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。肺炎链球菌不仅是引起肺炎的主要细菌，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，可能是世界上导致幼儿死亡的最大单一杀手。虽然肺炎链球菌是人类最重要的病原体之一，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。传统的微生物检测方法不仅费时费力，而且有很多局限性。迫切需要对肺炎球菌疾病进行新的实验室诊断测试，因为目前诊断测试的局限性阻碍了对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不利影响，而且对控制措施的有效性的评估也产生不利影响。适配子是一种小的单链核酸，它以高亲和力结合分子或细胞靶标，其生化特性在各种分析、诊断和潜在的治疗应用中可与抗体相媲美或优于抗体，特别是在生产简单性、批次一致性、保质期和成本方面。在这项为期两年的探索性拨款申请中，我们建议开发针对两种主要肺炎球菌毒力因子的适配子，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致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的胞外结构域，以及PSPA结合载脂蛋白和全乳铁蛋白(LF)和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配子，特殊目标2是定位毒力因子中适配子结合的位置，并开发适配子信标用于检测毒力因子。这项拟议的研究将为探索使用适配子开发肺炎球菌感染诊断试验的想法奠定基础，并为可视化肺炎球菌的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致病机制中的重要作用，以及开发的适配子中和两个最重要的毒力因子的能力，拟议的研究还将为验证抗毒力策略作为对抗肺炎球菌对经典抗生素耐药性的新的替代/补充策略的假说奠定基础。 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来源，而且还会引起额外的危及生命或侵袭性感染。这项拟议的研究将为探索使用适配子来开发肺炎球菌感染的诊断试验和可视化肺炎球菌发病机制的研究工具以及验证作为治疗中耳炎和其他肺炎球菌感染的潜在新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.