SUPPLEMENT ARTICLE

The Role of Clostridial Toxins

in the Pathogenesis of Gas Gangrene
Dennis L. Stevens1,3 and Amy E. Bryant1,2
1

Veterans Affairs Medical Center, Boise, Idaho; and 2University of Idaho, Moscow; and 3University of Washington School of Medicine, Seattle

Clostridium perfringens gas gangrene is, without a doubt, the most fulminant necrotizing infection that affects
humans. In victims of traumatic injury, the infection can become well established in as little as 68 h, and
the destruction of adjacent healthy muscle can progress several inches per hour despite appropriate antibiotic
coverage. Shock and organ failure are present in 50% of patients and, among these, 40% die. Despite modern
medical advances and intensive-care regimens, radical amputation remains the single best life-saving treatment.
Over the past century, much has been learned about the pathogenesis of this disease, and novel therapies are
on the horizon for patients with this devastating infection.
THE ORGANISM AND ITS TOXINS
Clostridium perfringens is a gram-positive, spore-forming, nonmotile, rod-shaped organism commonly found
in soil and in the intestines of humans and other animals. The species has been divided into 5 distinct types,
AE. Of these subgroups, C. perfringens type A causes
the majority of human infections. Although classified
as an anaerobe, C. perfringens is somewhat aerotolerant.
Under optimal conditions, its generation time can be
as little as 810 min, and growth is accompanied by
abundant gas production.
Gas gangrene caused by C. perfringens is characterized by extensive local destruction of muscle (myonecrosis), rapid destruction of viable tissue, shock, and,
ultimately, death. Of the many extracellular toxins produced by this organism, the a (phospholipase C, PLC)
and v (perfringolysin O, PFO) toxins are its major virulence factors. PLCs role as the major lethal factor in

This material is based on work supported by the Office of Research and

Development, Medical Research Service, Department of Veterans Affairs (D.L.S.).
Reprints or correspondence: Dr. Dennis L. Stevens, Infectious Diseases Section,
Veterans Affairs Medical Center, 500 West Fort St., Bldg. 45, Boise, ID 83702
(dlsteven@mindspring.com).
Clinical Infectious Diseases 2002; 35(Suppl 1):S93100
 2002 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2002/3505S1-0018$15.00

C. perfringens infections is supported by numerous

studies that have used a variety of approaches [1]. Both
active and passive immunization of animals against
PLC or its C-terminal fragment are protective in experimental wild-type infections. Similarly, experimental
infections established with genetic mutants of C. perfringens lacking PLC or with strains that produce less
PLC were markedly less fulminant, and mortality was
significantly reduced [2].
The importance of PFO in the pathogenesis of gas
gangrene has been largely controversial, despite the
early knowledge of its hemolytic nature and its serological and antigenic relationships to the cholesterolbinding cytolysins from Streptococcus pyogenes, Streptococcus pneumoniae, and Listeria monocytogenes.
Recently, the amino acid and nucleotide sequences of
pneumolysin, streptolysin O, and PFO have been determined [36]. Impressive homology exists among the
amino acid sequences of these toxins, particularly in
the region that contains the cysteine residue near the
amino terminus, where a highly conserved segment of
12 amino acids is identical for all 3 toxins. Some of
these toxins, including PFO, have been shown to facilitate the growth of their respective organisms within
mammalian phagocytic cells. Furthermore, experimental animal studies have demonstrated the protective efficacy of several antibody preparations against these

Clostridial Myonecrosis

CID 2002:35 (Suppl 1) S93

Figure 1. Effects of clostridial exotoxins on cardiac index (CI). Awake rabbits (n p 6 per group) were slowly infused (0.33 mL/min) via catheter
in the central vein with 50 mL of (1) sterile normal saline, (2) 150 hemolytic units (HU) of recombinant v toxin, (3) 4050 units of recombinant
phospholipase C (PLC), or (4) a crude clostridial toxin preparation that contained 150 HU v toxin activity and 50 units PLC activity. CI was measured
over a 3-h period by thermodilution. *Values significantly different from baseline. Data adapted from Asmuth et al. [9].

