Evaluating NanH Sialidase Contributions to Growth, Sporulation and Toxin Action for C. perfringens Type F Food Poisoning Strains

项目摘要 产气荚膜梭菌F型菌株是引起食物中毒和非食源性人类感染的主要原因 胃肠道（GI）疾病。F型FP是美国第二大常见的细菌性食源性疾病， 每年影响约100万人，造成经济损失> 3.1亿美元。肠溶 F型菌株的毒力需要产生C.产气荚膜杆菌肠毒素（CPE）。F型FP还可以开发 转化为致命的肠毒血症（其中肠中产生的CPE被吸收以损害内脏器官， 肝脏）。F型肠道疾病涉及F型菌株在肠道中的生长，随后是孢子形成。 孢子形成在这些疾病中起关键作用，即，CPE仅在F型菌株在培养基中形成孢子时表达。 当母细胞溶解释放成熟的孢子时，它就变成了细胞外的细胞。 C.产气荚膜杆菌还产生具有不同底物特异性的多达3种唾液酸酶（NanJ、NanI和NanH）。 在营养细胞中，NanJ和NanI总是分泌的，而NanH在对数生长期的早期是细胞质的，但在对数生长期的早期是细胞质的。 在对数生长期后期的营养培养物或孢子形成培养物中出现在细胞外。我们最近展示了- 艾德认为NanI唾液酸酶有助于F型非食源性胡萝卜素的体外生长、孢子形成和CPE产生。 人GI疾病菌株，当使用生殖相关底物如可溶性粘蛋白或肠细胞样人 Caco-2细胞。我们还表明，NanI可以通过增加这种毒素与培养细胞的结合来增强CPE作用。 细胞然而，大多数F型FP菌株中不存在nanI基因。因为，1）所有F型FP菌株都携带 nanH基因，2）在孢子形成培养物中NanH产量显著增加，和3）NanH是共存在的 在孢子形成培养物中，细胞外的CPE，我们假设NanH是一个重要的贡献者， F型菌株，特别是FP菌株在唾液酸中的生长、孢子形成和CPE产生/活性 富含酸的肠子当前提案将通过两个体外目标开始检验这一假设。具体地说， 目的1将通过比较NanH与FP菌株的生长/存活和孢子形成来评估NanH是否有助于FP菌株的生长/存活和孢子形成。 野生型FP菌株SM 101相对于其同基因nanH突变体的生长/存活、孢子形成和CPE产生， 一种补充菌株，其使用与生殖相关的粘液产生培养细胞或可溶性粘蛋白溶液作为 印刷受体.目的2将评估生理量的纯化NanH唾液酸酶是否可以增强CPE细胞毒性 使用肠细胞样细胞培养，产生或不产生大量粘液，如在肠道中发生的。 该目的还将使用这些细胞培养模型来确定纯化的NanH是否增强细胞旁渗透性。 作为肠毒血症的标志物。最后，目标2将测试NanH唾液酸酶抑制剂是否减少CPE act- 在相同的细胞培养模型中。总之，这项初步研究将进行体外研究，以辨别能力 提高CPE活性或促进生长、产孢和CPE产生作为前奏， 未来的动物模型研究，以评估NanH作为F型GI疾病的毒力因子和治疗靶点。

Project Summary Clostridium perfringens type F strains are a major cause of food poisoning (FP) and nonfoodborne human gastrointestinal (GI) disease. Type F FP is the 2nd most common bacterial foodborne illness in the United States, where it affects about 1 million people/year and causes economic losses >$310 million/year. The enteric virulence of type F strains requires production of C. perfringens enterotoxin (CPE). Type F FP can also develop into lethal enterotoxemia (where CPE produced in the intestines is absorbed to damage internal organs such as the liver). Type F enteric diseases involve growth, followed by sporulation, of type F strains in the intestines. Sporulation plays a critical role in these diseases, i.e., CPE is expressed only when a type F strain sporulates in the intestines and then becomes extracellular when the mother cell lyses to free its mature spore. C. perfringens also produces up to 3 sialidases (NanJ, NanI and NanH) with different substrate specificities. In vegetative cells, NanJ and NanI are always secreted, while NanH is cytoplasmic in early log phase but be- comes extracellular in late log-phase vegetative cultures or sporulating cultures. Our group recently demonstrat- ed that NanI sialidase contributes to in vitro growth, sporulation and CPE production by type F nonfoodborne hu- man GI disease strains when using intestinally-relevant substrates like soluble mucin or enterocyte-like human Caco-2 cells. We also showed that NanI can potentiate CPE action by increasing binding of this toxin to cultured cells. However, the nanI gene is ABSENT from most type F FP strains. Since, 1) all type F FP strains do carry the nanH gene, 2) NanH production significantly increases in sporulating cultures, and 3) NanH is co-present extracellularly with CPE in sporulating cultures, we hypothesize that NanH is an important contributor to growth, sporulation and CPE production/activity by type F strains, especially FP strains, in the sialic acid-rich intestines. The current proposal will begin testing this hypothesis via two in vitro aims. Specifically, Aim 1 will evaluate if NanH contributes to growth/survival and sporulation of FP strains by comparing the growth/survival, sporulation and CPE production for wild-type FP strain SM101 vs. its isogenic nanH mutant and a complemented strain using intestinally-relevant mucus-producing cultured cells or soluble mucin solution as substrates. Aim 2 will assess if physiological amounts of purified NanH sialidase can potentiate CPE cytotoxicity using enterocyte-like cell cultures that do or do not produce large amounts of mucus, as occurs in the intestines. This Aim will also use these cell culture models to determine if purified NanH enhances paracellular permeabil- ity/CPE transit, as markers for enterotoxemia. Last, Aim 2 will test if a NanH sialidase inhibitor reduces CPE act- ion in the same cell culture model. In summary, this pilot study will conduct in vitro studies to discern the ability of NanH to enhance CPE activity or promote growth, sporulation and CPE production as a prelude to justify future animal model studies to evaluate NanH as a virulence factor, and therapeutic target, for type F GI disease.