Clostridium perfringens type A strains producing the enterotoxin (CPE) are an important cause of gastro-intestinal (GI) diseases, including the 2nd most common bacterial foodborne illness and several nonfoodborne GI diseases. CPE is produced when CPE-positive type A strains are ingested and then sporulate in the GI tract. CPE action starts with binding of this toxin to claudin (cldn) receptors to form a small complex. CPE in small complex then oligomerizes on the plasma membrane of intestinal cells to form a surface prepore. Each of the six CPE molecules in the prepore extends a beta hairpin that inserts into the lipid bilayer to form the CH- 1 pore in the host cell plasma membrane. In vitro, this pore triggers a calcium influx that, by activating calpain, induces (at low toxin doses) apoptosis or (a high toxin doses) necrosis. CPE-induced cell death then causes intestinal damage that induces luminal fluid accumulation in the small intestine and colon. CPE can also cause an enterotoxemia when absorbed from the intestines to damage internal organs such as the liver and kidneys. Therapeutics against CPE-mediated GI disease could target either CPE action or CPE production during in vivo sporulation. For those efforts, it is 1st necessary to better understand CPE action at the molecular and intestinal levels and to improve knowledge of early steps in C. perfringens sporulation/CPE production. To accomplish these goals, four specific aims will be pursued:
Aim 1 will analyze CPE complex formation, composition and function by using peptide mass fingerprinting to identify the full repertoire of host proteins associated with the small CPE complex and CH-1 pore; proteoliposomes and siRNA knockdowns will be employed to evaluate those host protein's contributions to CPE pore formation.
This Aim will also assess if mepacrine can inhibit the activity of pores formed when CPE binds to cldn receptors.
Aim 2 will characterize CPE-induced death pathways in Caco-2 cells and animal models. This work will 1st examine if necrosis caused by high CPE doses in Caco-2 cells involves necroptosis by using RIP1 or RIP3 kinase inhibitors, along with RIP1 and RIP3 siRNA knockdowns.
Aim 2 will then assess if CPE induces apoptosis and/or necrosis in rabbit and mouse models of GI disease or enterotoxemia and whether this in vivo cell death induces inflammation.
Aim 3 will use inhibitors and knockout mice to test the importance of apoptosis and/or necrosis, as identified in Aim 2, for CPE-induced in vivo pathology. Since inhibitors can provide insights into therapeutic development against CPE-induced GI disease, Aim 3 will also explore whether mepacrine, which may block the CPE pore, or soluble cldn receptors, which bind CPE, can interfere with CPE pathology.
Aim 4 will use a -glucuronidiase (GUS) reporter construct and random mutagenesis to identify genes involved early in C. perfringens sporulation and CPE production. To demonstrate this library as an invaluable resource, it will be used to identify the kinase that phosphorylates Spo0A, a key initiating step in sporulation/CPE production and a potential therapeutic target.

Public Health Relevance

Clostridium perfringens enterotoxin (CPE) is responsible for the diarrhea and abdominal cramping symptoms of several very important intestinal diseases, including the 2nd most common bacterial foodborne illness in the USA. Three major gaps exist in understanding CPE-mediated intestinal diseases, namely: i) the process of CPE pore formation, ii) whether apoptosis and/or necrosis (possibly necroptosis) are activated by this toxin in vivo and if blocking those cell death pathways can inhibit CPE-induced pathology, and iii) early steps in C. perfringens sporulation, which is essential for CPE production. This application will address these three knowledge deficits by using a combination of in vitro and in vivo studies, with the eventual goal of identifying processes that could be inhibited by therapeutics.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Bacterial Pathogenesis Study Section (BACP)
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Ranallo, Ryan
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University of Pittsburgh
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