INFECTION AND IMMUNITY,0019-9567/01/$04.00�0 DOI: 10.1128/IAI.69.12.7904–7910.2001

Dec. 2001, p. 7904–7910 Vol. 69, No. 12

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Synergistic Effects of Alpha-Toxin and Perfringolysin O inClostridium 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, homolo-gous recombination was used to construct an alpha-toxin deficient derivative of a perfringolysin O mutant ofClostridium perfringens. The subsequent strain was complemented with separate plasmids that carried thealpha-toxin structural gene (plc), the perfringolysin O gene (pfoA), or both toxin genes, and the resultantisogenic strains were examined in a mouse myonecrosis model. Synergistic effects were clearly observed in theseexperiments. Infection with the control strain, which did not produce either toxin, resulted in very minimalgross pathological changes, whereas the isogenic strain that was reconstituted for both toxins produced apathology that was clearly more severe than when alpha-toxin alone was reconstituted. These changes weremost apparent in the rapid spread of the disease, the gross pathology of the footpad and in the rate at whichthe mice had to be euthanatized for ethical reasons. Elimination of both alpha-toxin and perfringolysin Oproduction removed most of the histopathological features typical of clostridial myonecrosis. These effects wererestored when the mutant was complemented with the alpha-toxin structural gene, but reconstituting onlyperfringolysin O activity produced vastly different results, with regions of coagulative necrosis, apparentlyenhanced by vascular disruption, being observed. Reconstitution of both alpha-toxin and perfringolysin Oactivity produced histopathology most similar to that observed with the alpha-toxin reconstituted strain. Thespreading of myonecrosis was very rapid in these tissues, and coagulative necrosis appeared to be restricted tothe lumen of the blood vessels. The results of these virulence experiments clearly support the hypothesis thatalpha-toxin and perfringolysin O have a synergistic effect in the pathology of gas gangrene.

Gas gangrene or clostridial myonecrosis is characterized bya rapid spread of tissue necrosis combined with a lack ofleukocyte infiltration at the site of infection. Left untreated, itsrapid and aggressive progression is almost always fatal. Themost common causative organism, Clostridium perfringens typeA, has the ability to produce numerous extracellular toxins (12)and is commonly found in the environment, in niches such assoil and sewage, and in the gastrointestinal tract of both hu-mans and animals.

Alpha-toxin is the most toxic extracellular enzyme producedby C. perfringens type A and is essential for virulence (3, 10,16). It is a phospholipase C that hydrolyzes both phosphatidyl-choline and sphingomyelin (12, 18), both of which are impor-tant constituents of eukaryotic cell membranes. Alpha-toxinmutants are avirulent in a mouse myonecrosis model (3, 10,17), with comparative histological examination of muscle tissuefrom mice infected with a series of isogenic strains indicatingthat alpha-toxin is required for tissue necrosis, inhibition of theinflux of polymorphonuclear leukocytes (PMNL) into the le-sion, and thrombosis formation (3, 10, 17). Thrombosis is apotentially important factor in the pathology of gas gangrene,since by reducing the oxygen tension within the tissues it pre-

sumably helps produce environmental conditions suitable forthe growth of the invading anaerobic bacterium.

Perfringolysin O, or theta-toxin, is a cholesterol-dependentcytolysin that lyses red blood cells (19). Since perfringolysin Omutants still cause murine myonecrosis, the toxin is not essen-tial for the disease, although it also has the ability to affect thehost inflammatory response, particularly PMNL influx into themyonecrotic lesion. Perfringolysin O has been implicated inthe vascular accumulation of leukocytes within blood vesselsand the extracellular matrix of host tissues (5, 8, 10).

There is now good evidence that exotoxin-mediated func-

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

TABLE 1. Complementation of the plc pfoA mutant JIR4444a

Strain Genotype

Plasmid-encoded

toxingene(s)

PerfringolysinO level

(log2 titer)

Mean alpha-toxin level �SD (U/mg ofprotein [102])

JIR325 Rifr Nalr 7.2 � 0.5 1.97 � 0.20JIR4069 JIR325 �pfoA::ermB �1 0.61 � 0.11JIR4444 JIR4069 plc�pJIR1774 �1 �0.09 � 0.01JIR4459 JIR4444(pJIR750) �1 �0.09 � 0.01JIR4460 JIR4444(pJIR871) pfoA� 6.7 � 0.3 �0.10 � 0.01JIR4461 JIR4444(pJIR1642) plc� �1 0.60 � 0.17JIR4462 JIR4444(pJIR1720) pfoA� plc� 6.7 � 0.3 1.90 � 0.06

a Perfringolysin O and phospholipase C assays were performed as previouslydescribed (3). The protein concentration was determined by using a microtiterplate protocol from the BCA Protein Assay Reagent Kit (Pierce), with bovineserum albumin as the standard. The results represent the average of duplicateassays carried out on preparations from three separate cultures of each strain.

