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抗？-内酰胺不依赖于青霉素酶和低亲和力青霉素结合蛋白（PBP）， PBP2a，两种已知的？金黄色葡萄球菌-内酰胺耐药性。这种新型耐药性是在实验中发现的，在实验中，对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——头孢双prole和头孢他林——的存在下传代。ceftobiprole传代突变体的全基因组测序显示，编码PBP4（一种非必需的低分子量PBP）的基因发生了突变；GdpP，一种假定的信号蛋白；以及AcrB，一种假定的转运蛋白。头孢他林也选择了PBP4和GdpP突变体，但没有选择AcrB突变体，这表明前两种蛋白的首要重要性。我们假设1)突变体PBP4的转肽酶功能的增加可以解释高水平？内酰胺阻力;2) GdpP通过上调pbp4表达的信号通路促进抗性。为了验证这些假设，提出了两个具体的目标。目的1：确定pbp4突变导致高水平？内酰胺阻力。Pbp4错义突变可以在突变体中修复或通过等位基因置换诱变引入亲本菌株。等基因菌株将进行测试？-内酰胺抗性鉴定突变的重要性。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，并测试羧基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，也将与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结构基础。目标2：确定gdp在调节对？内酰胺抗生素。GdpP是一种被认为具有磷酸二酯酶活性的信号蛋白，可抵抗最近发现的第二信使环二磷酸腺苷（c-di-AMP）。gdpP的突变与pbp4的表达增加和对-内酰胺的抗性有关。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-二- amp的积累。为了验证这一假设，细胞内c-di-AMP的浓度将通过gdpP突变或抑制dacA的表达来控制，dacA编码产生c-di-AMP的二腺苷酸环化酶，并确定对pbp4表达的影响。由于GdpP是一种信号分子，我们将通过芯片研究来识别其调控通路中潜在的下游蛋白。重组GdpP也将通过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？-内酰胺类抗生素的作用及耐药机制。

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.