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Infection and Immunity, December 2001, p. 7904-7910, Vol. 69, No. 12
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.12.7904-7910.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Synergistic Effects of Alpha-Toxin and Perfringolysin O in Clostridium perfringens-Mediated Gas Gangrene

Milena M. Awad,1 Darren M. Ellemor,2 Richard L. Boyd,2 John J. Emmins,2 and Julian I. Rood1,*

Bacterial Pathogenesis Research Group, Department of Microbiology, Monash University, Victoria 3800,1 and Department of Pathology and Immunology, Monash University Medical School, Alfred Hospital, Prahran 3181,2 Australia

Received 8 June 2001/Returned for modification 24 July 2001/Accepted 24 August 2001

To examine the synergistic effects of alpha-toxin and perfringolysin O in clostridial myonecrosis, homologous recombination was used to construct an alpha-toxin deficient derivative of a perfringolysin O mutant of Clostridium perfringens. The subsequent strain was complemented with separate plasmids that carried the alpha-toxin structural gene (plc), the perfringolysin O gene (pfoA), or both toxin genes, and the resultant isogenic strains were examined in a mouse myonecrosis model. Synergistic effects were clearly observed in these experiments. Infection with the control strain, which did not produce either toxin, resulted in very minimal gross pathological changes, whereas the isogenic strain that was reconstituted for both toxins produced a pathology that was clearly more severe than when alpha-toxin alone was reconstituted. These changes were most apparent in the rapid spread of the disease, the gross pathology of the footpad and in the rate at which the mice had to be euthanatized for ethical reasons. Elimination of both alpha-toxin and perfringolysin O production removed most of the histopathological features typical of clostridial myonecrosis. These effects were restored when the mutant was complemented with the alpha-toxin structural gene, but reconstituting only perfringolysin O activity produced vastly different results, with regions of coagulative necrosis, apparently enhanced by vascular disruption, being observed. Reconstitution of both alpha-toxin and perfringolysin O activity produced histopathology most similar to that observed with the alpha-toxin reconstituted strain. The spreading of myonecrosis was very rapid in these tissues, and coagulative necrosis appeared to be restricted to the lumen of the blood vessels. The results of these virulence experiments clearly support the hypothesis that alpha-toxin and perfringolysin O have a synergistic effect in the pathology of gas gangrene.

* Corresponding author. Mailing address: Bacterial Pathogenesis Research Group, Department of Microbiology, Monash University, P.O. Box 53, Victoria 3800, Australia. Phone: (613) 99054825. Fax: (613) 99054811. E-mail: Julian.Rood{at}med.monash.edu.au.

Infection and Immunity, December 2001, p. 7904-7910, Vol. 69, No. 12
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.12.7904-7910.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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