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tional modulation of the inflammatory cascade contributes sig-nificantly to the ineffective host response to C. perfringens in-fection (5, 8); vascular leukostasis and leukocyte paucitycannot be explained by the myonecrotic actions of the exotox-ins alone (10). This hypothesis is supported by studies (5, 9)which showed that exposure to these toxins upregulated theproduction of cell adhesion molecules, important factors in-volved in leukocyte migration across the endothelium duringinflammation. More recently, purified alpha-toxin was shownto strongly induce homotypic and heterotypic platelet aggre-gation, which would promote leukostasis within the vasculature(6, 7). Therefore, alpha-toxin seems to be of major importance

with respect to reducing blood flow and impeding the migra-tion of leukocytes to the site of infection. While work carriedout by using isogenic sets of genetically modified strains of C.perfringens that were defective in the production of either al-pha-toxin or perfringolysin O did not provide evidence to sup-port these claims, it did show that a perfringolysin O-deficientstrain produced no detectable leukocyte aggregation within theinfected tissue (10). Since thrombosis was present in manyblood vessels and thrombotic occlusions in these tissues werecompletely free of leukocyte accumulation, these studies pro-vided further evidence that leukocyte paucity is not purely dueto cytotoxic effects.

FIG. 1. Construction of a plc pfoA double mutant by homologous recombination. A single crossover event between the plc gene located on thechromosome of the pfoA mutant JIR4069 (A) and the truncated plc gene located on the suicide vector pJIR1774 led to the construction of theplc pfoA double mutant JIR4444 (B). The insert shows the results of Southern hybridization analysis of EcoRI-digested chromosomal DNA fromthe wild-type strain JIR325 and mutant strains JIR4069 and JIR4444 (lanes 1, 2, and 3, respectively), probed as indicated. Molecular size standardsare 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.

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FIG. 2. Gross pathology of infected mice. BALB/c mice were injected with an isogenic series of JIR4444-derived C. perfringens strains. For eachstrain, 10 6- to 8-week-old mice were injected intramuscularly in the right thigh with 100 �l 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, themice 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

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Previous studies have suggested that these toxins may worksynergistically to produce effects on the inflammatory system(5, 8, 10, 17). To gain a better understanding of this phenom-enon, we decided to construct a C. perfringens strain mutated inboth the alpha-toxin (plc) and perfringolysin O (pfoA) struc-tural genes and then to complement this mutant with variouscombinations of the wild-type genes.

To isolate a plc pfoA double mutant, the pfoA mutantJIR4069 (3) was transformed (14) to tetracycline resistancewith the plc-suicide vector pJIR1774 (Fig. 1). This plasmid is apUK21 (20) derivative that carries an internal 0.53-kb Sau3A-derived plc fragment from pTOX6 (13) and the tet(M) genefrom Tn916. The resultant tetracycline-resistant transformantswere screened on egg yolk agar and horse blood agar for theirability to produce alpha-toxin and perfringolysin O, respec-tively, and a mutant that was unable to produce either toxinwas chosen for further study. This mutant, JIR4444, was alsoresistant to rifampin, nalidixic acid, and erythromycin, as ex-pected for a derivative of JIR4069.

Southern hybridization analysis was used to confirm thatJIR4444 had the genotype expected from a single crossoverbetween the plc fragment carried on pJIR1774 and the plc genelocated on the JIR4069 chromosome (Fig. 1). ChromosomalDNA was purified (1, 3) from wild-type strain JIR325 and mu-tant strains JIR4069 and JIR4444, digested with EcoRI, sub-jected to electrophoresis on 0.8% agarose gels, and transferredto nylon membranes (Hybond N; Amersham). Southern blotswere hybridized overnight at 65°C and washed at high strin-gency (65°C with 0.1� SSC [1� SSC is 0.15 M NaCl plus 0.015M sodium citrate]) with either a 0.95-kb BamHI-HindIII frag-ment from pTOX6 (internal to the plc gene,); a 0.9-kb SpeI-HindIII fragment from pTS302 (internal to pfoA gene) (15);the 3.0-kb EcoRI fragment from pVB101 (V. Burdett, unpub-lished), which contains the tet(M) gene; or a 0.7-kb PCR prod-uct generated from pJIR870, which is internal to the regulatoryvirR gene (11). Each probe was labeled by using digoxigenin-labeled dUTP by random priming as specified by the manu-facturer (Boehringer Mannheim). As expected, the plc probehybridized to a 4.6-kb band in both JIR325 and the pfoA mu-tant JIR4069, whereas two bands (4.7 and 3.3 kb) were ob-served with JIR4444 (Fig. 1). This result was consistent withthe predicted single crossover event. When probed with thepfoA-specific fragment, the expected 6.7-kb hybridizing bandwas observed with JIR325, while JIR4069 and JIR4444 eachhad a 3.6-kb band and a 1.5-kb band, as predicted. To confirmthat the chromosomal rearrangements were specific for the plcgene, the DNA was probed with a virR-specific fragment. In allthree chromosomal preparations, a 2.5-kb band hybridizedwith the probe (data not shown). Finally, only JIR4444 hybrid-ized with the tet(M)-specific probe, with the expected 3.0-kbband being observed (Fig. 1). These results confirmed that

JIR4444 was a plc mutant derived from homologous recombi-nation between pJIR1774 and the JIR4069 chromosome.

