Clostridial Gas Gangrene: Evidence That a and u Toxins ...

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189 Clostridial Gas Gangrene: Evidence That a and u Toxins Differentially Modulate the Immune Response and Induce Acute Tissue Necrosis Dennis L. Stevens, Rodney K. Tweten, Milena M. Awad, Infectious Diseases Section, VA Medical Center, Boise, and Department of Microbiology, Molecular Biology and Biochemistry, University of Julian I. Rood, and Amy E. Bryant Idaho, Moscow, Idaho; Department of Medicine, University of Washington, Seattle, Washington; Department of Microbiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; Department of Microbiology, Monash University, Clayton, Australia The rapid extension of necrosis and an absence of polymorphonuclear leukocytes (PMNL) at the site of infection are two hallmarks of Clostridium perfringens gas gangrene. While both a and u toxins profoundly affect PMNL function and viability in vitro, their roles in muscle destruction and impairment of the inflammatory response in vivo have not been investigated. Comparative histopathologic examinations were performed on animals infected with either wild-type C. per- fringens, or isogenic, toxin-deficient mutants of C. perfringens. Tissue destruction was modest in animals infected with the a toxin – deficient mutant; destruction was more pronounced in tissues infected with the u toxin – deficient mutant or the wild-type strain. a and u toxins also displayed differing abilities to modulate the inflammatory response. Histopathologic studies in which recombi- nant toxins were injected together with killed, washed C. perfringens further substantiated these tissue-destructive and differential antiinflammatory effects. Clostridium perfringens is the most common organism iso- from degradative products released from skeletal muscle. Whatever the mechanisms, there is mounting evidence that the lated from patients with trauma-induced gas gangrene. Trauma establishes a low oxidation-reduction potential in tissues and clinical manifestations of gas gangrene are in fact related to the elaboration of potent extracellular protein toxins [1, 3, provides a portal of entry plus a readily accessible source of amino acids and peptides necessary for the growth of the toxi- 7, 8]. Historically, a toxin (phospholipase C, PLC) and u toxin genic clostridia. Initially, growth occurs within the devitalized anaerobic milieu; however, aggressive bacterial invasion and (perfringolysin O, PFO) have been considered the major viru- lence factors of C. perfringens [3, 9]. a toxin’s biologic effects destruction of healthy, living tissue rapidly ensues. Thus, the classic features of gas gangrene caused by C. are likely related to its ability to cleave lecithin, the major phospholipid in eukaryotic cell membranes, into phosphoryl- perfringens type A are extensive and fulminant tissue destruc- tion progressing to profound shock and death [1 – 4]. Several choline and diacylglycerol [10]. u toxin is a thiol-activated cytolysin that causes complete hemolysis of red blood cells theories have been proposed regarding the mechanisms of through, in part, its ability to bind to cholesterol [11]. shock and tissue destruction in clostridial gas gangrene. Bullen Previous studies in our laboratory and others have shown [5] concluded that shock and death were the result of progres- that both toxins contribute to the mortality associated with gas sive local and systemic hypoxia caused by dramatically en- gangrene [7, 8, 12, 13], yet the mechanisms responsible for hanced oxygen consumption within the infected tissue. In con- the widespread and progressive tissue destruction and for the trast, Ispolatovskaya [6] argued that shock and death resulted lack of a tissue inflammatory response have not been eluci- dated. We have previously demonstrated in vitro that both a and u toxin have profound effects on polymorphonuclear leukocyte (PMNL) functions of adherence and chemotaxis Received 15 October 1996; revised 13 January 1997. Presented in part: First International Conference on Molecular Biology and [14 – 16] and that these toxins stimulate both PMNL- and endo- Pathogenesis of the Clostridia, Rio Rico, Arizona, January 1995 (abstract B6). thelial cell – dependent mechanisms of leukocyte adherence [17, Animals were cared for according to guidelines established by the Animal 18]. We have hypothesized that toxin-induced dysregulation of Subjects Committee of the Research and Development Committee at the Boise these interactions could reduce the inflammatory response in VA Medical Center. Financial support: United States Department of Veterans Affairs (D.L.S.), infected tissues and may increase vascular leukostasis, leading Australian National Health and Medical Research Council (J.I.R.), and National to local and regional ischemia. In total, these toxin effects Institutes of Health (R.K.T.). would facilitate bacterial invasion of healthy muscle while ex- Reprints or correspondence: Dr. Dennis L. Stevens, Infectious Diseases Section (Bldg. 6), VA Medical Center, 500 West Fort Street, Boise, Idaho panding the margins of tissue necrosis [16, 18]. The present 83702. study investigated the antiinflammatory effects of a and u tox- The Journal of Infectious Diseases 1997; 176:189 – 195 ins in vivo and their roles in local tissue destruction (myo- q 1997 by The University of Chicago. All rights reserved. 0022–1899/97/7601 – 0024$02.00 necrosis), vascular leukostasis, and mortality. / 9d2b$$jy38 05-09-97 17:55:08 jinfa UC: J Infect Downloaded from https://academic.oup.com/jid/article-abstract/176/1/189/900953 by guest on 31 January 2018

