Molecular bssis of mef-mediated antibiotic resistance in Streptococcus pneumoniae

描述（由申请人提供）：肺炎链球菌中出现mefE相关的大环内酯类耐药性的基础是遗传元件mega（大环内酯外流基因组装）。 Mega最初出现在肺炎球菌血清型（例如14、6B、9V、19F和6A）中，现在已出现在血清型（例如33F、19A）中，导致替换携带和疾病。 我们鉴定并表征了肺炎链球菌中的大遗传位点。 mefE 基因以单拷贝形式存在于肺炎链球菌基因组中 5.5 kb 或 5.4 kb 染色体插入片段的 5' 端。 至少已鉴定出四个巨噬细胞插入位点。 除了mefE之外，在mega中还发现了组织成两个聚合簇的其他四个开放阅读框（ORF）。 mefE 的紧下游是一个 ORF，命名为 mel，它与表皮葡萄球菌的红霉素抗性 ATP 结合盒 (ABC) 蛋白 MsrA 具有显着的同源性。 mega 的 3' 端包含三个聚合转录的 ORF（ORF5、ORF4、ORF3），与 47.5 kb 转座子 Tn5252 中的 ORF 同源。 在含有 mefE 的侵袭性肺炎链球菌分离株中，在流行病学和遗传上不相关的菌株的肺炎球菌基因组中至少有四个不同位点发现了 mega。 mefE和mel的基因产物都是大环内酯类抗药性所必需的，它们协同工作、协调调节，并且可由大环内酯类诱导。 目前已在肺炎链球菌的 Tn1207.1、Tn1207.3、Tn2009 中以及化脓性链球菌、B 族链球菌和其他革兰氏阳性菌中的相关元素中发现了巨量元素同系物。 在两个具体目标中，我们将进一步描述肺炎链球菌中 mef 介导的大环内酯类耐药的巨量元素和遗传基础。将检验两个假设：首先，mega 的 Mef 和 Mel 代表革兰氏阳性细菌中的一种新型外排泵，由两种不同的外排蛋白、ABC 膜结合转运蛋白和质子动力转运蛋白组成。其次，mega是与接合转座子相关的缺陷或“搭便车”遗传元件。 Tn5252 或其他接合转座子增强了 mega 的水平转移和外排活性。 此外，ORF 3-5通过能力诱导和诱导肺炎球菌SOS应激反应的药物（例如磺胺甲氧苄氨嘧啶、氟喹诺酮类药物）促进了mega的快速出现及其在肺炎链球菌中的持续传播。 在具体目标 1 中，将确定外排泵的分子基础、调节和底物特异性，并评估外排泵作为毒力因子的潜在作用。 在具体目标 2 中，将确定影响肺炎链球菌群体中巨量传播（水平转移）和流出功能的因素。这些研究应进一步深入了解这种新型遗传元件和外排泵传播的分子基础和机制，并更好地了解肺炎链球菌抗生素耐药性的出现。

DESCRIPTION (provided by applicant): The basis for the emergence of mefE-associated macrolide resistance in Streptococcus pneumoniae is a genetic element, mega (macrolide efflux genetic assembly). Mega initially emerged in pneumococcal serotypes (e.g., 14, 6B, 9V, 19F and 6A) and has now appeared in serotypes (e.g., 33F, 19A) causing replacement carriage and disease. We identified and characterized the mega genetic locus in S. pneumoniae. The mefE gene is found in single copy on the 5' end of a 5.5 kb or 5.4 kb chromosomal insert in the S. pneumoniae genome. At least four sites of insertion of mega have been identified. In addition to mefE, four other open reading frames (ORFs) organized into two convergent clusters are found in mega. Immediately downstream of mefE is an ORF, designated mel, that had significant homology to the erythromycin resistance ATP-binding cassette (ABC) protein, MsrA, of Staphylococcus epidermidis. The 3' end of mega contains three convergently transcribed ORFs (ORF5, ORF4, ORF3), homologous to ORFs in the 47.5 kb transposon Tn5252. In mefE-containing invasive S. pneumoniae isolates, mega is found in at least four distinct sites in the pneumococcal genome in epidemiologically and genetically unrelated strains. The gene products of mefE and mel are both required for macrolide resistance, work in concert, are coordinately regulated, and are inducible by macrolides. Mega element homologs have now been found in Tn1207.1, Tn1207.3, Tn2009 in S. pneumoniae and related elements in S. pyogenes, group B streptococci and in other Gram-positive bacteria. In two Specific Aims, we will further characterize the mega element and the genetic basis of mef-mediated macrolide resistance in S. pneumoniae. Two hypotheses will be examined: first, Mef and Mel of mega represent a novel efflux pump in Gram-positive bacteria composed of two distinct efflux proteins, an ABC membrane-bound transporter and a proton motive force transporter. Second, mega is a defective or "hitchhiker" genetic element related to conjugative transposons. The horizontal transfer and efflux activity of mega is enhanced by Tn5252 or other conjugative transposons. Also, the rapid emergence of mega and its continued spead in S. pneumoniae has been facilitated through ORFs 3-5 by competence induction and by agents, e.g., trimethoprim-sulfa, fluoroquinolones, that induce an SOS stress response in pneumococci. In Specific Aim 1, the molecular basis, regulation and substrate specificity of the efflux pump will be determined and the pump's potential role as a virulence factor assessed. In Specific Aim 2, the factors that impact the dissemination of mega (horizontal transfer) and efflux functions in the S. pneumoniae population will be determined. These studies should provide further insights into the molecular basis and mechanism(s) of dissemination of this novel genetic element and efflux pump and provide greater understanding of the emergence of antibiotic resistance in S. pneumoniae.