The genus Clostridium consists of a diverse group of Gram-positive, anaerobic and heat resistant spore forming bacteria. They are widely distributed in soil, sewage and water. In addition, some species are normal inhabitant of gastrointestinal tract of mammals. Usually bacterial numbers remain small in intestine but due to some abrupt changes in diet or other factors, bacterial number increases upto 109 cells per gram of ileum contents and secretes large amounts of toxin (Payne and Oyston, 1997). They cause severe diseases in animals and human such as botulism, tetanus, gas gangrene and enterotoxemia. One of the members of this genus, Clostridium perfringens causes wide variety of diseases in human such as gas gangrene, food poisoning and necrotic enteritis. The bacterium also causes some severe gastrointestinal and enterotoxemia diseases in domestic animals (Frank, 1956; Songer, 1997a).
The etiology of diseases caused by Clostridium perfringens suggests mainly to the production of various extracellular toxins. The bacteria have been divided into five distinct types, A through E, on the basis of production of four major toxins (alpha, beta, epsilon and iota). Epsilon toxin is secreted by type B and D strains (Brooks et al., 1957). Clostridium perfringens type B is associated particularly with dysentery in lambs, while type D is associated with necrotic enteritis and enterotoxemia in sheep and lambs, along with a condition known as pulpy kidney or overeating disease (Bullen, 1970). These diseases are usually fatal and are characterized by a short period of time between the first appearance of symptoms and death. The mortality rate can be as high as 100%, and the diseases are of major economic significance, particularly in the area where animals is used for the economic purposes (Buxton and Fraser, 1977). The symptoms of disease, including neurological dysfunction and pulmonary edema, generally appear within an hour of the administration of purified epsilon toxin (Uzal and Kelly, 1997; Uzal and Kelly, 1998) whereas peritoneal and pericardial effusions are common in naturally infected sheep (Jubb et al., 1993). Many animals die per acutely, without premonitory signs (Niilo, 1993; Popoff, 1984). The epsilon toxin can cross the blood-brain barrier (Jover et al., 2007; Worthington and Mulders, 1975) and accumulates in the brain as well as in the kidney, causing widespread osmotic alterations by disrupting vascular endothelia.
Due to their devastating effect, epsilon toxin has been considered as the second most potent toxin after botulinum and titanus (McClane et al., 2005). This toxin has also been included in the Centers for Disease Control list of selected agents that might be used as biological weapons (Atlas, 1998). Epsilon toxin is secreted as an inactive prototoxin of 311 amino acids length (McDonel, 1986), which got activated to a lethal toxin by proteolytic cleavage (Bhown and Habeeb, 1977). The proteases for activation of epsilon toxin are provided by either host or the bacterium, such as trypsin and chymotrypsin by host (Bhown and Habeeb, 1977; Hunter et al., 1992) and lambda protease by bacterium (Jin et al., 1996; Minami et al., 1997). Maximum lethality with an LD50 70 ng/kg occurs when cleavage is done by trypsin and chymotrypsin combination, resulting in the loss of 13 N-terminal residues and 29 C-terminal residues. If the cleavage occurs due to trypsin alone, resulting in the loss of 13 N-terminal residues and 23 C-terminal residues, then the lethality was slight less with an LD50 320 ng/kg. As mentioned, the toxin can also be activated by a lambda protease secreted by Clostridium perfringens. This cleaves the 10 residues from N-terminus and 29 residues from C-terminus, which results the activity close to maximal with an LD50 of 100 ng/kg (Minami et al., 1997). The cleavage also causes a marked shift of pI from 8.02 for the prototoxin to 5.36 in the mature toxin (Worthington and Mulders, 1977). Madin-Darby canine kidney (MDCK) cell line of endothelial origin from the distal convoluted tubule is the most sensitive cell line to epsilon toxin (Payne et al., 1994). In-vitro exposure of MDCK cells with epsilon toxin results cytoskeleton changes and irreversible damage to plasma membrane (Donelli et al., 2003). Cells subsequently swell, develop membrane bleb (Borrmann et al., 2001; Petit et al., 2001). However, there is no evidence of internalization of the toxin (Petit et al., 1997). The binding of epsilon toxin to MDCK cells and rat synaptosomal membrane is associated with formation of stable and SDS resistant high molecular weight complex (Nagahama et al.; 1992; Petit et al.; 1997). The similar large molecular weight complexes has also been observed with other pore forming toxins, such as Staphylococcus aureus α-hemolysin (Song and Gouaux, 1998), C. septicum α-toxin (Melton et al., 2004), Pseudomonas aeruginosa cytotoxin (Ohnishi et al., 1994), and Aeromonas hydrophila aerolysin (Wilmsen et al., 1992). Epsilon toxin as many other pore-forming toxins, has been shown to interact specifically with detergent resistant micro-domains (DRMs) of the membrane and form pore (Miyata et al., 2002). This suggests that a putative receptor located in DRMs is responsible for toxin binding and subsequent heptamerization.
In spite of its sequential dissimilarity epsilon toxin has high structural similarity with Aeromonas hydrophila aerolysin (Cole et al., 2004) as well as with alpha-toxin of Clostridium septicum (Melton-Witt et al. 2006). Because of the structural similarity of Epsilon toxin to aerolysin and other β-pore forming toxins, it seems likely that epsilon toxin shares a related mechanism of pore formation including conformational changes from its secreted water-soluble form. By chemical modification of epsilon toxin, several essential amino acids have been identified for its activity, such as tryptophan, tyrosin and histidine and three or four aspartic or glutamic acid (Sakurai and Nagahama, 1985; Sakurai and Nagahama, 1987a; Sakurai and Nagahama, 1987b; Payne and Osten, 1997; Sakurai and Nagahama, 1987c). Substitution of the two histidine residues either with alanine or serine did not abolished lethal activity of the protein, suggesting that imidazole side-chain does not play a role in activity of the toxin; however, the change with proline results a loss in lethal activity (Oyston et al., 1998). This suggests that structural motif in this region is essential for biological activity, which undergo a conformation change in proline substitution.
There are some crude vaccines existing for the prevention of disease associated with Clostridium perfringens type B and D strains. These vaccines are based on formaldehyde-treated cell filtrates or bacterial cells and an equine derived antitoxin. The immunogenicity of these vaccines is variable and the vaccine may not provide complete protection (Percival et al. 1990).
Thus both the existing approaches to combat illness would be of limited significance in case of epsilon (ε) toxin bio-terrorism. Due to rapid progression of the disease, treatment is generally not possible, and the emphasis is placed on prevention either by vaccination or by administration of antitoxin to unvaccinated animals in occurrence of enterotoxemia. Therefore an alternative preventive measures are needed that can inhibit the activity of epsilon toxin.
The present invention discloses generation and high level expression of recombinant non-toxic mutant of epsilon toxin of Clostridium perfringens and its uses as a recombinant vaccine against Clostridium perfringens infection.
USPTO Backgrounds
USPTO Backgrounds