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189

Clostridial Gas Gangrene: Evidence That a and u Toxins DifferentiallyModulate the Immune Response and Induce Acute Tissue Necrosis

Dennis L. Stevens, Rodney K. Tweten, Milena M. Awad, Infectious Diseases Section, VA Medical Center, Boise, and Departmentof Microbiology, Molecular Biology and Biochemistry, University ofJulian I. Rood, and Amy E. Bryant

Idaho, Moscow, Idaho; Department of Medicine, University ofWashington, Seattle, Washington; Department of Microbiology,

University of Oklahoma Health Sciences Center, Oklahoma City,Oklahoma; Department of Microbiology, Monash University,

Clayton, Australia

The rapid extension of necrosis and an absence of polymorphonuclear leukocytes (PMNL) at thesite of infection are two hallmarks of Clostridium perfringens gas gangrene. While both a and u

toxins profoundly affect PMNL function and viability in vitro, their roles in muscle destructionand impairment of the inflammatory response in vivo have not been investigated. Comparativehistopathologic examinations were performed on animals infected with either wild-type C. per-fringens, or isogenic, toxin-deficient mutants of C. perfringens. Tissue destruction was modest inanimals infected with the a toxin–deficient mutant; destruction was more pronounced in tissuesinfected with the u toxin–deficient mutant or the wild-type strain. a and u toxins also displayeddiffering abilities to modulate the inflammatory response. Histopathologic studies in which recombi-nant toxins were injected together with killed, washed C. perfringens further substantiated thesetissue-destructive and differential antiinflammatory effects.

Clostridium perfringens is the most common organism iso- from degradative products released from skeletal muscle.Whatever the mechanisms, there is mounting evidence that thelated from patients with trauma-induced gas gangrene. Trauma

establishes a low oxidation-reduction potential in tissues and clinical manifestations of gas gangrene are in fact related tothe elaboration of potent extracellular protein toxins [1, 3,provides a portal of entry plus a readily accessible source of

amino acids and peptides necessary for the growth of the toxi- 7, 8].Historically, a toxin (phospholipase C, PLC) and u toxingenic clostridia. Initially, growth occurs within the devitalized

anaerobic milieu; however, aggressive bacterial invasion and (perfringolysin O, PFO) have been considered the major viru-lence factors of C. perfringens [3, 9]. a toxin’s biologic effectsdestruction of healthy, living tissue rapidly ensues.

Thus, the classic features of gas gangrene caused by C. are likely related to its ability to cleave lecithin, the majorphospholipid in eukaryotic cell membranes, into phosphoryl-perfringens type A are extensive and fulminant tissue destruc-

tion progressing to profound shock and death [1–4]. Several choline and diacylglycerol [10]. u toxin is a thiol-activatedcytolysin that causes complete hemolysis of red blood cellstheories have been proposed regarding the mechanisms ofthrough, in part, its ability to bind to cholesterol [11].shock and tissue destruction in clostridial gas gangrene. Bullen

Previous studies in our laboratory and others have shown[5] concluded that shock and death were the result of progres-that both toxins contribute to the mortality associated with gassive local and systemic hypoxia caused by dramatically en-gangrene [7, 8, 12, 13], yet the mechanisms responsible forhanced oxygen consumption within the infected tissue. In con-the widespread and progressive tissue destruction and for thetrast, Ispolatovskaya [6] argued that shock and death resultedlack of a tissue inflammatory response have not been eluci-dated. We have previously demonstrated in vitro that botha and u toxin have profound effects on polymorphonuclearleukocyte (PMNL) functions of adherence and chemotaxisReceived 15 October 1996; revised 13 January 1997.

