Novel Mechanisms of Beta-lactam Resistance in Staph Aureus

描述(申请人提供)：我们发现了一种新的金黄色葡萄球菌对β-内酰胺类抗生素耐药的机制，该机制不依赖于青霉素酶和低亲和力青霉素结合蛋白PBP2a，这两种机制是金黄色葡萄球菌对β-内酰胺类抗生素的已知耐药机制。这种新的耐药性是在对甲氧西林敏感的金黄色葡萄球菌菌株分别在头孢比洛尔和头孢他林这两种所谓的第五代抗MRSA头孢菌素存在下传代的实验中发现的。对一个头孢比洛尔通过的突变体的全基因组测序发现，编码PBP4、GdpP和AcrB的基因发生了突变。PBP4是一种非必需的低分子PBP，GdpP是一种假定的信号蛋白，AcrB是一种假定的转运蛋白。头孢他林也被选为PBP4和GdpP突变体，但不是AcrB突变体，这表明前两种蛋白的首要重要性。我们假设1)突变的pbp4获得转肽酶功能是导致高水平的β-内酰胺类耐药性的原因；2)gdpp通过上调pbp4的表达的信号通路参与了耐药性的产生。为了检验这些假说，本文提出了两个具体目标。目的1：探讨PBP4基因突变导致高水平β-内酰胺类抗生素耐药的机制。Pbp4错义突变将在突变株中修复或通过等位基因置换突变导入亲本菌株。将对同基因菌株进行β-内酰胺类耐药性测试，以确定重要的突变。将进行PBP结合分析和肽聚糖结构分析，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。结合和酶活性分析，包括β-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射线结晶学将被用来确定功能变化的结构基础，特别是那些与转肽酶活性相关的变化。目的：探讨gdpP在β-内酰胺类抗生素耐药中的作用。GdpP是一种可能的信号蛋白，具有针对新近发现的第二信使环二腺苷一磷酸(c-di-AMP)的磷酸二酯酶活性。Gdpp基因突变与PBP4表达增加和对β-内酰胺类抗生素耐药有关。我们假设这些突变通过丧失GdpP磷酸二酯酶活性而导致c-diAMP在细胞内积聚。为了验证这一假设，细胞内c-di-AMP的浓度将通过gdpP的突变或通过抑制DACA的表达来控制，DACA编码产生c-di-AMP的二腺苷环化酶，并确定对pbp4表达的影响。由于GdpP是一种信号分子，将进行微阵列研究，以确定其调控途径中潜在的下游蛋白。重组GdpP还将被提纯，并用X射线结晶学进行分析，以确定其关键的结构性质。实现这些目标将增加对β-内酰胺类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 β-lactams that is independent of penicillinase and the low affinity penicillin bindin protein (PBP), PBP2a, the two known mechanisms of β-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 β-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 β-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 β-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 β-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 β-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 β-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 β-lactam antibiotic effects and mechanisms of resistance.