toxins [1]. Thus, these studies support a principal role for thiolactivated cytolysins in the pathogenesis of their respective
diseases.
PLC and PFO each contribute to the morbidity and morality
of gas gangrene by uniquely different mechanisms, which are
detailed in the following sections. PLC is hemolytic, is cytotoxic
to platelets and leukocytes, and increases capillary permeabilityeffects that are likely related to its ability to cleave sphingomyelin and the phosphoglycerides of choline, ethanolamine,
and serine present in eukaryotic cell membranes. PLC requires
calcium for optimal activity. Zinc enhances a-toxin production
in culture and is essential for its activity in vivo. Histidine
residues have been shown to be essential for the binding of
zinc ions. Basak et al. [7] have recently crystallized a-toxin and
have provided preliminary X-ray diffraction analysis of the protein. Titball et al. [8] have determined that the protein is composed of 2 functional domains: the N-terminal domain possesses the phospholipase C activity and the C-terminal domain
binds to eukaryotic cell membranes.
PATHOGENESIS OF SHOCK AND ORGAN
FAILURE
Hemodynamic collapse is a common occurrence in patients
with gas gangrene caused by C. perfringens. In experimental
studies, both PLC and PFO uniquely contribute to the develS94 CID 2002:35 (Suppl 1) Stevens and Bryant

opment of shock. For example, a prompt reduction in cardiac

index (CI) occurred in rabbits that received either PLC or a
crude toxin preparation [9] (figure 1). Although many physiological mechanisms could contribute to such a reduction, we
subsequently demonstrated a direct reduction in myocardial
contractility (dF/dt) in isolated atrial strips bathed with recombinant PLC (rPLC) [9]. As reflected by the increased mortality
in the rabbits that received rPLC and crude toxin, a greater
reduction in CI was also measured in these groups compared
with those that received recombinant PFO (rPFO) or normal
saline [9]. Similarly, a marked decline in mean arterial pressure
(MAP) was observed in rabbits treated with rPLC and crude
toxin, although these effects were delayed until the later stages
of the experiment (figure 2) [9]. Thus, rabbits that received
PLC-containing toxin preparations maintained MAP in the face
of a falling CI by as-yet-uncharacterized compensatory mechanism for a brief period before hypotension ultimately occurred. The physiological mechanisms that maintained MAP
did not include significant changes in heart rate or central
venous pressure until the terminal stages of the experiments,
if at all [9].
In contrast, rabbits treated with rPFO demonstrated changes
most characteristic of warm shock, with profound falls in
peripheral vascular resistance after 1 h of toxin infusion [9,
10]. Of interest, rabbits that received crude toxin (containing
both PLC and PFO activity) demonstrated peripheral vascular

Figure 2. Effects of clostridial exotoxins on mean arterial pressure. Awake rabbits (n p 6 per group) were slowly infused (0.33 mL/min) via catheter
in the central vein with 50 mL of (1) sterile normal saline, (2) 150 hemolytic units (HU) of recombinant v toxin, (3) 4050 units of recombinant
phospholipase C (PLC), or (4) a crude clostridial toxin preparation that contained 150 HU v toxin activity and 50 units PLC activity. Mean arterial
pressure was monitored continuously for 3 h via a catheter placed in the carotid artery. *Values statistically different from baseline. Data adapted
from Asmuth et al. [9].

resistance (PVR) values intermediary to the other toxin groups.

