Targeting Pneumococcal Virulence Factors in Otitis Media

描述（由申请人提供）：拟议研究的长期目标是开发中耳炎（OM）和其他肺炎球菌感染的诊断工具以及研究肺炎球菌发病机制的研究工具。在美国和世界范围内，OM是幼儿的一个主要公共卫生问题，是一个巨大的经济负担。肺炎链球菌不仅是OM的主要细菌病因，而且还会引起其他危及生命或侵袭性感染。肺炎球菌感染是四大致命传染病之一，可能是世界上幼儿的最大杀手。尽管肺炎链球菌是最重要的人类病原体之一，但肺炎球菌感染的实验室诊断仍然依赖于传统的微生物学方法，很少有新的方法能够足够可靠地用于肺炎球菌感染的实验室诊断。传统的微生物学方法和测试不仅费力而且有许多局限性。目前迫切需要对肺炎球菌疾病进行新的实验室诊断测试，因为现有诊断测试的局限性阻碍了肺炎球菌感染的及时和准确诊断，这反过来不仅对疾病的治疗产生负面影响，而且对控制措施有效性的评估也产生负面影响。适配体是一种小的单链核酸，其结合分子或细胞靶标具有高亲和力，其生化特性在各种分析、诊断和潜在治疗应用中与抗体相媲美或优于抗体，特别是在易于生产、批量均匀性、保质期和成本方面。在这项为期两年的探索性资助申请中，我们建议开发针对两种主要肺炎球菌毒力因子的适配体，胆碱结合蛋白A （CbpA，也称为PspC或SpsA）和肺炎球菌表面蛋白A （PspA）。这两种毒力因子都是多结构域多功能蛋白，在肺炎球菌逃避宿主免疫和发病过程中发挥重要作用。其功能的分子基础是CbpA结合人补体因子H （FH）和pIgR外畴的能力，以及PspA结合载脂蛋白和全乳铁蛋白（LF）并抑制补体沉积的能力。特异性目标1是开发针对毒力因子的适配体，特异性目标2是绘制适配体与毒力因子结合的位置，并开发用于检测毒力因子的适配体信标。该研究将为探索利用适体开发肺炎球菌感染诊断测试的想法奠定基础，并为肺炎球菌发病机制的可视化提供研究工具。基于肺炎球菌毒力因子在肺炎球菌发病机制中的重要作用，以及所开发的适体对两种最重要的毒力因子的中和能力，本研究还将为检验抗毒力策略作为对抗肺炎球菌对经典抗生素耐药性的一种新的替代/补充策略的假设奠定基础。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to develop diagnostic tools for otitis media (OM) and other pneumococcal infections and research tools for studying pneumococcal pathogenesis. OM is a major public health problem in young children with an enormous economic burden in the US as well as in the world. S. pneumoniae not only is a major bacterial cause of OM but also causes additional life-threatening or invasive infections. Pneumococcal infection is one of the four killer infectious diseases and probably the world's single biggest killer of young children. Although S. pneumoniae is one of the most important human pathogens, the laboratory diagnosis of pneumococcal infections is still dependent on traditional microbiological methods and very few new methods are reliable enough for the laboratory diagnosis of pneumococcal infections. The traditional microbiological methods and tests not only are laborious but also have many limitations. There is an urgent need for new laboratory diagnostic tests for pneumococcal diseases, as the limitations of current diagnostic tests hinder the timely and accurate diagnosis of pneumococcal infections, which in turn negatively affects not only the treatment of the diseases but also the assessment of the effectiveness of control measures. Aptamers are small single-stranded nucleic acids that bind a molecular or cellular target with high affinity and their biochemical properties rival or are superior to those of antibodies in a variety of analytical, diagnostic, and potential therapeutic applications, particularly in ease of production, batch uniformity, shelf life, and cost. In this exploratory two-year grant application, we propose to develop aptamers against two major pneumococcal virulence factors, choline-binding protein A (CbpA, also known as PspC or SpsA) and pneumococcal surface protein A (PspA). Both virulence factors are multidomain multifunction proteins and play major roles in pneumococcal evasion of host immunity and pathogenesis. The molecular bases for their functions are the ability of CbpA to bind human complement factor H (FH) and the ectodomain of pIgR and the ability of PspA to bind apo- and holo-lactoferrin (LF) and to inhibit complement deposition. Specific Aim 1 is to develop aptamers against the virulence factors and Specific Aim 2 is to map where in the virulence factors the aptamers bind and develop aptamer beacons for the detection of the virulence factors. The proposed research will set up the stage for exploring the idea of using aptamers for the development of diagnostic tests for pneumococcal infections and provide research tools for visualizing pneumococcal pathogenesis. Based on the essential roles of pneumococcal virulence factors in pneumococcal pathogenesis and the ability of the developed aptamers to neutralize the two most important virulence factors, the proposed research will also set up the stage for testing the hypothesis of antivirulence strategy as a novel alternative/complementary strategy for combating pneumococcal resistance to classical antibiotics. PUBLIC HEALTH RELEVANCE:Otitis media, or middle ear infection, is a major public health problem in young children both in the US and in the world. Streptococcus pneumoniae not only is the major bacterial cause of otitis media but also causes additional life-threatening or invasive infections. The proposed research will set up the stage for exploring the idea of using aptamers for the development of diagnostic tests for pneumococcal infections and research tools for visualizing pneumococcal pathogenesis and for testing the hypothesis of antivirulence strategy as a novel potential strategy against otitis media and other pneumococcal infections.