Characterization of Clostridium perfringens Beta2 toxin
Open Access
- Author:
- Gurjar, Abhijit Arun
- Graduate Program:
- Pathobiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 04, 2007
- Committee Members:
- Bhushan M Jayarao, Committee Chair/Co-Chair
Avery August, Committee Member
Blake Peterson, Committee Member
Arthur Hattel, Committee Member
Brenda C Love, Committee Member - Keywords:
- beta2
perfringens
Clostridium
toxin - Abstract:
- Clostridium perfringens, a gram positive, sporulating anaerobic bacterium is responsible for a wide spectrum of diseases in animals and humans. Clostridium perfringens-mediated hemorrhagic enteritis and enterotoxemias in livestock, domestic animals, aquatic species and birds have imposed significant economic impact on animal husbandry. The anaerobic, nutrient-enriched environment of the mammalian gastrointestinal tract serves as an ideal habitat for the C. perfringens to proliferate and attain a disease inducing potential. The pathogenesis of the C. perfringens mediated enteric diseases can be attributed to the well characterized, lethal membrane active toxins such as alpha, beta, epsilon, iota, enterotoxin and perfringolysin O. In recent years, a variant of the C. perfringens beta toxin known as beta2 has been widely reported as one of the causative agents in C. perfringens related enteric diseases. The clinical symptoms of beta2 toxin induced enterotoxemias are characterized by watery sporadic diarrhea, edema of affected tissues and loss of body weight. The role of beta2 toxin in the causation of enterotoxemias and or haemorrhagic/necrotic enteritis in humans, domestic animals, birds and aquatic species has generated considerable interest in the field of human and veterinary medicine. Clostridium perfringens beta2 has been reported as an accessory toxin and may act in synergy with other major toxins of the C. perfringens in the production of necrotic and hemorrhagic enteritis. The beta2 toxin may play a role in hindering the process of nutrient absorption in the intestine and thereby debilitating affected animals. It is strongly speculated that a sudden change in the gut Microbiolflora or the intestinal physiological equilibrium may lead to clostridial disease outbreak. Studies that report on C. perfringens beta2 producing isolates from healthy animals suggest that beta2 toxin as a potential health hazard. Very few studies have reported the biophysical and molecular biological properties of the beta2 toxin in detail. It has been hypothesized that the mechanism of the beta2 toxin induced enteritis could include direct effects on transport of ions in the intestinal epithelial cells through membrane binding or pore formation. It is felt that experiments that focus on studying the cytotoxicity of beta2 toxin could lead to better understanding of beta2 toxin in context of C. perfringens related enterotoxemias. Based on these reported findings, elucidation of the molecular and biological properties of beta2 toxin would be of significant value to researchers towards developing a targeted epitope vaccine. Clostridium perfringens are toxinotyped into types A-E by conventional bacteriology and PCR based confirmation techniques. Owing to the widespread distribution of C. perfringens toxinotypes and its epidemiological significance a highly sensitive diagnostic tool that replaces the conventional PCR is required. In food animal practice, active immunoprophylaxis against C. perfringens is achieved by use of commercial toxoid vaccines against types B, C and D. There are no published reports on vaccines conferring immunity against beta2 toxin. The primary objectives of this dissertation are (1) develop a molecular based toxin typing technique for C. perfringens which has practical application in a diagnostic laboratory and aid in conducting molecular epidemiological studies, (2) Elucidate the structure and function of beta2 toxin, 3) Evaluate in vitro the cytotoxic properties of beta2 toxin, and 4) Evaluate the role of beta2 synthetic lipopeptide vaccine in a challenge study in the mouse model. The results of our study revealed a high prevalence and distribution of C. perfringens beta2 toxin encoding strains in feces of lactating cattle. Among the five toxinotypes, toxinotype A was widely distributed among seven dairy herds screened by multiplex Real time PCR assay. We successfully used a GST fusion system for expression of recombinant beta2 toxin. The purified recombinant toxin was shown to be cytotoxic to CaCo2 cell line at concentration of more than 10 g/ml. the toxin treated cells exhibited cytopathic effects such as cell rounding, membrane blebbing and leakage of cytoplasm. These results could suggest that the C. perfringens beta2 toxin could act as one of the pore forming toxins. A monoclonal antibody raised against beta2 toxin completely neutralized the cytopathic effects of the toxin on CaCo2 cells. To study the physical structure of the beta2 toxin, recombinant beta2 toxin was crystallized in buffer comprising of 32-36% PEG in one week. Preliminary crystallographic analysis of the recombinant beta2 toxin suggested the crystals of the toxin belonged to space group R3. The triangular prism shaped crystals diffracted up to 2.9Å resolution and measured up to 200 Microbiolns in size. A novel approach of developing and evaluating a synthetic lipopeptide vaccine against C. perfringens beta2 toxin was demonstrated as part of this study. The lipopeptide conjugate chemically linked to Pam2Cys, successfully elicited a strong protective response in subcutaneously immunized mice. The immune response was characterized as mixed Th1 and Th2 response to the synthetic lipopeptide vaccine. It is anticipated that the results of our study will provide animal agriculture with an alternate approach to protect food producing animals against C. perfringens infections. The study will lead to the development and the standardization of a real-time PCR based technique to identify and quantify the various toxigenic strains of C. perfringens directly from clinical samples. The structure and function studies demonstrated in our study will provide a better understanding of the molecular pathogenesis of beta2 toxin and the development of a safe and effective vaccine against beta2 toxin.