Presented in part: First International Conference on Molecular Biology and [14–16] and that these toxins stimulate both PMNL- and endo-Pathogenesis of the Clostridia, Rio Rico, Arizona, January 1995 (abstract B6). thelial cell–dependent mechanisms of leukocyte adherence [17,

Animals were cared for according to guidelines established by the Animal18]. We have hypothesized that toxin-induced dysregulation ofSubjects Committee of the Research and Development Committee at the Boisethese interactions could reduce the inflammatory response inVA Medical Center.

Financial support: United States Department of Veterans Affairs (D.L.S.), infected tissues and may increase vascular leukostasis, leadingAustralian National Health and Medical Research Council (J.I.R.), and National

to local and regional ischemia. In total, these toxin effectsInstitutes of Health (R.K.T.).would facilitate bacterial invasion of healthy muscle while ex-Reprints or correspondence: Dr. Dennis L. Stevens, Infectious Diseases

Section (Bldg. 6), VA Medical Center, 500 West Fort Street, Boise, Idaho panding the margins of tissue necrosis [16, 18]. The present83702.

study investigated the antiinflammatory effects of a and u tox-The Journal of Infectious Diseases 1997;176:189–195 ins in vivo and their roles in local tissue destruction (myo-q 1997 by The University of Chicago. All rights reserved.0022–1899/97/7601–0024$02.00 necrosis), vascular leukostasis, and mortality.

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Table 1. Influence of a and u toxins on the dynamics of inflamma-Materials and Methodstion and myonecrosis in experimental gas gangrene.

Exotoxins. Purification and analysis of native C. per-PMNL Tissue

fringens a and u toxins from log-phase culture supernatants influx destructionhave been described previously in detail [7]. Recombinant u Type of Clostridium perfringenstoxin was prepared from Escherichia coli expressing the PFO injected Early Late Early Late

toxin gene as described [19, 20]. PFO prepared from this cloneViablewas functionally and immunologically identical to the native

Wild-type (ATCC 13124) 0 0 / ////protein [19]. Activity of stock recombinant u toxin was deter- JIR325 0 0 / ////mined by hemolysis of a 1% suspension of sheep red blood Killedcells [7] and found to be 12,800 hemolytic units (HU)/mL Alone /// //// 0 0

/ a toxin 0 0 // //(specific activity, 4.3 HU/mg protein). Recombinant a toxin/ u toxin 0 / // //(PLC; provided by J. Tso, Protein Design Labs, Palo Alto, CA)

a toxin–deficient (JIR4120) ND //// ND /[21] was purified from E. coli harboring pPLC 8, which con-

a toxin–reconstituted (JIR4121) 0 0 // ////tains the C. perfringens phospholipase C gene. Phospholipase u toxin–deficient (JIR4081) / / // ////activity (determined by a synthetic chromogenic substrate u toxin–reconstituted (JIR4082) 0 0 / ////assay) of stock a toxin was 284 PLC U/mL (specific activity,

NOTE. ND Å not done.0.284 PLC U/mg protein). These toxin assays have been de-scribed previously in detail [7]. Both recombinant toxin prepa-rations yielded a single band on SDS-polyacrylamide gel elec- with amino acids, salts, and vitamins as previously describedtrophoresis (PAGE) with silver staining. u toxin and a toxin [24]. This formulation is optimal for toxin production [10]. Forstocks were also assayed for the presence of contaminating strains harboring plasmids, this media included either erythro-endotoxin by a limulus amebocyte assay (Associates of Cape mycin (50 mg/mL), chloramphenicol (30 mg/mL), or a combina-Cod, Woods Hole, MA); they contained 0.001 ng/HU and 22.7 tion of these antimicrobials. Inocula were prepared as pre-ng/PLC U, respectively. Protein concentrations of all toxin viously described [25]. In brief, 4-h log-phase cultures werepreparations were measured by UV absorption at 280 and 260 pelleted by centrifugation, washed once in sterile normal salinenm [22]. (NS), resuspended in NS and diluted to the desired concentra-

Antibodies. The neutralizing monoclonal antibodies tion using a standard plot of absorbance at 650 nm versusagainst u toxin (3H10) and a toxin (1C6) were gifts from colony forming units (cfu). The bacterial concentration of theHiroko Sato (NIH, Tokyo) [23]. A polyclonal anti–C. per- inoculum was verified by duplicate platings of serial dilutionsfringens antibody preparation was generated in goats immu- on blood agar plates. The prepared inoculum was capped andnized against whole, unwashed, formalinized C. perfringens in placed on ice until use.Freund’s adjuvant, following a standard immunization protocol Experimental gas gangrene. Female Swiss Webster micefor antibody production. (10/group) were injected intramuscularly in the right upper