The mutated pfoA and plc genes located on the JIR4444chromosome were complemented by transformation with sep-arate C. perfringens-Escherichia coli shuttle vectors that eachcarried a chloramphenicol resistance gene. These plasmids in-cluded the negative control, pJIR750 (4); pJIR871, which car-ries the wild-type pfoA gene (3); pJIR1642, a pJIR750 deriva-tive that contains the wild-type plc gene from pTOX6; andpJIR1720, a pJIR871 derivative that carries the same fragmentfrom pTOX6 and therefore harbors the wild-type plc and pfoAgenes. Each of the JIR4444-derived transformants had thecorrect phenotypes when grown on egg yolk agar and horseblood agar . Quantitative assays were then performed to de-termine the toxin levels produced by each strain. The results(Table 1) were in agreement with the qualitative data. Nodetectable perfringolysin O or hemolytic activity was observedin culture filtrates derived from the pfoA mutants, unless theyhad been complemented with the wild-type pfoA gene, inwhich case hemolytic activity was similar to wild-type levels.Similar results were observed for alpha-toxin activity, althoughalpha-toxin levels in JIR4069 and JIR4444(pJIR1642) were alittle lower than in the wild type (Table 1), for reasons notunderstood at this time. Taken together, these data indicatethat the shuttle plasmids were able to complement the respec-tive plc and pfoA mutations. In particular, the double comple-mentation plasmid pJIR1720 was found to restore both per-fringolysin O and alpha-toxin activity back to wild-type levels.

To quantify the relative contributions of alpha-toxin andtheta-toxin to the pathology of clostridial myonecrosis, BALB/cmice were injected with this isogenic series of JIR4444-derivedC. perfringens strains (Fig. 2). Based upon previous work withsingle toxin knockout strains (3), it was expected that comple-mentation of the double mutant with the alpha-toxin structuralgene would result in a more severe pathology than comple-mentation with the perfringolysin O gene. The results (Fig. 2)were in agreement with this prediction except that blackeningof the thigh was observed to be equally as severe when eitheralpha-toxin or perfringolysin O was produced (Fig. 2E). Syn-ergistic effects of alpha-toxin and perfringolysin O were clearlyobserved in these experiments. Infection with the controlstrain, which did not produce either toxin, resulted in veryminimal pathological changes, whereas the isogenic strain thatwas reconstituted for both toxins produced a pathology thatwas clearly more severe than when alpha-toxin alone was re-constituted. These changes were most apparent in the rapidspread of the disease pathology to the footpad and in the rateat which the mice had to be euthanatized for ethical reasons(Fig. 2).

In separate experiments, mice were killed at different timesafter infection, and the histopathology of the infected muscle

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 ofinfection) and for the ability to move the infected limb (limping). The severity of each pathology parameter was scored as either 0 (no discerniblepathology), 1⁄2 (moderate pathology), or 1 (marked pathology apparent). The number of severely affected mice killed for animal ethics reasons wasalso recorded. Note that cumulative pathology on the y axis refers to the sum of the individual pathology scores from 10 mice, the number 10therefore 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; E, JIR4444(pJIR1642), the double mutant carrying a plc� plasmid; and �, JIR4444(pJIR1720), thedouble mutant carrying a plc� pfoA� plasmid.

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FIG. 3. Hematoxylin-and-eosin-stained sections from infected tissues. Mice were injected as described in the legend to Fig. 2, with the strainsindicated (see Fig. 2). Two mice were randomly chosen from each infected group at various times after injection. After each mouse was killed, theinfected limb was exposed by blunt dissection, allowing observation of the severity and spread of necrosis. Samples of the muscle were thenremoved and fixed overnight in a solution of 0.5% tannic acid (wt/vol) in methanol before paraffin embedding and sectioning for hematoxylin andeosin staining and analysis. Symbols and abbreviations: arrows, leukocyte aggregates; asterisks, bacterial aggregates; mn, myonecrosis; cn,coagulative necrosis; th, thrombosis; bv, blood vessel tissue.

