Defining the bacterial factors that modulate sensitivity to B-lactam in CA-MRSA

描述(由申请人提供)：最近由社区获得的耐甲氧西林金黄色葡萄球菌(CA-MRSA)菌株在健康个人中引起的感染案例引起了全世界的关注。CA-MRSA菌株不同于医院获得的MRSA(HA-MRSA)，因为它们的基因组背景和动物模型中增强的毒力。金黄色葡萄球菌对甲氧西林的耐药性是由mecA介导的，mecA嵌入一个称为SCCmec的可移动遗传元件(21-67kb)中。在MRSA中有5种SCCmec类型，它们在mec复合体(mecI-mecR1-mecA)、整合质粒、转座子、IS元件和其他辅助基因方面存在差异。最常见的两株CA-MRSA分离株USA300和MW2(USA400)携带IV型SCCmec，而常见的HA-MRSA USA100和USA200携带II型。我们实验室最近的研究表明，青霉素结合蛋白4(PBP4)的缺失足以导致CA-MRSA对苯唑西林和那福西林的耐药性减少16倍，但在HA-MRSA中则不然。这一发现得到了头孢西丁的证实，头孢西丁是一种与PBP4不可逆结合的半合成β-内酰胺，它与苯唑西林对CA-MRSA菌株有协同杀伤作用，但对HA-MRSA菌株没有协同杀伤作用。由于所有MRSA菌株都含有mecA，这些数据表明编码PBP2a的mecA不是CA-MRSA对甲氧西林耐药的唯一决定因素。根据SCCmec类型的差异，我们推测，CA-MRSA菌株USA300和USA400的IV型SCCmec中的辅助基因在没有pbp4或有头孢西丁存在的情况下对苯唑西林(也称为奈福西林)具有敏感性。为了确定细菌决定因素，我们开发了两个目标：i)定位在没有PBP4或存在PBP4结合的β-内酰胺(如头孢西丁)存在的情况下，对苯唑西林/那福西林敏感的细菌决定因素；ii)鉴定在CA-MRSA菌株USA300和USA400中，在头孢西丁存在的情况下，导致苯唑西林敏感性的基因决定因素。通过这些研究，我们将确定IV型SCCmec中与这种表型有关的基因(目标1)。将构建USA300和USA400中这些基因的突变体，并从抗生素敏感性、细胞壁代谢和细胞壁形态(AIM II)方面对其进行鉴定。在这些研究完成后，我们试图确定影响CA-MRSA苯唑西林耐药性的基因决定因素。这些基因决定因素可能是新的抗菌疗法开发的靶点。公共卫生相关性：社区获得性耐甲氧西林金黄色葡萄球菌(CA-MRSA)已成为一个主要的健康问题。与医院获得的耐甲氧西林金黄色葡萄球菌相反，CA-MRSA的毒力更强，可以影响健康的人。我们在CA-MRSA中发现了一个“跟腱”，即青霉素结合蛋白4(PBP4)是CA-MRSA对甲氧西林耐药所必需的。在pbp4基因缺失的情况下，CA-MRSA中存在与苯唑西林敏感性有关的基因。我们的目标是识别和表征这些赋予苯唑西林敏感性的基因。

DESCRIPTION (provided by applicant): Recent cases of infections caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strains in healthy individuals have raised concerns worldwide. CA-MRSA strains differ from hospital-acquired MRSAs (HA-MRSA) by virtue of their genomic background and increased virulence in animal models. Resistance to methicillin in S. aureus is mediated by mecA which is embedded within a mobile genetic element (21-67 kb) called SCCmec. There are five types of SCCmec in MRSA, which differ with respect to the mec complex (mecI-mecR1-mecA), integrated plasmids, transposons, IS elements and other accessory genes. USA300 and MW2 (USA400), the two most common CA-MRSA isolates, carry the SCCmec type IV while USA100 and USA200, which are common HA-MRSAs, contain type II. Recent studies from our lab have shown that a loss of penicillin-binding protein 4 (PBP4) is sufficient to cause a 16-fold reduction in oxacillin and nafcillin resistance in CA-MRSA, but not in HA- MRSA. This finding was confirmed with cefoxitin, a semi-synthetic beta-lactam that binds PBP4 irreversibly, which exhibits synergistic killing with oxacillin against CA-MRSA strains but not against HA-MRSA strains. As all MRSA strains contain mecA, these data indicate that that mecA encoding PBP2A is not the sole determinant of methicillin resistance in CA-MRSA strains. Based on the differences in SCCmec types, we hypothesize that the accessory genes within SCCmec type IV in CA-MRSA strains USA300 and USA400 confer sensitivity to oxacillin (also nafcillin) in the absence of pbp4 or in the presence of cefoxitin. To define the bacterial determinants, we have developed two aims: I) mapping the bacterial determinants that confer sensitivity to oxacillin/nafcillin in the absence of pbp4 or in the presence of PBP4-binding beta-lactam such as cefoxitin; II) characterization of the genetic determinants that confer oxacillin sensitivity in the presence of cefoxitin in CA-MRSA strains USA300 and USA400. With these studies, we will identity the genes in type IV SCCmec responsible for this phenotype (Aim 1). Mutants of these genes in USA300 and USA400 will be constructed and characterized with respect to antibiotic sensitivity, cell wall metabolism and cell wall morphology (Aim II). Upon completion of these studies, we seek to identify genetic determinants that impact oxacillin resistance in CA-MRSA. These genetic determinants may be targets for the development of novel antimicrobial therapy. PUBLIC HEALTH RELEVANCE: Community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) has become a major health problem. Contrary to hospital-acquired methicillin resistant S. aureus, CA-MRSAs are more virulent and can affect healthy individuals. We have found an "Achilles Heel" in CA-MRSA in that penicillin binding protein 4 (PBP4) is required for methicillin resistance in CA-MRSA. There are genes in CA-MRSA involved in this oxacillin sensitivity upon deletion of the pbp4 gene. Our goal is to identify and characterize these genes that confer oxacillin sensitivity.