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

项目摘要 F型产气荚膜梭菌是引起食物中毒和非食源性人类的主要原因 胃肠道(GI)疾病。F型FP是美国第二种最常见的细菌性食源性疾病， 每年影响约100万人，造成3.1亿美元的经济损失。肠胃病 F型菌株的毒力需要产生产气荚膜梭菌肠毒素(CPE)。F型FP也可以发展成 转化为致死性肠毒素血症(肠道中产生的CPE被吸收以损害内脏，如 肝脏)。F型肠道疾病涉及肠道中F型菌株的生长和产孢量。 孢子形成在这些疾病中起着关键作用，即CPE只有在F型菌株在 当母细胞裂解以释放其成熟的孢子时，肠道就变成了细胞外。 产气荚膜梭菌还产生3种不同底物特异性的唾液酸酶(NanJ、NanI和NanH)。 在营养细胞中，NanJ和NanI总是分泌的，而NanH在对数早期是细胞质的，但在对数生长期是胞质的。 在原木生长后期的营养培养或孢子培养中出现在细胞外。我们的团体最近示威- 认为NaNi唾液酸酶对F型非食源性HU-1菌株的体外生长、产孢量和CPE的产生有促进作用。 使用与肠道相关的底物，如可溶性粘蛋白或类肠道细胞时的MAN GI疾病菌株 Caco-2细胞。我们还表明，NaNi可以通过增加这种毒素与培养物的结合来增强CPE的作用。 细胞。然而，NANI基因在大多数FFP型菌株中缺失。因为，1)所有的FFP菌株都携带 NanH基因，2)在产孢子培养中显著增加NanH的产量，以及3)NanH是共存的 在胞外与产孢子菌的CPE，我们假设NanH是一个重要的贡献 F型菌株，特别是FP菌株在唾液中的生长、产孢量和CPE产生/活性 富含酸性的肠道。目前的提议将通过两个体外目标开始检验这一假说。具体来说， 目标1将通过比较NanH对FP菌株的生长/存活和产孢量的影响来评估NanH是否有助于FP菌株的生长/存活和产孢量 野生型FP菌株SM101与其同基因突变株SM101的生长存活、产孢量和CPE产量的比较 利用与肠道相关的产生粘液的培养细胞或可溶性粘蛋白溶液作为补充菌株 底物。Aim 2将评估生理剂量的纯化NaH唾液酸酶是否可以增强CPE的细胞毒性 使用能或不能产生大量粘液的类肠细胞培养物，就像在肠道中发生的那样。 该目标还将利用这些细胞培养模型来确定纯化的NaH是否增强细胞旁通透性。 作为肠毒血症标志物的IIT/CPE转运蛋白。最后，Aim 2将测试NanH唾液酸酶抑制剂是否会减少CPE作用- 离子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，以辨别这种能力 将NanH增强CPE活性或促进生长、产孢量和CPE产生作为前奏来证明 未来的动物模型研究将评估NanH作为F型胃肠道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.