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

项目摘要 F型产气荚膜梭菌是细菌性食物中毒的第二大常见原因 在美国，每年大约发生100万起病例。这些细菌还会导致许多非食源性HU-1病例。 人类肠道疾病(NFD)，如抗生素相关性腹泻。F型毒力测定结果表明，F型毒力较强。 停止生产产气荚膜梭菌肠毒素(CPE)。所有的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；这种毒素是 母细胞裂解后释放到肠腔内以释放其成熟的孢子。F型感染是 通常是一种腹泻疾病，但也可能涉及致命性肠毒素血症(肠道中产生的CPE是 吸收以损害肝脏等内脏器官)有某些诱因的医疗条件的患者。 除CPE外，所有F型NFD菌株和~50%的FFP菌株都能产生一种分泌型唾液酸酶，名为 纳尼。对于F型菌株，NaNi是其主要的外唾液酸酶，这种唾液酸酶在两种类型的菌株中都有产生 营养培养和孢子培养(NANI与CPE共同存在)。纳尼正在崛起为一名 对NaNi+型F菌株的重要毒力因子，例如，我们在体外表明NaNi唾液酸酶有助于生长， 产孢子和CPE的产生以及持续的肠道定植。我们还报道，i)NaNi增强了 CPE对Caco-2细胞的结合/细胞毒性和II)与小肠液的接触，如在F型肠道中发生的情况 感染，蛋白水解性将NaNI加工成一个60 kDa的片段，具有增加的唾液酸酶活性和 比天然NaNi更能促进肠细胞样Caco-2细胞的CPE结合/细胞毒作用。 在产生细胞表面唾液酸偶联物的同时，Caco-2细胞产生最少的粘液，这些粘液被严重唾液分解 并在肠道中大量存在。因此，我们假设，i)NaNi对CPE的促进作用更大 在大量粘液存在的情况下的活动，如在肠道中发生的，ii)这种作用通过蛋白分解而增强 NANI由肠道蛋白水解酶激活，以及iii)NANI促进F型感染/疾病。该项目将测试 这些重要的假设使用了CPE活性或F型感染/疾病的体外和体内模型。目标1 将使用能产生或不能产生大量粘液的肠细胞样细胞培养模型来比较 NaNi或蛋白水解性NaNi促进CPE细胞毒性或细胞旁转运的相对影响 (肠毒素血症时CPE从肠道吸收的体外替代物) 粘液。AIM 2将采用动物感染模型，即兔和小鼠小肠循环肠炎模型 或肠毒素血症，以测试NANI是否与F型感染/疾病有关，并表征NANI 可能有助于毒力的影响，即NaNi是否增加CPE活性/转运和/或F型菌株的生长， 在肠道中产生孢子还是产生CPE？如果纳米镍被证明可以增强F型感染，它会建议 未来的翻译方法，即使用唾液酸酶抑制剂，以改善这些疾病。

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.