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: 8586251
• 负责人: Henry F HENRY CHAMBERS
• 金额: $ 67.26万
• 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/8586251
• 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）：我们发现了一种新的耐药机制？-不依赖于青霉素酶和低亲和力青霉素结合蛋白（PBP）（PBP 2a）的内酰胺类，这两种已知的？金黄色葡萄球菌内酰胺耐药性。这种新型的耐药性是在对甲氧西林敏感的沙门氏菌的实验中发现的。金黄色葡萄球菌菌株在两种所谓的“第五代”抗MRSA头孢菌素头孢吡普和头孢洛林中的每一种存在下传代。头孢吡普传代突变体的全基因组测序显示编码PBP 4（一种非必需的低分子量PBP）、GdpP（一种推定的信号蛋白）和AcrB（一种推定的转运蛋白）的基因发生突变。头孢洛林还选择PBP 4和GdpP突变体，但不选择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PBP 4获得转肽酶功能是高水平？内酰胺抗性;和2）GdpP通过上调pbp 4表达的信号传导途径促成抗性。提出了两个具体的目标来测试这些假设。目的1：确定pbp 4突变赋予高水平？内酰胺抗性。PBP 4错义突变将在突变体中修复或通过等位基因置换诱变引入亲本菌株。将对等基因菌株进行？-内酰胺耐药性，以确定突变的重要性。将进行PBP结合试验和肽聚糖结构分析，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。结合和酶活性测定，包括？-内酰胺酶，也将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，特别是与转肽酶活性相关的功能变化。目的2：确定gdpP在介导？内酰胺类抗生素GdpP是一种推定的信号蛋白，其具有针对环二腺苷单磷酸（c-di-AMP）（最近鉴定的第二信使）的磷酸二酯酶活性。gdpP突变与pbp 4表达增加和对？内酰胺。我们推测这些突变通过GdpP磷酸二酯酶活性的丧失导致细胞内c-di-AMP的积累。为了检验这一假设，c-di-AMP的细胞内浓度将通过突变gdpP或通过抑制dacA的表达来操纵，dacA编码产生c-di-AMP的二腺苷酸环化酶，并确定对pbp 4表达的影响。由于GdpP是一种信号分子，因此将进行微阵列研究以鉴定其调控途径中的潜在下游蛋白。重组GdpP也将被纯化，并通过X射线晶体学分析，以确定其关键的结构特性。实现这些目标将增加知识？内酰胺类抗生素的作用和耐药机制。