Pathogenic Contributions of Clostridium perfringens NanI Sialidase

项目摘要 产气荚膜梭菌是人类和牲畜疾病的主要原因，起源于肠道 并且涉及肠炎和/或肠毒血症，其中C.产气荚膜杆菌在肠内生长并产生毒素 被吸收到血液循环中，然后损害大脑等器官。肠粘连和 生长在C.产气荚膜杆菌肠道感染，特别是当这些疾病可以是慢性的， 例如，在一个实施例中，腹泻相关性腹泻。我们发表的研究表明，C。A型和C型产气荚膜杆菌 引起人类慢性肠道感染的菌株产生NanI唾液酸酶。这些NanI+肠道疾病 菌株对Caco-2人肠细胞样细胞的粘附性也比NanI-C更强。产气荚膜杆菌菌株 引起急性肠道疾病。使用nanI无效突变体和两种NanI+肠杆菌的互补菌株， 疾病菌株，我们表明NanI生产对于那些NanI+菌株粘附于Caco-2人 肠细胞样细胞。使用相同的菌株，获得了额外的数据，表明NanI+肠道疾病 容易的菌株可以通过使用NanI从宿主来源获得唾液酸来生长，这在肠道中可能是重要的。 伊内斯。最后，我们发现唾液酸酶抑制剂siastatin B降低了NanI+菌株对Caco-2细胞的粘附。 鉴于我们强有力的初步数据，我们假设，i）NanI是肠道炎症的重要贡献者- NanI+ C引起的感染。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。目前的提案将直接测试这些hypo- 论文具体而言，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NanI+ C的体内（肠）附着和毒力。产气荚膜杆菌肠道疾病菌株。这项工作 将使用NanI+ C。产气荚膜杆菌肠道疾病菌株，它们的同基因nanI无效突变体和互补 菌株以解决NanI是否增强NanI+ C的肠粘附。产气荚膜菌性肠病 新开发的小鼠口服攻击模型中的菌株。目的2将探索NanI是否可以支持体外 和NanI+ C的体内生长。产气荚膜杆菌肠道疾病菌株。这些研究也将使用野生型 NanI+肠道疾病菌株、它们的同基因nanI无效突变体和互补菌株，以测试NanI原 微粒，i）使用从Caco-2细胞去除的唾液酸的NanI+肠道疾病菌株的体外生长，或 粘液蛋白Muc-2，作为生殖相关的唾液酸来源，或ii）这些的体内生长/存活 小鼠肠袢模型中的菌株。最后，基于我们的体外Caco-2研究，Aim 3将测试NanI是否 唾液酸酶抑制剂可降低NanI+ C的粘附、生长/存活和毒力。产气荚膜杆菌性小肠疾病 缓解目标1和2中使用的小鼠模型中的品系。拟议的研究，包括体内 工作，代表了逻辑的下一步，建立在强大的以前的工作，并将探索创新的潜力， 唾液酸酶抑制剂作为对抗几种重要肠道感染的新治疗方法的初步用途。

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.