Ampicillin resistance mechanisms in E. faecium

屎肠球菌的氨苄西林耐药机制

• 批准号: 7371920
• 负责人: Louis B. Rice
• 金额: $ 27.9万
• 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 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7371920
• 关键词: 5' Untranslated Regions; Affinity; Amino Acid Substitution; Ampicillin; Ampicillin Resistance; Binding; Bypass; Carboxypeptidase; Cell Wall; Cells; Class; Complex; Depth; Development; Enterococcus; Enterococcus faecalis; Enterococcus faecium; Environment; Enzymes; Failure; Funding; Gene Expression; Genes; Genetic Transcription; Genome; Hospitals; Infection; Investigation; Lactams; Mediating; Messenger RNA; Mutation; N-terminal; Nosocomial Infections; Open Reading Frames; Operon; Pathway interactions; Patients; Peptidoglycan; Peptidyltransferase; Phenotype; Predisposition; Production; Proteins; Regulation; Relative (related person); Resistance; Rice; Risk; Role; Structure; Structure-Activity Relationship; Testing; Theft; Therapeutic; Transcription Coactivator; Transcriptional Activation; Work; base; crosslink; gastrointestinal; glycosyltransferase; inhibitor/antagonist; mutant; novel; novel strategies; research study; resistance mechanism; 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（由申请方提供）：多重耐药屎肠球菌对医院感染治疗和控制造成重大问题。氨苄青霉素是对该药物敏感的肠球菌菌株的首选治疗药物。E.屎肠通过表达低亲和力PBP 5对低水平的氨苄青霉素具有内在抗性。增加的氨苄青霉素抗性与PBPS内的氨基酸取代和增加的蛋白质产量有关。氨苄青霉素耐药性与治疗失败和获得肠球菌胃肠道定植的风险增加有关。在该项目的前三年完成的工作已经确定了PBP 5介导的抗性中氨基酸取代的特定组合的作用。我们还提供了令人信服的证据表明，PBP 5的产量增加的结果从转录激活的双顺反子操纵子（psr-pbp 5）的扩散激活剂，特别是由高耐药突变体。最后，我们证明了E.屎肠菌可以通过激活肽聚糖交联的替代途径独立于PBP 5产生而获得氨苄青霉素抗性。这种旁路机制需要增加的细胞羧肽酶活性以提供L，D-转肽酶的底物（四肽）来替代所有的PBPs。在这个竞争性的更新，我们将继续我们的PBPS的结构-功能关系的分析，基于晶体结构的研究已经创建的突变体。我们还将分离和表征结合psr上游的转录激活因子，并更充分地探索另一个上游基因ftsWEfm在氨苄青霉素抗性表达中的参与。我们将结晶和表征新的L，D-转肽酶的结构，以破译这种新的酶类的催化机制。我们将研究两种假定的羧肽酶在产生L，D转肽所需的四肽底物中的作用。最后，我们将确定在E.粪菌基因组与PBPS协同合成成熟肽聚糖。这些实验的成功完成将使我们更深入地了解E。粪便变得对氨苄青霉素具有抗性，这是一种对医院环境内肠球菌的传播和重病患者中肠球菌感染的治疗具有重要意义的抗性表型。