Novel Mechanisms of Beta-lactam Resistance in Staph Aureus

描述(申请人提供)：我们发现了一种对β-内酰胺类抗生素耐药的新机制，该机制独立于葡萄球菌对β-内酰胺类抗生素的两种已知耐药机制：喷菌素酶和PBP2a。这种耐药是在对甲氧西林敏感的金黄色葡萄球菌菌株在所谓的第五代抗MRSA头孢菌素头孢比洛尔存在的情况下传代的实验中确定的。对一个抗药性突变体的全基因组测序发现，编码以下蛋白质的基因发生了突变：PBP4，一种非必需的低分子青霉素结合蛋白；GdpP，一种可能的信号蛋白；以及AcrB，一种可能的转运蛋白。我们假设，这些基因中的一个或多个突变会产生高水平的内酰胺类耐药性。为了验证这一假设，本文提出了三个具体目标。目的1：确定pbp4基因的哪些突变导致高水平的内酰胺类药物耐药。Pbp4错义突变将使用标准的等位基因替换突变方案进行修复；修复后的菌株将与亲本进行比较，以了解β-内酰胺类抗生素耐药性的变化。或者，将突变引入敏感的金黄色葡萄球菌菌株，以确定对敏感性的影响。？-内酰胺类抗生素结合和PBP4的酶功能也将被检测。目的：探讨gdpP在介导β-内酰胺类抗生素敏感性和耐药性中的作用。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。上述遗传学方法将被用来确定GdpP是否调节pbp4的表达，以及GdpP是否通过pbp4或其他机制影响抗性。目的3：鉴定介导β-内酰胺类抗生素耐药的其他非mecA基因。除pbp4和gdpP外，在抗性突变体的arcB中还发现了一个错义突变。它对抵抗的贡献尚不清楚。将进行等位基因替换实验，以确定AcrB是否调节pbp4的表达或影响抗性表型。将对另一种头孢比普利传代突变株SRB进行全基因组重测序，该突变株也有pbp4突变，但没有gdpP或AcrB突变，以确定导致耐药性的其他突变。总目标 这项研究的目的是确定β-内酰胺类药物非mecA非依赖性耐药的分子基础，从而更好地了解β-内酰胺类抗生素的抗菌作用，并确定新的药物靶点。

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.