Gastrointestinal Colonization of Diarrheagenic Clostridium difficile

描述（由申请人提供）： 我们目前的优异奖侧重于艰难梭菌毒力的因素，并且已经确定了许多介导细菌粘附宿主肠上皮细胞的表面层蛋白（SLP）。我们现在建议大大扩展这些研究，最终目标是将我们的研究结果转化为一种安全，易于利用，基于益生菌的预防C。艰难梭菌感染（CDI）。提出了三个具体目标。首先，我们将定义CD粘附宿主上皮细胞介导的表面层蛋白（SLP）的机制基础。CD诱变和CDI动物模型将用于这些研究。其次，我们将描述CD定植中涉及的非SLP因素。将研究参与和促成CD定植以及定植抗性的细菌和宿主应答蛋白。第三，我们将通过开发一种新的基于依从性的CDI干预措施，将我们的实验室研究结果转化为临床相关结果。这将涉及通过工程化益生菌（嗜酸乳杆菌）以表达CD定殖蛋白SlpA来构建靶向CD定殖抑制剂。我们将通过对在我们VA医院进行的监测研究中回收的CD的频繁分离的单次发作和复发菌株进行测试来验证以上开发的益生菌。对于本提案中的所有研究，我们将使用急性CDI和CD定植的仓鼠和小鼠模型。我们还将采用最先进的方法，包括自动质谱和活动物生物发光成像，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.