This latter observation provides insights into possible antagonistic interactions between rPFO and rPLC in terms of PVR.
In total, these experiments suggest that PLC initially augments
cardiac function, counteracting the vasodilatory effect of rPFO.
Later, decreased cardiac output, hypotension, and death occurred. In addition, PLC may contribute indirectly to shock by
stimulating production of endogenous mediators such as TNF
[11] (figure 3) and platelet-activating factor [12].
PFO likely contributes to septic shock through indirect
routes, including the augmented release of TNF, IL-1, and IL6 [13, 14], platelet-activating factor (PAF), and prostaglandin
I2 [15]. Perhaps PFO-induced synthesis of nitric oxide by host
cells such as macrophages or endothelial cells could also play
a role in early hypotension. In addition, PLC and PFO may act
synergistically in inducing hypotension, hypoxia, and reduced
cardiac output. This latter point should be considered when
interpreting results from experiments that have used isogenic
mutants or single toxins.
Thus, shock associated with gas gangrene may be attributable, in part, to direct and indirect effects of toxins. PLC directly
suppresses myocardial contractility [10], thereby contributing
to profound hypotension via a sudden reduction in cardiac
output [9]. PFO reduces systemic vascular resistance and markedly increases cardiac output [9, 10]. PFO-induced after-load

reduction occurs undoubtedly through the induction of endogenous mediators that cause relaxation of blood vessel wall
tension [15]. Reduced vascular tone develops rapidly, and, to
maintain adequate tissue perfusion, a compensatory host response is required to either increase cardiac output or rapidly
expand the intravascular blood volume. In contrast, patients
with gram-negative sepsis compensate for hypotension by
markedly increasing cardiac output; however, this adaptive
mechanism is abrogated in C. perfringensinduced shock because of direct suppression of myocardial contractility by PLC
[10].
THE PATHOGENESIS OF TISSUE NECROSIS
C. perfringens gas gangrene is an aggressive infection in which
viable tissue is rapidly destroyed despite appropriate antibiotic
therapy, leaving the modern physician with few treatment alternatives save the centuries-old practice of radical amputation.
Trauma introduces organisms into the deep tissues and produces an anaerobic niche with a sufficiently low redox potential
and acid pH for optimal clostridial growth. Histologically, clostridial myonecrosis is remarkable for both the absence of acute
inflammatory cells in tissues and the accumulation of leukocytes between fascial planes and within small vessels (leukostasis) [1, 16, 17]. This picture is distinctly different from inClostridial Myonecrosis CID 2002:35 (Suppl 1) S95

Figure 3. Phospholipase C (PLC) induces TNF production in human peripheral blood mononuclear cells. TNF-a levels were measured in supernatant
fluid from 106 human mononuclear cells stimulated with recombinant PLC. Samples were collected at 24 h and assayed in duplicate by commercial
enzyme-linked immunosorbent assay. From Stevens and Bryant [11].

fections caused by bacteria such as Staphylococcus aureus,

Haemophilus influenzae, or S. pneumoniae, which are characterized by minimal tissue destruction and a luxuriant leukocytic
response at the site of infection. The rapid progression of infection and tissue necrosis associated with clostridial gas gangrene is related to the absence of an acute tissue inflammatory
response, to tissue perfusion deficits resulting from toxin-mediated vascular dysfunction and injury, and to the elaboration
of potent cytotoxins and proteases.
Mechanisms of vascular dysfunction. We have recently
shown that the rapid destruction of muscle involves toxinmediated impairment of local and regional blood flow [18].
Intramuscular injection of either a clostridial toxin preparation
that contains both PFO and PLC activity or of recombinant
PLC into rat abdominal musculature caused a rapid, dosedependent, and irreversible decrease in blood flow (figure 4)
that was not due to vasoconstriction but did parallel the formation of freely moving intravascular aggregates initially in
venules (!2 min) and, later (8 min), in arterioles [18]. Immunohistochemistry demonstrated that these early aggregates
consisted of activated (i.e., P-selectin positive) platelets. Later
(2040 min), aggregates enlarged and consisted of platelets,
fibrin, and neutrophils [18]. These heterotypic aggregates became lodged within vessels, completely obstructing local and
regional blood flow. Flow cytometry of human whole blood
confirmed that PLC induced formation of both activated platelet/platelet (not shown) and platelet/neutrophil aggregates (figure 5) [19]. Neutralization of PLC activity completely abrogated

