Molecular bssis of mef-mediated antibiotic resistance in Streptococcus pneumoniae

描述（由申请方提供）：肺炎链球菌中出现mefE相关大环内酯类耐药的基础是遗传元件mega（大环内酯类外排遗传组装）。 Mega最初出现在肺炎球菌血清型中（例如，14，6B，9V，19F和6A），并且现在已经出现在血清型（例如，33F，19A）造成更换运输和疾病。 我们确定并表征了S.肺炎。 mefE基因以单拷贝存在于S. pneumoniae基因组。 至少有四个网站的插入兆已被确定。 除了mefE，在mega中发现了组织成两个会聚簇的其他四个开放阅读框架（ORF）。 紧接着mefE的下游是ORF，命名为mel，其与表皮葡萄球菌的红霉素抗性ATP结合盒（ABC）蛋白MsrA具有显著的同源性。 mega基因的3'端含有3个转录一致的ORF（ORF5、ORF4、ORF3），与47.5kb转座子Tn5252的ORF同源。 在含mefE的侵袭性S.在流行病学和遗传学上不相关的菌株中，在肺炎球菌基因组的至少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药物抗性所需的，协同工作，协调调节，并可由大环内酯类药物诱导。 目前已在S. pneumoniae及其相关元件。化脓性链球菌、B组链球菌和其它革兰氏阳性菌。 在两个具体目标中，我们将进一步描述mef介导的大环内酯类耐药的mega元件和遗传基础。肺炎。两个假设将被检查：首先，Mef和梅尔的兆代表一种新的外排泵在革兰氏阳性菌组成的两个不同的外排蛋白，ABC膜结合转运蛋白和质子动力转运蛋白。其次，mega是一个与接合转座子相关的缺陷或“搭便车”遗传元件。 通过Tn5252或其他接合转座子增强mega的水平转移和外排活性。 此外，巨型的迅速崛起和它在S。通过感受态诱导和试剂，例如，磺胺甲氧苄氨嘧啶，氟喹诺酮类药物，它们在肺炎球菌中诱导SOS应激反应。 在特定目标1中，将确定外排泵的分子基础、调节和底物特异性，并评估泵作为毒力因子的潜在作用。 在具体目标2中，对影响超级（水平转移）和外排功能在S。将确定肺炎菌群。这些研究将为进一步了解这种新的遗传元件和外排泵的分子基础和传播机制提供进一步的见解，并为S.肺炎。

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.