VIRULENCE STUDIES ON CHROMOSOMAL ALPHA-TOXIN AND THETA-TOXIN MUTANTS CONSTRUCTED BY ALLELIC EXCHANGE PROVIDE GENETIC-EVIDENCE FOR THE ESSENTIAL ROLE OF ALPHA-TOXIN IN CLOSTRIDIUM PERFRINGENS-MEDIATED GAS-GANGRENE

The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasi on and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an alleli c exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin-resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a w orkable level by increasing the amount of homologous DNA located on ei ther side of the inactivated gene. Allelic exchange was used to isolat e mutations in the chromosomal pfoA gene, which encodes an oxygen-labi le haemolysin known as theta-toxin or perfringolysin O, and in the chr omosomal pie gene, which encodes the alpha-toxin or phospholipase C. T he resultant mutants failed to produce detectable theta-toxin or alpha -toxin activity, respectively, and could be complemented by recombinan t plasmids that carried the respective wild-type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The result s showed that the pie mutants had demonstrably reduced virulence and t herefore provided definitive genetic evidence for the essential role o f a-toxin in gas gangrene or clostridial myonecrosis.