Novel Mechanisms of Beta-lactam Resistance in Staph Aureus

金黄色葡萄球菌β-内酰胺耐药的新机制

• 批准号: 8455851
• 负责人: Henry F HENRY CHAMBERS
• 金额: $ 27.8万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-12-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8455851
• 关键词: Accounting; Affinity; Antibiotics; Binding; Biological Assay; Carboxypeptidase; Cell Wall; Cephalosporins; Crystallography; Data; Epidemic; FDA approved; Gene Expression; Generations; Genes; Goals; Infection; Knowledge; Lactamase; Lactams; Lead; Mediating; Methicillin; Methicillin Resistance; Missense Mutation; Modeling; Molecular Weight; Monobactams; Muramoylpentapeptide Carboxypeptidase; Mutagenesis; Mutation; Parents; Penicillin-Binding Proteins; Penicillins; Peptidoglycan; Peptidyltransferase; Phenotype; Production; Property; Protein Binding; Proteins; Protocols documentation; Recombinants; Regulatory Pathway; Research; Resistance; Role; Second Messenger Systems; Signal Pathway; Signaling Molecule; Signaling Protein; Staphylococcus aureus; Structure; Testing; Variant; Work; X-Ray Crystallography; base; beta-Lactam Resistance; bindin; design; genome sequencing; in vitro Assay; methicillin resistant Staphylococcus aureus; mutant; novel; novel therapeutic intervention; phosphoric diester hydrolase; public health relevance; repaired; research study; resistance mechanism; resistant strain; response; second messenger

DESCRIPTION (provided by applicant): We have discovered a novel mechanism of resistance to ?-lactams that is independent of penicillinase and the low affinity penicillin bindin protein (PBP), PBP2a, the two known mechanisms of ?-lactam resistance in Staphylococcus aureus. This new type of resistance was identified during experiments in which methicillin- susceptible S. aureus strains were passaged in the presence of each of the two so-called "fifth generation" anti-MRSA cephalosporins, ceftobiprole and ceftaroline. Whole genome sequencing of a ceftobiprole- passage mutant revealed mutations in genes encoding PBP4, a non-essential, low-molecular weight PBP; GdpP, a putative signaling protein; and AcrB, a putative transporter. Ceftaroline also selected for PBP4 and GdpP mutants, but not AcrB mutants, indicating the primary importance of the former two proteins. We hypothesize 1) that a gain of transpeptidase function by mutant PBP4 accounts for high-level ?-lactam resistance; and 2) that GdpP contributes to resistance via a signaling pathway that up-regulates expression of pbp4. Two specific aims are proposed to test these hypotheses. Aim 1: To determine the mechanism by which mutations in pbp4 confer high-level ?-lactam resistance. pbp4 missense mutations will be repaired in mutants or introduced into parent strains by allelic replacement mutagenesis. Isogenic strains will be tested for ?-lactam resistance to identify mutations of importance. PBP binding assays and analyses of peptidoglycan structure will be performed to determine the effect of mutations on PBP binding and to test for functional changes in carboxypeptidase or transpeptidase activities. Binding and enzymatic activity assays, including ?-lactamase, also will be conducted with model substrates for recombinant wild-type and mutant proteins. X-ray crystallography will be used to identify the structural basis of functional changes, particularly those associated with transpeptidase activity. Aim 2: To determine the role of gdpP in mediating response to ?-lactam antibiotics. GdpP is a putative signaling protein that has phosphodiesterase activity against cyclic diadenosine monophosphate (c-di-AMP), a recently identified second messenger. Mutations in gdpP were associated with increased expression of pbp4 and with resistance to ?-lactams. We hypothesize that these mutations lead to intracellular accumulation c-di-AMP through loss of GdpP phosphodiesterase activity. To test this hypothesis intracellular concentrations of c-di-AMP will be manipulated by mutation of gdpP or by inhibition of expression of dacA, which encodes the diadenylate cyclase that generates c-di-AMP, and effects on pbp4 expression determined. As GdpP is a signaling molecule, microarray studies will be conducted to identify potential downstream proteins in its regulatory pathway. Recombinant GdpP also will be purified and analyzed by x-ray crystallography to identify its critical structural properties. Achieving these aims will increase knowledge of ?-lactam antibiotic effects and mechanisms of resistance.

描述（由申请人提供）：我们发现了一种对lactams的耐药机制，该机制独立于青霉酶和低亲和力青霉蛋白结合素蛋白（PBP），PBP2A，这是葡萄球菌葡萄球菌中的两种已知机制。在实验中鉴定了这种新型的抗性，其中在两个所谓的“第五代”抗Mrsa头孢菌素，ceftobipolole和ceftaroline的情况下，甲氧基易感性金黄色葡萄球菌菌株被转移。 Ceftobrole-传递突变体的整个基因组测序揭示了编码PBP4的基因突变，该基因是一种非必需的低分子重量PBP； GDPP，一种推定的信号蛋白；和ACRB，一种推定的运输车。 Ceftaroline还选择用于PBP4和GDPP突变体，而不是ACRB突变体，表明前两种蛋白质的主要重要性。我们假设1）突变体PBP4的转肽酶功能的增益是高水平的？-lactam抗性的； 2）GDPP通过信号通路上调PBP4表达的信号通路有效。提出了两个具体的目的来检验这些假设。目的1：确定PBP4中突变赋予高级耐药性的机制。 PBP4错义突变将在突变体中修复或通过等位基因替代诱变将其引入父菌株。异源性菌株将被测试吗？ - lactam耐药性以识别重要性的突变。将进行PBP结合测定和肽聚糖结构的分析，以确定突变对PBP结合的影响并测试羧肽酶或转肽酶活性的功能变化。结合和酶促活性测定，包括？ - 乳糖酶，也将使用重组野生型和突变蛋白的模型底物进行。 X射线晶体学将用于确定功能变化的结构基础，尤其是与转肽酶活性相关的变化。目标2：确定GDPP在介导对-lactam抗生素反应中的作用。 GDPP是一种推定的信号蛋白，它具有磷酸二酯酶活性，抗循环丁丁二磷酸（C-DI-AMP），这是最近被鉴定出的第二信使。 GDPP中的突变与PBP4的表达增加以及对lactams的抗性有关。我们假设这些突变通过丧失GDPP磷酸二酯酶活性导致细胞内积累C-DI-AMP。为了检验该假设的C-DI-AMP细胞内浓度，将通过GDPP突变或抑制DACA的表达来操纵，该表达编码会产生C-DI-AMP的二氨基甲基酸环化酶以及对确定PBP4表达的影响。由于GDPP是一种信号分子，将进行微阵列研究，以鉴定其调节途径中的潜在下游蛋白。重组GDPP还将通过X射线晶体学纯化和分析，以识别其关键结构特性。实现这些目标将增加对lactam抗生素效应和抗性机制的了解。