Organisms. C. perfringens (ATCC 13124) was purchased thigh with either 2.5–5.0 1 108 cfu of washed, log-phasein lyophilized form (American Type Culture Collection, Rock- ATCC 13124 C. perfringens or 1.0–3.0 1 109 cfu of C. per-ville, MD). Mutant strains of C. perfringens were constructed fringens JIR325 or with mutant derivatives of this strain in 100as described below. All organisms were maintained either in mL of NS. Infected animals were observed carefully for thechopped meat–glucose broth alone or, in the case of organisms first 24 h for signs and symptoms of infection, and toxicitycarrying antibiotic resistance plasmids, in the presence of eryth- and mortalities were noted. For histopathology studies, 10 addi-romycin or chloramphenicol. The purity and phenotype of each tional mice were included in each group, and 2 mice per groupstrain were verified before each experiment by inoculating were sacrificed by cervical dislocation at 2, 4, 8, 12, and 24 hblood and egg yolk agar plates, which contained appropriate after infection. The infected muscle tissue was exposed andantibiotics as necessary. photographed, and a portion was formalin-fixed, embedded in

Chromosomal mutants of C. perfringens strain JIR325 that paraffin, stained with hematoxylin-eosin, and examined by mi-did not produce either a or u toxin (table 1) were constructed by croscopy.homologous recombination with suicide plasmids containing In a separate series of experiments, mice were injected ininsertionally inactivated a or u toxin structural genes, respec- the right upper thigh muscle with either washed, UV-killed C.tively [12]. Toxin production was reconstituted in toxin-nega- perfringens (ATCC 13124) alone or together with recombinanttive mutants by the introduction of recombinant plasmids con- u toxin (32 HU) or a toxin (1 PLC U). Animals were sacrificedtaining the respective structural genes [12]. These strains have at various times, and the muscle tissue surrounding the injectionbeen characterized previously [12]. site was prepared for routine histopathology.

Growth conditions and inoculum preparation. Organisms Active and passive immunization studies. In protectionstudies, animals were pretreated 3–5 h prior to infection withwere grown in basal proteose peptone media supplemented

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191JID 1997;176 (July) Clostridial Toxins in Gas Gangrene

a single dose (500 mL) of neutralizing monoclonal antibodyagainst either u or a toxin or with goat polyclonal anti–wholeC. perfringens antisera delivered intraperitoneally at two sepa-rate sites.

Alternatively, mice were actively immunized against C. per-fringens a or u toxin, using an adjuvant system (MPL/TDM;Ribi ImmunoChem Research, Hamilton, MT) according to themanufacturer’s recommendations. This system has been shownto be superior to Freund’s adjuvant both in eliciting an antibodyresponse and in minimizing animal discomfort [26]. Each vialof prewarmed adjuvant emulsion was reconstituted with 2.1mL of NS containing recombinant u (7.5 mg/mL) or a (0.4mg/mL) toxin. These concentrations were sufficient to induceantibody production as determined by Western blot (not shown)but were not toxic to the animals. Animals received 100 mL

Figure 1. Micrograph showing histopathology of experimental gasof this emulsion intraperitoneally in each of two sites. Control gangrene in hematoxylin-eosin–stained murine tissues obtained 4 hanimals received adjuvant alone in the same fashion. Three after intramuscular injection of viable wild-type Clostridium per-weeks after the initial immunization, animals were boosted fringens (ATCC 13124). Extensive myonecrosis is evident, and scant

no. of inflammatory cells are present at margin between healthy andusing the same procedure. One week after boosting, animalsnecrotic tissue, especially within small vessels (arrow).were challenged with an LD100 of C. perfringens ATCC 13124

as described above.

improve outcome (not shown). Similarly, passive immunizationResults

of mice with monoclonal antibody against a toxin containingsufficient antibody to neutralize 226 PLC U provided signifi-Clinical features and histopathology of experimental gas

gangrene caused by wild-type C. perfringens infection. Mice cant protection (figure 2B). Passive immunization with antiseraraised against whole, unwashed C. perfringens provided theinjected intramuscularly with ú3.0 1 108 cfu of log-phase C.

perfringens ATCC 13124 developed marked swelling within greatest protection (figure 2). The stringency of these experi-mental infections is very high because antibiotics were not used3–6 h, followed by blackening of the foot, sloughing of fur,

crepitus beneath the skin, and severe muscle destruction with and devitalized tissue was not removed, allowing continuedbacterial growth and toxin production. Thus, complete protec-extension of infection onto the abdominal wall and thorax.

