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

NanI 唾液酸酶：对产气荚膜梭菌肠毒素活性的影响以及对产气荚膜梭菌 F 型感染的贡献

基本信息

批准号：
10183154
负责人：
Bruce A Mc Clane
金额：
$ 23.51万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-08 至 2024-05-31
项目状态：
已结题

项目摘要

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.

项目摘要 F型F型F菌株是细菌食物中毒（FP）的第二个最常见原因在美国，发生约100万例案件。这些细菌还会引起许多非食源性hu- 人肠道疾病（NFD），例如抗生素相关的腹泻。 F型菌株的毒力重新刺激性孔毒素肠毒素（CPE）的Quires产生。所有类型的F疾病都是真正的感染 F型F菌株最初在肠道中繁殖，但在体内孢子时会产生CPE；这种毒素是母细胞裂解以释放其成熟的孢子后释放到肠腔中。 F型感染是通常是腹泻性疾病在患有某些易感性医疗状况的患者中，吸收会损害内部器官（例如肝脏）。除CPE外，所有类型的F NFD菌株和〜50％的F FP菌株产生了一个分泌的唾液酸酶纳尼。对于F菌株，NANI是其主要的外氨基苷酶，并且这种唾液酸酶都是在两者中产生的营养培养和散发性培养物（Nani与CPE共同展示）。 Nani正在作为一个 NANI+ F型菌株的重要毒力因子，例如，我们显示nani sialidase在体外有助于生长，孢子形成和CPE产生以及持续的肠道定殖。我们还报告，i）nani增强 CPE结合/CACO-2细胞的细胞毒性和ii）与小肠液体接触，如F型肠型期间发生的那样感染，蛋白水解将nani处理为60 kDa的片段，该片段具有增加的唾液酸酶活性和比天然NANI促进肠类细胞样CACO-2细胞的CPE结合/细胞毒性的能力更大。在产生细胞表面siAllyl结合物的同时，CACO-2细胞产生的粘液最少，粘液质量很大肠道丰富。因此，我们假设，i）nani对促进CPE的影响更大在存在大量粘液存在下的活性，如肠中所发生的那样，ii）蛋白水解增强了这种作用肠蛋白酶激活NANI，而III）NANI促进了F型感染/疾病。这个项目将测试那些使用体外和体内CPE活性或F型感染/疾病的体内模型的重要假设。目标1 将采用类似肠细胞的细胞培养模型，这些模型会产生或不产生大量粘液来比较 NANI或蛋白水解激活的NANI对促进CPE细胞毒性或CPE细胞细胞传输的相对影响（肠毒素期间肠道吸收CPE吸收的体外替代物）在不存在的情况下与存在粘液。 AIM 2将使用动物感染模型，即兔和小鼠小肠肠炎模型或分别测试NANI是否有助于F型感染/疾病并表征NANI 可能导致毒力的影响，即nani会增加CPE活性/过境和/或F型菌株生长，肠中孢子或CPE产生？如果显示纳尼会增强F型感染，则建议一种未来的翻译方法，即使用唾液酸酶抑制剂来改善这些疾病。