。项目前三年完成的工作已经确定了氨基酸取代的特定组合在pbp5介导的抗性中的作用。我们还提供了令人信服的证据，表明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）的转录激活增加了PBP5的产生。最后，我们已经证明，通过激活肽聚糖交联的替代途径，粪肠杆菌可以独立于PBP5的产生获得氨苄西林耐药性。这种旁路机制需要增加细胞羧肽酶活性来提供L， d -转肽酶的底物（四肽）来替代所有的PBPs。在这个竞争性更新中，我们将在已经创建的突变体晶体结构研究的基础上继续分析PBPS的结构-功能关系。我们还将分离和表征结合psr上游的转录激活因子，并更全面地探索另一个上游基因ftsWEfm在氨苄西林耐药表达中的作用。我们将结晶和表征新型L， d -转肽酶的结构，以破译这种新型酶类的催化机制。我们将研究两种假定的羧基肽酶在产生L，D转肽化所需的四肽底物中的作用。最后，我们将确定在大肠杆菌基因组中鉴定的三种A型PBPs中的哪一种与PBPs合作合成成熟的肽聚糖。这些实验的成功完成将使我们更深入地了解粪肠杆菌对氨苄西林产生耐药性的复杂机制，这种耐药表型对肠球菌在医院环境中的传播和重症肠球菌感染的治疗具有重要意义。