Hematuria developed by 6–8 h, and mortality was 100% by tion was not realized because the finite amount of neutralizingantibodies administered was likely consumed.9–12 h. Histopathology revealed localized myonecrosis as

early as 1 h (not shown), becoming extensive by 4–6 h (figure In an attempt to circumvent this problem, we actively immu-nized separate groups of animals against either u or a toxin1). A corresponding lack of a tissue inflammatory response

was seen in the areas of intense muscle necrosis. When present, prior to bacterial challenge. Active immunization against eithertoxin also afforded protection: 14 h after infection, 30% ofinflammatory cells were visible at the margins between healthy

and necrotic tissue, and marked leukostasis was seen in adja- sham-immunized controls survived, versus 70% of animals im-munized against u toxin, and 90% of animals immunizedcent small capillaries and venules (figure 1, arrow). The physio-

logic and histologic findings in these experiments are in agree- against PLC. However, the overall course of infection, includ-ing the time required to reach 50% mortality, was the samement with our previous work utilizing this model [24, 25, 27]

and with clinical reports describing the histopathology of gas for animals actively immunized against these exotoxins andthose receiving passive immunization.gangrene in humans [28].

Attenuation of wild-type infection by immunization against Toxin-induced attenuation of the inflammatory response tokilled organisms. Intramuscular injection of washed, UV-u and a toxins. Mice were passively immunized prior to bac-

terial challenge with either monoclonal antibody against u toxin killed C. perfringens in mice resulted in an intense granulocyticinflammatory response as early as 1–4 h after injection (figureor polyclonal anti–whole C. perfringens antisera containing

sufficient antibody to neutralize 53 or 800 HU of u toxin activ- 3A), becoming maximal by 18–24 h (not shown).Next, killed, washed C. perfringens were injected intramus-ity, respectively. Compared with mortality rates and time to

death in nonimmunized control animals, mortality in animals cularly together with recombinant u (56 HU) or a (1 PLCU) toxin in an effort to determine if these toxins altered theimmunized with either of the antibody preparations was re-

duced and time to death was delayed (figure 2A). Subsequent inflammatory response observed with killed C. perfringensalone. Of interest, in both cases, pathologies were markedlyexperiments in which higher concentrations or multiple doses

of monoclonal anti–u toxin antibody were given did not further different than the response to killed organisms alone and very

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the experiments described above, and induction of infectionwith C. perfringens JIR325 required an 8- to 10-fold higherinoculum. Further, the clinical course was less fulminant andmortality was lower at 24 h (50%–70%, table 2). Despitethese differences, the extent of myonecrosis and absence ofinfiltrating PMNL were similar to those induced by the standardATCC strain (table 1). Mutant strains lacking either u or a

toxin induced myonecrosis (table 1), but the course was moreindolent than that produced by strain JIR325, as judged byvisible signs of illness and toxicity and by mortality (table 2)[12]. In fact, the a toxin–deficient mutant (JIR4120) did notcause a lethal infection (table 2). Nonetheless, myonecrosiswas extensive, but unlike with the wild-type infection, an im-pressive tissue inflammatory response was observed in re-sponse to this strain (figure 4A). Conversely, significant myo-necrosis and a markedly attenuated inflammatory reaction atthe site of infection were observed in animals infected with astrain in which the ability to produce a toxin had been geneti-cally restored (JIR4121, table 2). Compared with neutrophilinflux from parent strain JIR325, neutrophil influx into tissuesharboring u toxin–deficient organisms (JIR4081) was modestlyincreased early in the course of infection (figure 4B) but didnot increase over time (table 1). These results further supportthe concept that both u and a toxins cause myonecrosis andattenuate the acute inflammatory reaction, although a toxin isclearly the more potent toxin.

Discussion

Histologically important features of clostridial gas gangreneFigure 2. Effect of passive immunization on mortality of mice withof humans and experimental animals include the rapid exten-experimental gas gangrene. Animals were pretreated (3–5 h before

infection) with 500 mL of neutralizing monoclonal antibody against sion of necrosis into healthy tissue and an aberrant cellularu (A) or a (B) toxin or with polyclonal anti–whole Clostridium immune response characterized by a paucity of PMNL in in-perfringens anti-serum. Results were compared (Fisher’s exact test, fected soft tissues together with marked leukostasis in adjacent1-tailed) with those of controls receiving 500 mL of normal saline.

