Enterococcus faecium virulence: A molecular and therapeutic approach

描述（由申请人提供）：我的近期目标是在导师阶段获得分子和转化研究方面的必要技能和专业知识，以在抗生素耐药性和细菌分子发病机制领域发展职业过渡到独立。这项工作的长期目标是将与毒力和发病机制相关的体外和体内观察转化为新的有用策略，以在抗生素耐药性时代对抗人类严重的肠球菌感染。屎肠球菌（EFM）由于对多种抗生素的耐药性，已成为美国医院最难治疗的微生物之一。先前的研究已经确定了质粒编码的推定透明质酸酶基因，该基因仅在医院分离的Efm中发现，并且在美国分离株中也更常见（与欧洲分离株相反）。透明质酸酶组是革兰氏阳性菌中众所周知的致病决定因素。在小鼠腹膜炎模型中的实验证实，含有hyl样质粒的EFM比缺乏该质粒的同基因分离株显著更致命。在相同的动物模型中减弱Efm hyl缺失突变体。抗“Hyl”抗体已显示在动物模型中对Efm的攻击具有保护性。该提议的假设是：i）hyl样基因（或质粒的一些其他组分）有助于最近观察到的Efm在医院获得性感染中的成功，以及ii）毒力决定因素（如“Hyl”，如果其在毒力中的作用被证实的话）可以用作开发新的治疗方法的靶标。本项目指导部分的具体目的是明确hyl样基因在Efm毒力中的作用和贡献。本提案的独立部分的目的包括：i）在动物模型中使用翻译工具评估治疗Efm感染的抗体-抗生素方法，以及ii）探索含hyl质粒中除hyl以外的基因在毒力中的可能作用（如果hyl不能解释质粒的所有毒力特性）。这些目标将通过开发用于Efm分离株的遗传操作的工具和在动物模型中测试突变体来描述hyl敲除（或其他适当的基因）在毒力中的作用来解决。将使用导师实验室建立的动物模型（小鼠腹膜炎和大鼠感染性心内膜炎）的转化方法来探索治疗方法。

DESCRIPTION (provided by applicant): My immediate goal is to acquire the necessary skills and expertise in molecular and translational research during the mentor phase to develop a career transition to independence in the area of antibiotic resistance and molecular pathogenesis of bacteria. The long term goals of this work are to translate in vitro and in vivo observations relating to virulence and pathogenesis into new and useful strategies to combat severe enterococcal infections in humans in the era of antibiotic resistance. Enterococcus faecium (Efm) have emerged as one of most difficult organisms to treat in US hospitals due to resistance to multiple antibiotics. Previous research has identified a plasmid-encoded putative hyaluronidase gene which is exclusively found in hospital isolates of Efm and is also more frequently present in isolates from the USA (as opposed to European ones). The hyalurodinase group of enzymes are well known pathogenic determinants in Gram-positive bacteria. Experiments in a mouse peritonitis model confirmed that an Efm containing the hyl-like plasmid was significantly more lethal than isogenic isolates lacking this plasmid. An Efm hyl-deletion mutant was attenuated in the same animal model. Anti-"Hyl" antibodies have been shown to be protective against challenge of Efm in animal models. The hypotheses for this proposal are: i) the hyl-like gene (or some other component of the plasmid) contributes to the recently observed success of Efm in hospital acquired infections and ii) virulence determinants (like "Hyl", if its role in virulence is confirmed) could be used as targets to develop novel therapeutic approaches. The specifc aim for the mentored part of this project is to define the role and contribution of the hyl-like gene to virulence in Efm. The aims for the independent part of this proposal include to: i) evaluate an antibody-antibiotic approach to treat Efm infections using translational tools in animal models and also, ii) explore the possible role in virulence of genes other than hyl in the hyl-containing plasmid (if hyl turns out not to explain all the virulence properties of the plasmid). These aims will be addressed by means of developing tools for genetic manipulation of Efm isolates and testing the mutants in animal models to characterize the role of hyl-knockouts (or other genes as appropriate) in virulence. A translational approach using the mentor's laboratory established animal models (mouse peritonitis and rat infective endocarditis) will be utilized to explore therapeutic approaches.