Gastrointestinal Colonization of Diarrheagenic Clostridium difficile

描述(由申请人提供)： 我们目前的优秀奖集中在艰难梭菌毒力的影响因素上，已经确定了一些表层蛋白(SLP)，它们介导细菌与宿主肠道上皮细胞的黏附。我们现在建议大幅扩展这些研究，最终目标是将我们的发现转化为一种安全、易用、基于益生菌的干预措施，用于预防艰难梭菌感染(CDI)。提出了三个具体目标。首先，我们将确定表面层蛋白(SLP)介导的CD与宿主上皮细胞黏附的机制基础。CD诱变和CDI的动物模型将被用于这些研究。第二，我们将描述与CD定植有关的非SLP因素。细菌和宿主反应蛋白参与和贡献CD定植以及定植抗性将被研究。第三，我们将通过开发一种新的基于依从性的CDI干预方法，将我们的长凳研究结果转化为临床相关的结果。这将涉及通过工程益生菌(嗜酸乳杆菌)表达Cd定植蛋白SLpA来构建靶向Cd定植抑制物。我们将通过对从我们退伍军人医院进行的监测研究中发现的频繁分离的单发和复发的CD菌株进行测试，来验证上面开发的益生菌。对于本方案中的所有研究，我们将使用仓鼠和小鼠的急性CDI和CD定植模型。我们还将采用最先进的方法，包括自动质谱仪和活体动物生物发光成像，以跟踪摄入的CD孢子的命运。

DESCRIPTION (provided by applicant): Our current Merit Award focuses on factors contributing to virulence in Clostridium difficile, and a number of surface-layer proteins (SLPs) have been identified, that mediate bacterial adhesion to host intestinal epithelial cells. We now propose to significantly expand on these studies with the ultimate goal of translating our findings to a safe, easily utilizable, probiotic-based intervention for prevention of C. difficile infection (CDI). Three Specific Aims are proposed. First, we will define mechanistic bases of CD adherence to host epithelia mediated by surface-layer proteins (SLPs). CD mutagenesis and animal models of CDI will be exploited for these studies. Second, we will characterize non-SLP factors involved in CD colonization. Both bacterial and host- response proteins involved in, and contributing to, CD colonization as well as colonization resistance will be studied. Third, we will translate our bench-research findings to a clinically relevant outcome by developing a new adherence-based CDI intervention. This will involve construction of a targeted CD colonization inhibitor, by engineering a probiotic bacterium (Lactobacillus acidophilus) to express the CD colonization protein SlpA. We will validate the probiotic developed above by testing it against frequently isolated single-episode and relapse strains of CD recovered from surveillance studies performed at our VA hospital. For all studies in this proposal, we will use both the hamster and mouse models of acute CDI and CD colonization. We will also incorporate state-of-the-art methodologies including automated mass spectrometry and live-animal bioluminescence imaging to track the fate of ingested CD spores.