S96 CID 2002:35 (Suppl 1) Stevens and Bryant

human platelet/neutrophil responses and reduced perfusion deficits in the rat model [18].
Although P-selectin binding of polymorphonuclear leukocyte (PMNL) glycoproteins has been the paradigm for platelet/
PMNL interactions, other investigators have demonstrated that
the platelet fibrinogen receptor, gpIIbIIIa (CD41/CD61), also
participates in the adhesion of activated platelets to PMNL in
vitro [2022]. These studies have shown that this interaction
is fibrinogen-dependent [21, 22], that CD11b/CD18 serves as
the PMNL ligand for fibrinogen [23, 24], and that this interaction is further enhanced when the functionally active conformation of CD11b/CD18 is expressed [25]. The observation
that injection of PLC into muscle caused the rapid formation
of freely mobile intravascular aggregates consisting of activated
platelets suggested that PLC stimulated the conformational
change in gpIIbIIIa necessary for platelets in circulation to bind
soluble fibrinogen. Indeed, PLC-induced platelet/neutrophil aggregation could be neutralized by antibody against gpIIbIIIa or
competitively inhibited by peptides and proteins that mimic
the fibrinogen molecule binding site (figure 6) [19]. In contrast,
strategies that targeted P-selectin had no effect (fucoidan, figure
6) [19]. Thus, it is likely that PLC-induced activation of gpIIbIIIa is responsible for PLC-induced platelet/platelet and
platelet/neutrophil aggregation in vivo.
Mechanisms of toxin-induced suppression of the acute inflammatory response. Several plausible mechanisms exist to
explain the lack of a tissue inflammatory response in clostridial
gas gangrene. First, an absence of bacterial- or host-derived

Figure 4. Phospholipase C (PLC) induces a rapid and irreversible decrease in skeletal muscle blood flow. Rat abdominal muscles were injected
with 0.1 mL of (1) sterile normal saline, (2) 10 mM phenylephrine, or (3) a clostridial toxin preparation that contained 8 units of PLC activity. Blood
flow was measured for 40 min by laser Doppler blood perfusion monitor and is expressed as the mean percentage (SE) of baseline perfusion. From
Bryant et al. [18].

chemoattractants could account for the paucity of leukocytes

in the tissues; however, studies have shown that both an extracellular component of bacterial culture and serum incubated
with killed bacilli were potent chemoattractants [17]. Second,
both PLC and PFO are cytolytic for leukocytes in high concentrations [26], and the destruction of any infiltrating phagocytes at the site of active bacterial proliferation and toxin
elaboration likely contributes to the marked reduction of inflammatory cells in these areas. However, were this the only
mechanism responsible for the absence of a tissue inflammatory
response, abundant phagocytes should be found in tissues distal
to the nidus of infection, but, as the infiltrating phagocytes
approached the site of infection, they would be abruptly halted
at the point where toxin concentrations reached cytolytic proportions. Thus, cytotoxicity alone could account for what is
classically observed in both human and experimental cases of
gas gangrenethe paucity of inflammatory cells in the tissuesbut it does not explain the characteristic leukostasis in
adjacent vasculature.
A third possible mechanism involves the effects of sublytic
amounts PLC and PFO on the function and interaction of
leukocytes and endothelial cells. PLC and PFO each uniquely
affect the normal, physiological mechanisms of leukocyte accumulation, adherence, and extravasation (see below). Toxininduced dysregulation of these events, which orchestrate the
pyogenic responses with other infections, could, in part, explain

the leukostasis and anti-inflammatory response characteristic

of clostridial gas gangrene. Furthermore, such dysregulation
could lead to local and regional ischemia, thereby extending
the region for optimal clostridial proliferation.
Effects of clostridial toxins on endothelial cell function.
Successful transmigration of leukocytes through the vessel and
to the site of infection is the culmination of a complex cascade
of both leukocyte- and endothelial cell (EC)dependent adherence and activational events. Initially, the circulating, inactivated leukocyte is tethered to the activated EC and rolls
along the vessels luminal surfaceprocesses mediated by selectins. Tethering results in juxtacrine activation of the leukocyte by PAF, functional up-regulation of leukocyte CD11b/
CD18, and firm adhesion to intercellular adhesion molecule1
(ICAM-1; CD54) constitutively expressed on the EC [27]. Local
production of cytokines (e.g., TNF and IL-1) augments the
inflammatory response by stimulating EC to produce the neutrophil chemoattractant/activator, IL-8, to increase ICAM-1 expression, and to transiently express endothelial leukocyte adhesion molecule1 (E-selectin). Strongly adherent, activated
leukocytes move to EC junctions and emigrate between endothelial cells, aided by platelet-endothelial cell adhesion
molecule1.
Our work has shown that PLC strongly induces the expression of E-selectin and ICAM-1 on cultured human umbilical
vein endothelial cells [28]. The magnitude and duration of these

