C3-Binding and -Degrading Proteins in S. pneumoniae

描述(申请人提供)：肺炎链球菌仍是最主要的 社区获得性呼吸道感染的发病和死亡原因。 补体的第三组分C3是宿主的中心调节因子 对缺乏抗囊膜抗体的易感患者的防御。在过去的时间里 5年来，我们已经从肺炎链球菌中鉴定出两种C3降解酶：CPPA、 它降解C3β链；以及PHPA，它裂解C3α链 变成了以前无法辨认的碎片。两种蛋白水解酶都没有同源基因 这两个数据库都是由各种封装的临床数据表示的 分离株。重组rCppA减毒鼻腔免疫小鼠的研究 鼻咽定植与血清型3型生物。小鼠的免疫 使用rPhpA显著减少菌血症并提高存活率；在单独的 实验表明，rPhpA免疫比3型更有效。 结合疫苗在减少鼻咽定植中的作用。除 CPPA和PHPA在体内的保护作用，CPPA-和PHPA-突变体更多 在体外对C3介导的吞噬细胞的易感性高于同基因 家长。这项修订后的提案侧重于CPPA和PHPA 使肺炎链球菌能够逃脱C3在血液和肺部的杀伤作用。 在特定的目标一中，我们将描述蛋白质分解的机制，通过 CPPA利用发色底物和标准物质降解C3β链 蛋白酶抑制剂。在乳酸乳球菌中表达的截短结构 用于绘制活动站点的地图。包膜4型血清中的CPPA突变体 将会被建造。具体目标二将集中在PHPA，一种79 kDa的蛋白酶 这将C3阿尔法链切割成97和83 kDa的新片段。可能的 这些C3片段抑制C3或中性粒细胞的生物活性 接受化验。生化技术将被用来理解如何 全长PHPA释放出一个内部20 kDa的多肽，似乎 占C3裂解活动的大部分。一个phpa突变体在一个 将构建囊化的4型血清。具体目标三将使用 标准杀灭试验和双变异体以测试添加剂或协同作用 CPPA和PHPA的影响。血液和肺中的其他调理素(纤维连接蛋白) (表面活性蛋白A)将被评估为CPPA和 PHPA。特定目标四将在封装的CPPA和PHPA突变中使用 菌株了解CPPA和PHPA是否对C3介导的 在肺炎和菌血症的兔模型中，杀戮有助于致病。 这项修订后的提案将定义两种有效的免疫原在 肺炎链球菌的发病机制。

DESCRIPTION (provided by applicant): Streptococcus pneumoniae remains a leading cause of morbidity and mortality in community acquired respiratory infections. The third component of complement, C3, stands as the central mediator of host defense in susceptible patients who lack anti-capsular antibody. Over the past 5 years, we have identified two C3-degrading enzymes from S. pneumoniae: CppA, which degrades the C3 beta-chain; and PhpA, which cleaves the C3 alpha-chain into previously unrecognized fragments. Neither proteinase has any homolog in the database, and both are expressed by a wide variety of encapsulated clinical isolates. Intranasal immunization of mice with recombinant rCppA reduced nasopharyngeal colonization with a serotype 3 organism. Immunization of mice with rPhpA significantly reduced bacteremia and increased survival; in separate experiments, immunization with rPhpA was more effective than the serotype 3 conjugate vaccine in reducing nasopharyngeal colonization. In addition to the protective effects of CppA and PhpA in vivo, cppA- and phpA- mutants are more susceptible to C3-mediated opsonophagocytosis in vitro than is the isogenic parent. This revised proposal focuses on the mechanisms by which CppA and PhpA enable S. pneumoniae to elude C3-mediated killing in blood and lung. In Specific Aim One, we will characterize the mechanism of proteolysis by which CppA degrades the C3 beta-chain using chromogenic substrates and standard protease inhibitors. Truncation constructs expressed in Lactococcus lactis will be used to map the active site. A cppA- mutant in an encapsulated serotype 4 will be constructed. Specific Aim Two will focus on PhpA, a 79 kDa proteinase that cleaves the C3 alpha-chain into novel fragments of 97 and 83 kDa. Possible biologic activities of these C3 fragments in inhibiting C3 or neutrophils will be assayed. Biochemical techniques will be employed to understand how full-length PhpA liberates an internal 20 kDa polypeptide that appears to account for the majority of C3-cleaving activity. A phpA- mutant in an encapsulated serotype 4 will be constructed. Specific Aim Three will use a standard killing assay and a double mutant to test for additive or synergistic effects of CppA and PhpA. Other opsonins in blood (fibronectin) and lung (surfactant protein A) will be assessed as potential substrates for CppA and PhpA. Specific Aim Four will employ cppA- and phpA- mutants in the encapsulated strain to understand whether the effects of CppA and PhpA on C3-mediated killing contribute to virulence in a rabbit model of pneumonia and bacteremia. This revised proposal will define the role of two potent immunogens in pneumococcal pathogenesis.