vessels [24, 25, 27, 28]. Results from the present study suggest* P õ .05.that the major clostridial virulence factors, a and u toxins, havedirect and unique roles in these pathologies. While a luxuriousinflux of PMNL occurred at the site of intramuscular injectionsimilar to that seen in the viable, wild-type infection (figure 3B,

C). Specifically, both toxins induced significant myonecrosis as of killed, washed C. perfringens, the addition of either a or utoxin attenuated this tissue inflammatory response, stimulatedearly as 4 h after injection (table 1). At 4 h after injection of

killed organisms plus u toxin, PMNL were absent from the accumulation of PMNL in adjacent vessels, and induced tissuedestruction characteristic of wild-type infection. Although in-tissues (not shown), but by 16 h, a modest inflammatory re-

sponse was noted (figure 3C, table 1). PMNL were also absent jection of a and u toxins produced similar pathologies, themagnitude and duration of the antiinflammatory effects of aat 4 h in tissue injected with killed organisms plus a toxin

(table 1); however, this attenuation of the PMNL response was toxin were greater than those elicited by u toxin. Studies utiliz-ing toxin-deficient strains of C. perfringens confirmed thissustained for 16 h (figure 3B). These results demonstrate that

both u and a toxins, but not killed bacilli, can induce myo- finding. Taken together, these results support the concept thatboth toxins play important roles in myonecrosis and attenuationnecrosis in the mouse model and that both a and u toxins

attenuate the accumulation of PMNL at the site of infection, of the acute inflammatory reaction and suggest that the toxinsmay act via different mechanisms.albeit to different degrees.

Comparison of the gross anatomic and histopathologic as- In vitro evidence suggests that a and u toxins contribute tothe paucity of PMNL in gas gangrene via direct toxin-inducedpects of gas gangrene caused by wild-type and genetically

altered C. perfringens. The parent strain used in the genetic alterations of PMNL viability and function [7, 16]. Indeed,destruction of emigrating PMNL at the site of infection wherestudies (JIR325) was different from the ATCC strain used in

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193JID 1997;176 (July) Clostridial Toxins in Gas Gangrene

Figure 3. Histopathology of tissue injected with killed Clostridiumperfringens. Mice were injected intramuscularly with killed, washedC. perfringens alone (A) or together with u (C) or a (B) toxin. A,Intense influx of polymorphonuclear leukocytes (PMNL) 4 h afterinjection. Inflammation was attenuated by u toxin at 4 h (not shown),but modest inflammatory response was apparent at 16 h (C). In con-trast, PMNL influx was attenuated by a toxin at both 4 (not shown)and 16 h (B). Both toxins caused extensive myonecrosis (B, C).

high concentrations of cytotoxic C. perfringens exotoxins are late in blood vessels near the site of infection cannot beexplained by these mechanisms. In fact, PMNL accumulationelaborated together with toxin-induced deficits in the ability of

PMNL to undergo directed migration could account for the within the vasculature (leukostasis) is a consistent findingwhenever a or u toxin is present within the tissues (i.e., inabsence of inflammatory cells in the tissues [7, 16].

Results from the present study are compatible with these human cases of gas gangrene, when experimental infectionin animals is initiated with either wild-type or toxin-restoredpossibilities; however, the observation that PMNL accumu-

Table 2. Virulence of wild-type and toxin-deficient mutants of Clostridium perfringens in experimentalgas gangrene.

Toxinprofile

Inoculum size Time (h) to 50% Mortality (%) atOrganism (cfu in 100 mL) a u mortality 18–24 h

Wild-type (ATCC 13124) 1.0–3.21 108 / / 5–8 100JIR325 (parent) 2.3–3.11 109 / / 15.5–24.0 50–70JIR4120 (plc-inactivated) 3.45 1 109 0 / N/A 0JIR4121 (plc-reconstituted) 1.55 1 109 / / 12 100JIR4081 (pfo-inactivated) 2.75 1 109 / 0 18 40JIR4082 (pfo-reconstituted) 2.15 1 109 / / 18 30

NOTE. plc Å a toxin gene; pfo Å u toxin gene.

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194 Stevens et al. JID 1997;176 (July)

1) on endothelial cells in vitro [17, 18]. Further, we have shownthat a toxin up-regulates both endothelial leukocyte adhesionmolecule 1 and ICAM-1 on endothelial cells [18]. These find-ings support our hypothesis that reduced inflammation at thesite of infection is due to toxin-induced dysregulation of PMNLand endothelial cell adherence mechanisms.