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tissue was examined (Fig. 3). Infection with the wild-type strainproduced the typical pathology of advanced myonecrosis,thrombosis and vascular leukostasis (Fig. 3A). Leukocyte in-filtration of these tissues was minimal and was typically re-stricted to small aggregates at the borders of myonecrosis.Elimination of both alpha-toxin and perfringolysin O produc-tion removed most of the features typical of clostridial myo-necrosis (Fig. 3B). Little or no vascular leukostasis was appar-ent, and numerous leukocytes infiltrated these tissues andmigrated fully to the seat of infection. Indeed, tissues frommice infected with these strains presented almost identicalhistopathology to that observed in previous experiments frommice injected with washed, heat-killed C. perfringens cells (datanot shown).

Reconstituting only perfringolysin O activity in the doublemutant produced vastly different results (Fig. 3C and D).While some areas of these tissues displayed the expected myo-necrosis (Fig. 3C), this pathology was quickly replaced or ob-scured by vast regions of coagulative necrosis, apparently en-hanced by vascular disruption (Fig. 3D). As a result, thepresence or absence of vascular leukostasis was difficult todetermine. In contrast, tissue samples from mice infected withthe alpha-toxin reconstituted double mutant displayed pathol-ogy more typical of clostridial myonecrosis (Fig. 3E and F).Extensive myonecrosis and thrombosis were observed in areassurrounding the seat of infection. Some vascular leukostasiswas apparent but was primarily restricted to arterioles and notto capillaries and venules (Fig. 3E). Numerous leukocytes wereobserved to infiltrate the tissue by 8 h (Fig. 3F), although thisinfiltration appeared to be restricted to nonnecrotic areas.Note that, in a recent study, leukostasis was found to predom-inate in the venular system, particularly early after injection ofcrude toxin preparations or purified alpha-toxin, and wasfound to be triggered by the alpha-toxin-mediated aggregationof activated platelets (6, 7).

Reconstitution of both alpha-toxin and perfringolysin O ac-tivity produced histopathology most similar to that observedwith the alpha-toxin reconstituted strain (Fig. 3G and H).Some vascular leukostasis was observed (Fig. 3G), although itwas not as severe as that observed when infecting with thewild-type strain. The spreading of myonecrosis was very rapidin these tissues, and the coagulative necrosis previously ob-served upon infection with the perfringolysin O reconstitutedstrain appeared to be restricted to the lumen of the bloodvessels (Fig. 3H). Some thrombosis was observed, but it wasmostly replaced or obscured by the coagulative necrosis.

The results of these virulence experiments clearly supportthe hypothesis that alpha-toxin and perfringolysin O have asynergistic effect in the pathology of gas gangrene, particularlywhen the rate at which pathology became apparent in thefootpad and the rate at which the mice were killed due toethical intervention are taken into consideration. Indeed, nomice injected with the doubly reconstituted strain survivedbeyond 10 h in these experiments, so rapid and severe were thecombined effects of both toxins.

It was hoped that the mechanism of this synergy could beelucidated from these experiments, but a complicating factor inthe toxin reconstitution process became apparent during thehistopathology studies. Specifically, the prominence of coagu-lative necrosis in the pathology of infection by the perfringo-

lysin O reconstituted C. perfringens strain was markedly differ-ent from that expected from previous experiments with singletoxin knockout mutants (3, 10, 17). This difference appears tobe a result of the absence of alpha-toxin, since coagulativenecrosis seems to be confined to the lumen of blood vesselswith the double reconstituted strain. Despite this complication,the results clearly show that perfringolysin O and alpha-toxinhave markedly different roles in an infected lesion. Havingmany of the distinctive features of gas gangrene, infection withthe alpha-toxin reconstituted double mutant provides yet moreevidence for the primary role that alpha-toxin plays in theprogression of disease. However, vascular leukostasis and apaucity of leukocyte infiltration, both of which are very typicalin gas gangrene lesions, were absent when perfringolysin O wasnot present. Unfortunately, it was very difficult to show thatthese effects were reversed in the presence of just perfringoly-sin O, due to the massive coagulative necrosis and vasculardisruption that occurs when alpha-toxin is not present to iso-late these effects to the lumen of the vasculature.

Previous studies provided evidence that the ability to pro-duce extracellular collagenase was not a major virulence factorin clostridial myonecrosis (2). Our current results show that adouble mutant unable to produce either alpha-toxin or per-fringolysin O had a disease pathology almost identical to thatseen in mice injected with washed, heat-killed C. perfringenstype A cells. That is, a pathology consistent with a normalinflammatory response that successfully clears a bacterial in-fection. These studies provide further evidence that alpha-toxin and perfringolysin O are the major C. perfringens extra-cellular toxins involved in the pathology of gas gangrene.

J.I.R. gratefully acknowledges research grant support from the Aus-tralian National Health and Medical Research Council.

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Editor: D. L. Burns

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