Pathogenic Contributions of Clostridium perfringens NanI Sialidase

项目摘要 产气荚膜梭菌是人类和家畜肠道疾病的主要原因。 并涉及肠炎和/或肠毒素血症，其中产气荚膜梭菌在肠道中生长并产生毒素 这些物质被吸收到循环中，然后损害器官，如大脑。肠粘连和 生长在产气荚膜梭菌肠道感染中起关键作用，特别是当这些疾病可以是慢性的， 例如，抗生素相关性腹泻。我们已发表的研究表明，A型和C型产气荚膜梭菌 引起慢性人类肠道感染的菌株产生纳米唾液酸酶。这些Nani+肠道疾病 菌株对Caco-2人肠细胞样细胞的黏附能力也比纳尼-C-产气荚膜杆菌菌株更强。 导致急性肠道疾病。利用Nani零突变体和两株Nani+肠道的互补菌株 结果表明，NaNi的产生对这些NaNi+菌株与Caco-2细胞的黏附至关重要。 肠细胞样细胞。使用相同的菌株，获得了更多的数据，表明NaNi+肠道疾病。 Ease菌株可以通过使用NaNi从宿主来源获得唾液酸来生长，这在试验中可能是重要的。 伊内斯。最后，我们发现唾液酸酶抑制剂siastatin B减少了NaNi+菌株对Caco-2细胞的黏附。 鉴于我们强劲的初步数据，我们假设，i)NaNi是肠道内的重要贡献者- NANI+产气荚膜梭菌引起的感染和II)影响NANI的抑制剂代表了一种潜在的新的 针对这些肠道感染的治疗方法。目前的提案现在将直接测试这些次要- 论文。具体地说，基于我们的体外Caco-2细胞研究，目标1将评估NaNi是否重要 用于纳尼+产气荚膜梭菌肠道疾病菌株的体内(肠道)附着和毒力。这部作品 将使用纳尼+产气荚膜梭菌肠病菌株，其等基因纳尼缺失突变体和补充 NANI是否增强NANI+产气荚膜梭菌肠道疾病肠道粘附性的菌株 一种新开发的小鼠口腔挑战模型中的菌株。Aim 2将探索NaNi是否可以支持体外 以及纳尼+产气荚膜梭菌肠道疾病菌株的体内生长。这些研究还将使用野生型 NANI+肠道疾病菌株及其同基因NANI缺失突变体和补体菌株检测NANI-PRO Motes，I)用从Caco-2细胞中去除的唾液酸体外生长NaNi+肠病菌株 粘液蛋白MUC-2，作为肠道相关唾液酸源，或II)这些细胞在体内的生长/存活 小鼠肠道循环模型中的菌株。最后，基于我们的体外Caco-2研究，Aim 3将测试是否有Nani 唾液酸酶抑制剂可降低纳尼+产气荚膜梭菌的黏附、生长/存活和毒力。 AIMS 1和AIMS 2中使用的小鼠模型中的易感菌株。拟议的研究，包括它们在体内的 工作，代表着顺理成章的下一步，以强大的先前工作为基础，并将探索创新潜力- 使用唾液酸酶抑制剂作为一种治疗几种重要肠道感染的新方法。

Project Summary Clostridium perfringens is a major cause of human and livestock diseases that originate in the intestines and involve enteritis and/or enterotoxemia, where C. perfringens grows in the intestines and produces toxins that are absorbed into the circulation and then damage organs such as the brain. Intestinal adherence and growth play critical roles in C. perfringens intestinal infections, particularly when these diseases can be chronic, e.g., antibiotic-associated diarrhea. Our published studies established that the C. perfringens type A and C strains causing chronic human intestinal infections produce NanI sialidase. These NanI+ intestinal disease strains are also more adherent to Caco-2 human enterocyte-like cells than are the NanI- C. perfringens strains causing acute intestinal disease. Using nanI null mutants and complementing strains of two NanI+ intestinal disease strains, we showed that NanI production is critical for those NanI+ strains to adhere to Caco-2 human enterocyte-like cells. Using the same strains, additional data was obtained suggesting that NanI+ intestinal dis- ease strains can grow by using NanI to obtain sialic acid from a host source, as may be important in the intest- ines. Last, we showed that the sialidase inhibitor siastatin B reduces NanI+ strain adherence to Caco-2 cells. Given our strong preliminary data, we hypothesize that, i) NanI is an important contributor to intestinal in- fections caused by NanI+ C. perfringens strains and ii) inhibitors affecting NanI represent a potentially novel therapeutic approach against these intestinal infections. The current proposal will now directly test these hypo- theses. Specifically, based upon our in vitro Caco-2 cell studies, Aim 1 will evaluate whether NanI is important for the in vivo (intestinal) attachment and virulence of NanI+ C. perfringens intestinal disease strains. This work will employ NanI+ C. perfringens intestinal disease strains, their isogenic nanI null mutants and complementing strains to address whether NanI enhances the intestinal adherence of NanI+ C. perfringens intestinal disease strains in a newly-developed mouse oral challenge model. Aim 2 will explore if NanI can support the in vitro and in vivo growth of NanI+ C. perfringens intestinal disease strains. These studies will also use wild-type NanI+ intestinal disease strains, their isogenic nanI null mutants and complementing strains to test if NanI pro- motes, i) the in vitro growth of NanI+ intestinal disease strains using sialic acid removed from Caco-2 cells or mucus protein Muc-2, as intestinally-relevant sialic acid sources, or ii) the in vivo growth/survival of these strains in a mouse intestinal loop model. Last, based upon our in vitro Caco-2 studies, Aim 3 will test if a NanI sialidase inhibitor can reduce the adhesion, growth/survival and virulence of NanI+ C. perfringens intestinal dis- ease strains in the mouse models used in Aims 1 and 2. The proposed studies, with their inclusion of in vivo work, represent the logical next step to build upon strong previous work and will explore the innovative poten- tial use of sialidase inhibitors as a novel therapeutic approach against several important intestinal infections.