Interference of PMNL migration to the site of clostridialproliferation could impair the host’s ability to arrest the spreadof infection. Further, hyperadhesion of PMNL to endothelialsurfaces in the presence of bacterial or host-derived neutrophilactivators (e.g., bacterial toxins or endogenous cytokines andlipid autocoids, respectively) could result in the unconstrainedgeneration and release of oxygen radicals and intracellular hy-drolases, thereby compromising vascular integrity and reducingeffective tissue perfusion. Declining oxygen concentrations intissue would favor anaerobic glycolysis and lactic acid produc-tion by the involved muscles, resulting in reduced oxidation-reduction potential and enhanced conditions for growth of theclostridia. Thus, once initiated and left unchecked, this cycleof events could result in rapid progression of tissue necrosis.

In summary, the absence of PMNL at the site of infectionin gas gangrene caused by C. perfringens is related to theeffects of both u and a toxins. Specifically, both toxins impededirected migration of PMNL and destroy PMNL at the site ofinfection. In addition, toxin-induced dysregulation of PMNL–endothelial cell adhesive interactions contribute to vascularleukostasis, microvascular injury, perfusion deficits, and therapid invasion of viable tissues.

Figure 4. Histopathology of gas gangrene caused by genetic mu-tants of Clostridium perfringens. Mice were injected intramuscularly Referenceswith mutant strains that lacked ability to express a toxin. A, a toxin–deficient strain (JIR4120) induced extensive tissue necrosis and was 1. Smith LDS. Clostridium perfringens. In: Smith LDS, ed. The pathogenicassociated with marked inflammatory response at 16 h. a toxin– anaerobic bacteria. Springfield, IL: CC Thomas, 1975:115–76.reconstituted strain (JIR4121) induced marked tissue destruction and 2. Finegold SM, George WL, Mulligan ME. Anaerobic infections. Part I.impaired PMNL influx comparable to that induced by wild-type strain Dis Mon 1985;31:50–7.(see figure 1A). Polymorphonuclear leukocyte influx into tissues har- 3. MacLennan JD. The histotoxic clostridial infections of man. Bacteriologicboring u toxin–deficient organisms (JIR4081) was modestly increased Rev 1962;26:177–276.early in course of infection (6 h, B) but did not increase over time. 4. Darke SG, King AM, Slack WK. Gas gangrene and related infection:

classification, clinical features and aetiology, management and mortal-ity. A report of 88 cases. Br J Surg 1977;64:104–12.

5. Bullen JJ. Role of toxins in host-parasite relationships. In: Montie TC,genetic mutants, or when muscle is injected with killed organ-Kadis S, Ajl SJ, eds. Microbial toxins. London: Academic Press, 1970:

isms plus toxins). 233–75.Thus, we suggest that these C. perfringens exotoxins impede 6. Ispolatovskaya MV. Type A Clostridium perfringens toxin. In: Kadis S,

Montie TC, Ajl SJ, eds. Microbial toxins. New York: Academic Press,the egress of PMNL via direct deleterious effects upon neutro-1972:109–58.phil margination, diapedesis, and chemotaxis and that leuko-

7. Stevens DL, Troyer BE, Merrick DT, Mitten JE, Olson RD. Lethal effectsstasis begins with enhancement of the interaction of PMNLand cardiovascular effects of purified a- and u-toxins from Clostridium

with the vascular endothelium adjacent to the site of infection. perfringens. J Infect Dis 1988;157:272–9.Disruption or dysregulation of this delicate adherence mecha- 8. Asmuth DA, Olson RD, Hackett SP, et al. Effects of Clostridium per-

fringens recombinant and crude phospholipase C and u toxins on rabbitnism could result in tight binding of PMNL to endothelium,hemodynamic parameters. J Infect Dis 1995;172:1317–23.thus preventing diapedesis and contributing to the accumulation

9. Smith LDS. Virulence factors of Clostridium perfringens. Rev Infect Disof PMNL within vessels. For example, we have recently dem-1979;1:254–60.

onstrated that u toxin enhanced the expression of proadhesive 10. Mollby RC, Wadstrom T. Purification of phospholipase C (alpha-toxin)molecules, such as CD11b/CD18, on PMNL [16] and platelet- from Clostridium perfringens. Biochim Biophys Acta 1973;321:569–

84.activating factor and intracellular adhesion molecule 1 (ICAM-

/ 9d2b$$jy38 05-09-97 17:55:08 jinfa UC: J Infect

Downloaded from https://academic.oup.com/jid/article-abstract/176/1/189/900953by gueston 31 January 2018

Page 7: Clostridial Gas Gangrene: Evidence That a and u Toxins ...

