The First Strain of Clostridium perfringens Isolated from an Avian Source Has an Alpha-Toxin with Divergent Structural and Kinetic Properties^,

Abstract

Clostridium perfringens alpha-toxin is a 370-residue, zinc-dependent, phospholipase C that is the key virulence determinant in gas gangrene. It is also implicated in the pathogenesis of sudden death syndrome in young animals and necrotic enteritis in chickens. Previously characterized alpha-toxins from different strains of C. perfringens are almost identical in sequence and biochemical properties. We describe the cloning, nucleotide sequencing, expression, characterization, and crystal structure of alpha-toxin from an avian strain, SWan C. perfringens (SWCP), which has a large degree of sequence variation and altered substrate specificity compared to these strains. The structure of alpha-toxin from strain CER89L43 has been previously reported in open (active site accessible to substrate) and closed (active site obscured by loop movements) conformations. The SWCP structure is in an open-form conformation, with three zinc ions in the active site. This is the first example of an open form of alpha-toxin crystallizing without the addition of divalent cations to the crystallization buffer, indicating that the protein can retain three zinc ions bound in the active site. The topology of the calcium binding site formed by residues 269, 271, 336, and 337, which is essential for membrane binding, is significantly altered in comparison with both the open and closed alpha-toxin structures. We are able to relate these structural changes to the different substrate specificity and membrane binding properties of this divergent alpha-toxin. This will provide essential information when developing an effective vaccine that will protect against C. perfringens infection in a wide range of domestic livestock.

Bacterial protein toxins and lipids: role in toxin targeting and activity

“…The N-terminal domain is related (29% identity) to the non-toxic Bacillus cereus PC-PLC (phosphatidylcholine PLC), which does not possess the C-terminal domain. Both proteins show similar folding patterns (Naylor et al, 1998;Titball et al, 1999;Justin et al, 2002).…”

Highly Conserved Alpha-Toxin Sequences of Avian Isolates ofClostridium perfringens

“…The alpha-toxin has been implicated in several diseases (18), including necrotic enteritis in chickens (3,10). The alphatoxin structural gene (plc or cpa) has been isolated from several strains of C. perfringens and characterized (4), and the encoded proteins were found to be highly conserved in all but one recently identified strain (8). This strain (SWCP) was isolated from a diseased swan.…”

Cloning of α-β fusion gene from Clostridium perfringens and its expression

“…Clostridium perfringens enterotoxin (CPE) serves as an additional virulence factor which is believed to cause enteritis in pigs, cats and dogs [12] . The Gram-positive pathogen Clostridium perfringens is a major cause of human and veterinary enteric disease largely because this bacterium can produce several toxins when present inside the gastrointestinal tract [13] . Biotype A strains of Clostridium perfringens are of particular importance as the aetiological agents of gas gangrene in humans.…”