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.
Clostidium perfringens type F isolates, which produce C. perfringens enterotoxin (CPE), are a major cause of enteric disease, including a very common food poisoning (FP) that can lead to lethal enterotoxemia (systemic infection due to absorption of toxins produced in the intestines). While CPE clearly plays a major role in these infections, NanH sialidase represents another potentially important virulence factor in type F enteric diseases, including FP. Consequently, this project will, 1) test the contributions of NanH to FP strain growth, sporulation and CPE production using both host cell and mucin models and 2) test the contributions of NanH to CPE activity using in vitro host cell models and explore if a sialidase inhibitor can reduce CPE activity, which might suggest a potentially novel therapeutic approach against type F enteric infections.