195JID 1997;176 (July) Clostridial Toxins in Gas Gangrene

11. Harris RW, Sims PJ, Tweten RK. Evidence that Clostridium perfringens 19. Tweten RK. Cloning and expression in Escherichia coli of the perfringo-theta-toxin induces colloid-osmotic lysis of erythrocytes. Infect Immun lysin O (theta-toxin) gene from Clostridium perfringens and character-1991;59:2499–501. ization of the gene product. Infect Immun 1988;56:3228–34.

12. Awad MM, Bryant AE, Stevens DL, Rood JI. Virulence studies on chromo- 20. Tweten RK. Nucleotide sequence of the gene for perfringolysin O (theta-somal a-toxin and u toxin mutants constructed by allelic exchange toxin) from Clostridium perfringens: significant homology with theprovide genetic evidence for the essential role of a-toxin in Clostridium genes for streptolysin O and pneumolysin. Infect Immun 1988;56:3235–perfringens–mediated gas gangrene. Mol Microbiol 1995;15:191–202. 40.

13. Ninomiya M, Matsushita O, Minami J, Sakamoto H, Nakano M, Okabe 21. Tso JY, Siebel C. Cloning and expression of the phospholipase C geneA. Role of alpha-toxin in Clostridium perfringens infection determined from Clostridium perfringens and Clostridium bifermentans. Infect Im-by using recombinants of C. perfringens and Bacillus subtilis. Infect mun 1989;57:468–76.Immun 1994;62:5032–9. 22. Layne E. Spectrophotometric and turbidimetric methods for measuring

14. Stevens DL, Mitten J, Henry C. Effects of a and u toxins from Clostridium proteins. In: Colowick SP, Kaplan ND, eds. Methods in enzymology.perfringens on human polymorphonuclear leukocytes. J Infect Dis 1987; New York: Academic Press, 1957:447–54.156:324–33. 23. Sato H. Monoclonal antibodies against bacteria. 3rd ed. New York: Aca-

15. Stevens DL, Gibbons AE, Bergstrom RA. Ultrastructural changes in hu- demic Press, 1986.man granulocytes induced by purified exotoxins from Clostridium per- 24. Stevens DL, Maier KA, Laine BM, Mitten JE. Comparison of clindamycin,fringens. Washington, DC: American Society for Microbiology, 1989.

rifampin, tetracycline, metronidazole, and penicillin for efficacy in pre-16. Bryant AE, Bergstrom R, Zimmerman GA, et al. Clostridium perfringens

vention of experimental gas gangrene due to Clostridium perfringens.invasiveness is enhanced by effects of theta toxin upon PMNL structure

J Infect Dis 1987;155:220–8.and function: the roles of leukocytotoxicity and expression of CD11/

25. Stevens DL, Laine BM, Mitten JE. Comparison of single and combinationCD18 adherence glycoprotein. FEMS Immunol Med Microbiol 1993;

antimicrobial agents for prevention of experimental gas gangrene caused7:321–36.by Clostridium perfringens. Antimicrob Agents Chemother 1987;31:17. Whatley RE, Zimmerman GA, Stevens DL, Parker CJ, McIntyre TM,312–6.Prescott SM. The regulation of platelet activating factor synthesis in

26. Rudbach JA, Cantrell JL, Ulrich JT. Molecularly engineered immuno-endothelial cells—the role of calcium and protein kinase C. J Biolstimulators. Tech Adv Vaccine Dev 1988;443–54.Chem 1989;11:6325–33.

27. Stevens DL, Bryant AE, Adams K, Mader JT. Evaluation of hyperbaric18. Bryant AE, Stevens DL. Phospholipase C and perfringolysin O from Clos-oxygen therapy for treatment of experimental Clostridium perfringenstridium perfringens upregulate endothelial cell– leukocyte adherenceinfection. Clin Infect Dis 1993;17:231–7.molecule 1 and intercellular leukocyte adherence molecule 1 expression

28. McNee JW, Dunn JS. The method of spread of gas gangrene into livingand induce interleukin-8 synthesis in cultured human umbilical veinendothelial cells. Infect Immun 1996;64:358–62. muscle. Br Med J 1917;1:727–9.

/ 9d2b$$jy38 05-09-97 17:55:08 jinfa UC: J Infect

Downloaded from https://academic.oup.com/jid/article-abstract/176/1/189/900953by gueston 31 January 2018