Novel Mechanisms of Beta-lactam Resistance in Staph Aureus

描述（由申请人提供）：我们发现了一种新的β-内酰胺耐药机制，该机制独立于青霉素酶和低亲和力青霉素结合蛋白（PBP）、PBP2a，这是金黄色葡萄球菌中已知的两种已知的β-内酰胺耐药机制。这种新类型的耐药性是在实验中发现的，在实验中，甲氧西林敏感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（头孢比普罗和头孢洛林）的存在下进行传代。 ceftobiprole 传代突变体的全基因组测序揭示了编码 PBP4 的基因发生突变，PBP4 是一种非必需的低分子量 PBP； GdpP，一种假定的信号蛋白；和 AcrB，一种假定的转运蛋白。 Ceftaroline 还选择了 PBP4 和 GdpP 突变体，但没有选择 AcrB 突变体，表明前两种蛋白的首要重要性。我们假设 1) 突变体 PBP4 获得转肽酶功能是高水平的 β-内酰胺抗性的原因； 2) GdpP 通过上调 pbp4 表达的信号通路促进耐药性。提出了两个具体目标来检验这些假设。目标 1：确定 pbp4 突变赋予高水平 β-内酰胺抗性的机制。 pbp4错义突变将在突变体中被修复或通过等位基因置换诱变引入亲本菌株中。将测试同基因菌株的β-内酰胺抗性，以确定重要的突变。将进行 PBP 结合测定和肽聚糖结构分析，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β-内酰胺酶）也将使用重组野生型和突变蛋白的模型底物进行。 X 射线晶体学将用于识别功能变化的结构基础，特别是与转肽酶活性相关的变化。目标 2：确定 gdpP 在介导 β-内酰胺抗生素反应中的作用。 GdpP 是一种假定的信号蛋白，具有针对最近发现的第二信使环二腺苷 (c-di-AMP) 的磷酸二酯酶活性。 gdpP 突变与 pbp4 表达增加和β-内酰胺抗性相关。我们假设这些突变通过 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，通过 gdpP 突变或抑制 dacA（编码产生 c-di-AMP 的二腺苷酸环化酶）的表达来控制 c-di-AMP 的细胞内浓度，并确定对 pbp4 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确定其调节途径中潜在的下游蛋白质。重组 GdpP 还将通过 X 射线晶体学进行纯化和分析，以确定其关键结构特性。实现这些目标将增加对β-内酰胺抗生素作用和耐药机制的了解。

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.