Streptococcal virulence factors in extra-oral disease

在美国，感染性心内膜炎每年影响大约22,000人，并导致超过2,000人死亡。这种疾病主要由绿僵菌引起。 链球菌，在绿色链球菌中，主要由链球菌 桑吉斯。关于链球菌毒力的先前研究 心内膜炎主要集中在可疑致病因素的检查上。 拟议的研究采取了一种完全不同的方法，即直接 确定新的致病因子。这将通过调整技术来实现 用于血链球菌的签名标记突变(STM)。这种方法 不需要事先假设任何特定基因的重要性 或疾病因果关系中的活动。相反，它依赖于感染过程 确定疾病所需的细菌基因及其相关基因 贡献。一种血链球菌菌株，也是另一种 正在进行的基因组测序项目将由STM使用一种 体外转位方法。由此产生的突变体将被汇集在一起 接种于作为心内膜炎模型的大鼠体内。STM方法将 允许在池中识别拥有增加的或 降低了毒力。这些突变体的明显毒力变化将是 通过体内竞争试验进行验证。突变体的进一步特征 将包括DNA序列分析以识别突变基因，进一步 在体内进行基因操作以验证突变基因的贡献 感染性试验和体外功能试验。据预计， 毒力改变的突变体的特征将导致更好的 了解心内膜炎的发病机制并将发现有希望的 疫苗或治疗开发的新靶点。

Infective endocarditis affects about 22,000 people per year in the U.S. and kills over 2,000. This disease is caused primarily by viridans streptococci, and among viridans streptococci, primarily by Streptococcus sanguis. Previous studies concerning the virulence of streptococci for endocarditis have centered upon the examination of suspected virulence factors. The proposed research takes an entirely different approach, that of directly identifying new virulence factors. This will be done by adapting the technique of signature-tagged mutagenesis (STM) for use with S. sanguis. This approach requires no prior assumptions concerning the importance of any particular gene or activity in disease causation. Instead, it relies on the infection process to identify the bacterial genes needed for disease and their relative contributions. An S. sanguis strain that is also the subject of a separate ongoing genome sequencing project will be randomly mutagenized by STM using an in vitro transposition approach. The resulting mutants will be pooled and inoculated into rats serving as models for endocarditis. The STM approach will allow for the identification of mutants within the pool possessing increased or decreased virulence. The apparent altered virulence in these mutants will be verified by in vivo competition assays. Further characterization of mutants will include DNA sequence analysis to identify the mutated genes, further genetic manipulation to verify the contribution of the mutated genes, in vivo infectivity assays, and in vitro functional assays. It is anticipated that the characterization of mutants with altered virulence will result in a better understanding of the pathogenesis of endocarditis and will identify promising novel targets for vaccine or therapeutic development.