Identification and Characterization of Clostridium perfringens Beta Toxin Variants with Differing Trypsin Sensitivity and in vitro Cytotoxicity Activity.

By producing toxins, Clostridium perfringens causes devastating diseases of both humans and animals. C. perfringens beta toxin (CPB) is the major virulence determinant for type C infections and is also implicated in type B infections, but little is known about the CPB structure vs function relationship. Amino acid sequence comparisons of CPB made by 8 randomly-selected isolates identified two natural variant toxins with four conserved amino acid changes, including an E168K switch that introduces a potential trypsin cleavage site into the CPB protein of strain JGS1076. To investigate if this potential trypsin cleavage site impacts trypsin sensitivity, a primary host defense against this toxin, the two CPB variants were assayed for their trypsin sensitivity. Results demonstrated a significant difference in trypsin sensitivity, which was linked to the E168K switch using recombinant CPB site-directed mutants. The natural CPB variants also displayed significant differences in their cytotoxicity to human endothelial cells. This cytotoxicity difference was mainly attributable to increased host cell binding rather than the ability to oligomerize or form functional pores. Using rCPB site-directed mutants, differences in cytotoxicity and host cell binding were linked to an A300V amino acid substitution in the strain JGS1076 CPB variant possessing more cytotoxic activity. Mapping sequence variations on a CPB structure modeled using related toxins suggests the E168K substitution is surface-localized, so it can interact with trypsin, and the A300V substitution is located in a putative binding domain of the CPB toxin.Copyright © 2015, American Society for Microbiology. All Rights Reserved.