Synergistic effects of alpha-toxin and perfringolysin O in Clostridium perfringens-mediated gas gangrene

Abstract

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.

FIG. 1

Construction of a plc pfoA double mutant by homologous recombination. A single crossover event between the plc gene located on the chromosome of the pfoA mutant JIR4069 (A) and the truncated plc gene located on the suicide vector pJIR1774 led to the construction of the plc pfoA double mutant JIR4444 (B). The insert shows the results of Southern hybridization analysis of EcoRI-digested chromosomal DNA from the wild-type strain JIR325 and mutant strains JIR4069 and JIR4444 (lanes 1, 2, and 3, respectively), probed as indicated. Molecular size standards are also shown (lane S). The arrow indicates the position of the faint 1.5-kb band detected in lanes 2 and 3 with the pfoA-specific probe.

FIG. 2

Gross pathology of infected mice. BALB/c mice were injected with an isogenic series of JIR4444-derived C. perfringens strains. For each strain, 10 6- to 8-week-old mice were injected intramuscularly in the right thigh with 100 μl of a washed cell suspension that contained ca. 10⁹ CFU (10). The identity of each strain was independently encoded prior to injection to ensure that the study was carried out blind. After injection, the mice were monitored regularly and detailed observations of the gross pathological changes were noted over a 24-h period, as previously described (2). These observations involved scoring each mouse for outward signs of illness, which included matting of the fur and lack of response to environmental stimuli, as well as for the severity of swelling and blackening (scored for both the thigh and foot as an indicator of the spread of infection) and for the ability to move the infected limb (limping). The severity of each pathology parameter was scored as either 0 (no discernible pathology), ½ (moderate pathology), or 1 (marked pathology apparent). The number of severely affected mice killed for animal ethics reasons was also recorded. Note that cumulative pathology on the y axis refers to the sum of the individual pathology scores from 10 mice, the number 10 therefore represents a maximal score. Key: □, JIR4444(pJIR750), the plc pfoA double mutant carrying a vector plasmid; ×, JIR4444(pJIR871), the double mutant carrying a pfoA⁺ plasmid; ○, JIR4444(pJIR1642), the double mutant carrying a plc⁺ plasmid; and ⋄, JIR4444(pJIR1720), the double mutant carrying a plc⁺ pfoA⁺ plasmid.

FIG. 3

Hematoxylin-and-eosin-stained sections from infected tissues. Mice were injected as described in the legend to Fig. 2, with the strains indicated (see Fig. 2). Two mice were randomly chosen from each infected group at various times after injection. After each mouse was killed, the infected limb was exposed by blunt dissection, allowing observation of the severity and spread of necrosis. Samples of the muscle were then removed and fixed overnight in a solution of 0.5% tannic acid (wt/vol) in methanol before paraffin embedding and sectioning for hematoxylin and eosin staining and analysis. Symbols and abbreviations: arrows, leukocyte aggregates; asterisks, bacterial aggregates; mn, myonecrosis; cn, coagulative necrosis; th, thrombosis; bv, blood vessel tissue.