NanI sialidase: Effects on Clostridium perfringens enterotoxin activity and contributions to C. perfringens type F infection

项目摘要 F型产气荚膜梭菌菌株是细菌性食物中毒（FP）的第二大常见原因 在美国，每年发生约100万例。这些细菌也会引起许多非食源性胡萝卜素的病例。 人类肠道疾病（NFD），如腹泻相关性腹泻。F型菌株的毒力为 需要产气荚膜梭菌肠毒素（CPE）的生产。所有F型疾病都是真正的感染， F型菌株最初在肠道中繁殖，但当它们在体内形成孢子时产生CPE;这种毒素 在母细胞裂解以释放其成熟孢子时释放到肠腔中。F型感染是 通常是一种肠道疾病，但也可能涉及致命的肠毒血症（其中肠内产生的CPE是 吸收以损害内部器官，如肝脏）。 除CPE外，所有F型NFD菌株和~50%的F型FP菌株产生一种分泌型唾液酸酶， 奶奶对于那些F型菌株，NanI是其主要的外唾液酸酶，并且这种唾液酸酶在两种菌株中都产生 营养培养物和孢子形成培养物（其中NanI与CPE共存）。纳尼正在成为一个 NanI+ F型菌株的重要毒力因子，例如，我们显示NanI唾液酸酶在体外有助于生长， 孢子形成和CPE产生以及持续的肠定植。我们还报道，i）NanI增强了 Caco-2细胞的CPE结合/细胞毒性，以及ii）与小肠液接触，如在F型肠溶期间发生的 感染后，蛋白水解将NanI加工成具有增加的唾液酸酶活性的60 kDa片段， 比天然NanI更大的促进肠细胞样Caco-2细胞的CPE结合/细胞毒性的能力。 在产生细胞表面唾液酸结合物的同时，Caco-2细胞产生最少的粘液，其是高度唾液酸化的 并在肠道中大量存在。因此，我们假设，i）NanI对促进CPE更有影响力 在大量粘液存在下的活性，如在肠中发生的，ii）这种作用通过蛋白水解增强 肠蛋白酶激活NanI，和iii）NanI促进F型感染/疾病。该项目将测试 使用CPE活性或F型感染/疾病的体外和体内模型的那些重要假设。要求1 将采用肠细胞样细胞培养模型，这些模型产生或不产生大量粘液， NanI或蛋白水解活化的NanI对促进CPE细胞毒性或CPE细胞旁转运的相对影响 (an肠毒血症期间肠内CPE吸收的体外替代物） 粘液。目标2将使用动物感染模型，即，兔和小鼠小肠袢肠炎模型 或肠毒血症，以测试NanI是否导致F型感染/疾病并表征NanI 可能导致毒力的影响，即，NanI是否增加CPE活性/转运和/或F型菌株生长， 孢子形成或CPE生产在肠道？如果NanI被证明能增强F型感染， 未来的翻译方法，即，使用唾液酸酶抑制剂来改善这些疾病。

Project Summary Clostridium perfringens type F strains are the 2nd most common cause of bacterial food poisoning (FP) in the USA, where ~1 million cases/year occur. These bacteria also cause many cases of nonfoodborne hu- man intestinal diseases (NFD), such as antibiotic-associated diarrhea. The virulence of type F strains re- quires production of Clostridium perfringens enterotoxin (CPE). All type F diseases are true infections where type F strains initially multiply in the intestines but then produce CPE when they sporulate in vivo; this toxin is released into the intestinal lumen upon lysis of the mother cell to free its mature spore. Type F infections are typically a diarrheal disease but can also involve lethal enterotoxemia (where CPE produced in the intestines is absorbed to damage internal organs like the liver) in patients with certain predisposing medical conditions. In addition to CPE, all type F NFD strains and ~50% of type F FP strains produce a secreted sialidase named NanI. For those type F strains, NanI is their predominant exosialidase and this sialidase is produced in both vegetative cultures and sporulating cultures (where NanI is co-present with CPE). NanI is emerging as an important virulence factor for NanI+ type F strains, e.g., we showed NanI sialidase contributes in vitro to growth, sporulation and CPE production and to persistent intestinal colonization. We also reported that, i) NanI enhances CPE binding/cytotoxicity for Caco-2 cells and ii) contact with small intestinal fluid, as occurs during type F enteric infections, proteolytically processes NanI to a 60 kDa fragment that possesses increased sialidase activity and greater ability than native NanI to promote CPE binding/cytotoxicity for enterocyte-like Caco-2 cells. While producing cell surface sialyl-conjugates, Caco-2 cells make minimal mucus, which is heavily sialylated and abundant in the intestines. Therefore, we hypothesize, i) NanI is even more impactful for promoting CPE activity in the presence of substantial mucus, as occurs in the intestines, ii) this effect is enhanced by proteolytic activation of NanI by intestinal proteases, and iii) NanI promotes type F infection/diseases. This project will test those important hypotheses using in vitro and in vivo models of CPE activity or type F infection/diseases. Aim 1 will employ enterocyte-like cell culture models that do or do not produce substantial mucus to compare the relative impact of NanI or proteolytically-activated NanI on promoting CPE cytotoxicity or CPE paracellular transit (an in vitro surrogate for CPE absorption from the intestines during enterotoxemia) in the absence vs. presence of mucus. Aim 2 will use animal infection models, i.e., rabbit and mouse small intestinal loop models of enteritis or enterotoxemia, respectively, to test if NanI contributes to type F infection/diseases and characterize NanI effects that could contribute to virulence, i.e., does NanI increase CPE activity/transit and/or type F strain growth, sporulation or CPE production in the intestines? If NanI is shown to potentiate type F infections, it would suggest a future translational approach, i.e., using sialidase inhibitors, to ameliorate these diseases.