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耐药机制（或多种机制）？-葡萄球菌对β-内酰胺类抗生素的耐药性与葡萄球菌对β-内酰胺类抗生素的两种已知耐药机制（青霉素酶和PBP 2a）无关。这种类型的耐药性是在实验中发现的，其中甲氧西林敏感的S。金黄色葡萄球菌菌株在所谓的“第五代”抗MRSA头孢菌素头孢吡普存在下传代。耐药突变体的全基因组测序揭示了编码以下蛋白质的基因突变：PBP 4，一种非必需的低分子量青霉素结合蛋白; GdpP，一种推定的信号蛋白; AcrB，一种推定的转运蛋白。我们假设这些基因中的一个或多个突变赋予高水平的？内酰胺抗性。为了检验这一假设，提出了三个具体目标。目的1：确定pbp 4中的哪些突变赋予高水平？内酰胺抗性。pbp 4错义突变将使用标准等位基因置换诱变方案进行修复;修复的菌株将与亲本菌株比较？内酰胺抗性。或者，将突变引入易感的S。金黄色葡萄球菌菌株，以确定对敏感性的影响。?-还将测定PBP 4的内酰胺抗生素结合和酶功能。目的2：确定gdpP在介导？内酰胺类抗生素gdpP突变与pbp 4表达增加和对？-的耐药性相关内酰胺。上述遗传方法将用于确定GdpP是否调节pbp 4的表达以及GdpP是否通过pbp 4或其他机制影响抗性。目的3：鉴定介导？的其他非mecA基因。内酰胺类抗生素耐药性。除pbp 4和gdpP外，在arcB中还发现了一个错义突变。它对抵抗的贡献尚不清楚。将进行等位基因置换实验以确定AcrB是否调节pbp 4表达或影响抗性表型。将对另一种头孢吡普传代突变体SRB（也具有pbp 4突变，但没有gdpP或acrB突变）进行全基因组重测序，以鉴定导致耐药性的其他突变。总目标 这项研究的目的是确定mecA非依赖性耐药的分子基础。内酰胺，这可能会导致更好地了解？内酰胺类抗生素作用和新药物靶点的鉴定。

DESCRIPTION (provided by applicant): We have discovered a novel mechanism (or mechanisms) of resistance to ?-lactams that is independent of the two known mechanisms of staphylococcal resistance to beta-lactams: pencillinase and PBP2a. This type of resistance was identified during experiments in which a methicillin-susceptible S. aureus strain was passaged in the presence of a so-called "fifth generation" anti-MRSA cephalosporin, ceftobiprole. Whole genome sequencing of a resistant mutant revealed mutations in genes encoding the following proteins: PBP4, a non-essential, low-molecular weight penicillin-binding protein; GdpP, a putative signaling protein; and AcrB, a putative transporter. We hypothesize that mutations in one or more of these genes confer high-level ?-lactam resistance. To test this hypothesis, three specific aims are proposed. Aim 1: To determine which mutations in pbp4 confer high-level ?-lactam resistance. pbp4 missense mutations will be repaired using a standard allelic replacement mutagenesis protocol; repaired strains will be compared to the parent for changes in ?- lactam resistance. Alternatively, mutations will be introduced into susceptible S. aureus strains to determine the effect on susceptibility. ?-lactam antibiotic binding and enzymatic functions of PBP4 will also be assayed. Aim 2: To determine the role of gdpP in mediating susceptibility and resistance to ?-lactam antibiotics. Mutation in gdpP is associated with increased expression of pbp4 and with resistance to ?-lactams. The genetic approach described above will be used to determine whether GdpP regulates expression of pbp4 and whether GdpP impacts resistance by pbp4 or other mechanisms. Aim 3: To identify other non-mecA genes that mediate ?-lactam antibiotic resistance. Besides pbp4 and gdpP, a missense mutation was identified in arcB of the resistant mutant. Its contribution to resistance is not known. Allelic replacement experiments will be performed to determine whether AcrB regulates pbp4 expression or influences resistance phenotype. Whole genome resequencing of another ceftobiprole passage mutant, SRB, which also has pbp4 mutations but no mutations in gdpP or acrB, will be performed to identify other mutations that contribute to resistance. The overall goal of the proposed research is to define the molecular basis for mecA-independent resistance to ?-lactams, which could lead to a better understanding of ?-lactam antibiotic effects and identification of novel drug targets.