Pathogenic Contributions of Clostridium perfringens NanI Sialidase

产气荚膜梭菌 NanI 唾液酸酶的致病作用

• 批准号: 9163296
• 负责人: Jihong Li
• 金额: $ 18.98万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-17 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9163296
• 关键词: Acute; Address; Adherence; Adhesions; Affect; Anaerobic Bacteria; Antibiotics; Bacteria; Blood Circulation; Brain; Caco-2 Cells; Cell Adhesion; Cell Culture Techniques; Cell-Matrix Junction; Cells; Chronic; Classification; Classification Scheme; Clostridium perfringens; Complement; Data; Development; Diarrhea; Disease; Enterocytes; Enterotoxemia; Enterotoxins; Genes; Growth; Human; In Vitro; Infection; Intestinal Diseases; Intestinal Mucosa; Intestines; Livestock; Modeling; Muc 2 protein; Mucous body substance; Mus; Nature; Neuraminidase; Nutrient; Oral; Organ; Pathogenesis; Play; Process; Production; Publishing; Reproduction spores; Research; Role; Sialic Acids; Source; Testing; Therapeutic; Toxin; Virulence; Work; base; cell growth; enteritis; gut microbiota; in vivo; inhibitor/antagonist; innovation; mouse model; mutant; novel therapeutic intervention; pathogen; purge

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.

项目摘要 灌注梭状芽胞杆菌是人类和牲畜疾病的主要原因，起源于肠道 并涉及肠炎和/或肠毒性血症，肠道刺激在肠中生长并产生毒素 被吸收到循环中，然后损坏器官，例如大脑。肠道依从性和 生长在灌注肠道内感染中起关键作用，尤其是当这些疾病可能是慢性病时， 例如，抗生素相关的腹泻。我们已发表的研究确定，C. perfringens A型和C型 引起慢性人肠道感染的菌株会产生nani唾液酸酶。这些nani+肠道疾病 菌株也比纳米C菌株更坚持Caco-2人肠肠细胞样细胞 引起急性肠道疾病。使用NANI无效突变体并补充两个Nani+肠的菌株 疾病菌株，我们表明NANI的产生对于那些nani+菌株至关重要 肠细胞状细胞。使用相同的菌株，获得了其他数据，表明NANI+肠道疾病 轻松菌株可以通过使用NANI从宿主来源获得唾液酸来生长，这在最重要的情况下可能很重要 ines。最后，我们表明唾液酸酶抑制剂s抑菌蛋白B降低了NANI+菌株对CACO-2细胞的依从性。 鉴于我们的强大初步数据，我们假设，i）nani是肠内的重要贡献者 由NANI+ C.刺激性菌株和II）影响NANI的抑制剂代表了潜在的新型抑制剂，这可能是新颖的 针对这些肠道感染的治疗方法。当前的提案现在将直接测试这些不足 这些。具体而言，基于我们的体外CACO-2细胞研究，AIM 1将评估NANI是否重要 用于体内（肠道）的附着和NANI+ C.灌注肠疾病菌株的毒力。这项工作 将采用NANI+ C.灌注肠道疾病菌株，其等源性NANI NULL突变体和补充 菌株以解决NANI是否增强了Nani+ C.灌注肠疾病的肠道粘附 新开发的鼠标口服挑战模型中的菌株。 AIM 2将探索NANI是否可以支持体外 Nani+ C.灌注肠道疾病菌株的体内生长。这些研究也将使用野生型 NANI+肠道疾病菌株，其等源性NANI无效突变体，并补充菌株以测试NANI Pro-pro- motes，i）使用从Caco-2细胞或 粘液蛋白MUC-2，作为与无关紧要的唾液酸来源，或II）体内生长/存活 小鼠肠环模型中的菌株。最后，根据我们的体外CACO-2研究，AIM 3将测试NANI是否 唾液酸酶抑制剂可以降低Nani+ C.灌注肠道肠道的粘附，生长/存活和毒力 在目标1和2中使用的小鼠模型中的轻松菌株。拟议的研究，包括体内 工作，代表着建立在以前的强大工作的逻辑下一步，并将探索创新的潜在 将唾液酸酶抑制剂作为一种新型的治疗方法，以抗几种重要的肠道感染。