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。OM是幼儿的主要公共卫生问题，在美国和世界上都有巨大的经济负担。S.肺炎不仅是OM的主要细菌原因，而且还导致其他危及生命或侵入性感染。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S.肺炎球菌是人类最重要的病原体之一，肺炎球菌感染的实验室诊断仍然依赖于传统的微生物方法，很少有新的方法足以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试验不仅费力，而且有许多局限性。迫切需要新的肺炎球菌疾病实验室诊断测试，因为目前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，而且对控制措施的有效性评估也产生负面影响。适体是小的单链核酸，其以高亲和力结合分子或细胞靶标，并且在各种分析、诊断和潜在的治疗应用中，特别是在易于生产、批次均匀性、保质期和成本方面，其生物化学性质与抗体的生物化学性质相当或上级于抗体的生物化学性质。在这项探索性的两年拨款申请中，我们建议开发针对两种主要肺炎球菌毒力因子的适体，即胆碱结合蛋白A（CbpA，也称为PspC或SpsA）和肺炎球菌表面蛋白A（PspA）。这两种毒力因子是多结构域多功能蛋白，在肺炎球菌逃避宿主免疫和致病中起主要作用。其功能的分子基础是CbpA结合人补体因子H（FH）和pIgR的胞外域的能力以及PspA结合载脂蛋白和全乳铁蛋白（LF）并抑制补体沉积的能力。具体目标1是开发针对毒力因子的适体，具体目标2是绘制毒力因子中适体结合的位置并开发用于检测毒力因子的适体信标。拟议的研究将为探索使用适体开发肺炎球菌感染诊断测试的想法奠定基础，并为可视化肺炎球菌发病机制提供研究工具。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中和两种最重要的毒力因子的能力，拟议的研究还将为测试抗毒力策略作为对抗肺炎球菌对经典抗生素耐药性的新型替代/补充策略的假设搭建舞台。 公共卫生相关性：中耳炎或中耳感染是美国和世界上幼儿的主要公共卫生问题。肺炎链球菌不仅是中耳炎的主要细菌病因，而且还会引起其他危及生命或侵袭性感染。拟议的研究将为探索使用适体开发肺炎球菌感染诊断测试的想法和可视化肺炎球菌发病机制的研究工具以及测试抗毒力策略作为对抗中耳炎的新型潜在策略的假设奠定基础。和其他肺炎球菌感染。

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.