Clostridial Myonecrosis CID 2002:35 (Suppl 1) S97

Figure 5. Platelet/granulocyte complex formation is induced by phospholipase C (PLC). Heparinized whole blood was activated by (1) PBS, (2) nformyl-methionyl-leucyl-phenylalanine (fMLP), (3) a crude clostridial toxin preparation that contained both PLC and perfringolysin O activity, or (4)
recombinant PLC in the presence of fluorescein isothiocynateconjugated anti-human CD11b (granulocyte marker) and phycoerythrin-conjugated antihuman CD62P (platelet marker). Dual color-flow cytometry was performed on the CD11b events located within the granulocyte gate. Data are the
mean (SD) fluorescence intensity of granulocyte-associated CD62P from 4 experiments done in duplicate. fMLP was included as a granulocyte
activator that does not induce complex formation. From Bryant et al. [19].

responses were similar to that reported in other studies that

used either TNF or IL-1 or lipopolysaccharide from gramnegative organisms. In addition, PLC also stimulated production of endothelial cellderived IL-8 [28]. The local production
of physiological concentrations of IL-8 in gas gangrene could
both amplify the recruitment of leukocytes and prime them
for enhanced respiratory burst activity. Alternatively, high concentrations of IL-8 attenuate transmigration of leukocytes
through an endothelial cell monolayer in response to chemoattractantsa process termed heterologous desensitization [29]. This desensitization results from inhibition of neutrophil F-actin polymerization in response to chemotactic
factor stimulation [30]. Of interest, we have shown that PFO,
but not PLC, at sublytic concentrations also prevents F-actin
polymerization in response to chemotactic factor stimulation
(see below) [17]. Finally, toxin-induced expression of PMNL
and endothelial cell adhesion molecules could result in reduced
diapedesis [17].
Effects of clostridial toxins on neutrophil function. In
contrast to PLC, PFO caused a modest, but significant, increase
in endothelial ICAM-1, had no effect on E-selectin expression,
and did not induce detectable IL-8 synthesis [28]. Yet intramuscular injection of PFO in mice produces marked vascular
leukostasis adjacent to the site of toxin injection [17], which
suggests that PFO may impair the inflammatory response by
primarily affecting neutrophil, rather than endothelial cell,
S98 CID 2002:35 (Suppl 1) Stevens and Bryant

function. Indeed, sublytic concentrations of PFO dose-dependently stimulated random migration of neutrophils but decreased directed migration toward n-formyl-methionyl-leucylphenylalanine or a complement-derived chemoattractant [26]
and prevented F-actin polymerization by leukocytes in response
to chemotactic factor stimulation [17]. Thus, in vivo, desensitization of neutrophils could contribute to the lack of phagocyte emigration into tissues infected with C. perfringens.
SUMMARY
These pieces of evidence can be assimilated into a molecular
and cellular model of pathogenesis that is initiated by direct
toxin effects on venous capillary EC function, leading to the
expression of proinflammatory mediators and adhesion molecules and initiation of platelet aggregation. Toxin-induced hyperadhesion of leukocytes with enhanced respiratory burst activity due to toxins directly or to toxin-induced IL-8 or PAF
synthesis by host cells and toxin-induced chemotaxis deficits
could result in neutrophil-mediated vascular injury. Direct
toxin-induced cytopathic effects on EC may also contribute to
vascular abnormalities associated with gas gangrene. Over prolonged incubation periods, PLC at sublytic concentrations
causes EC to undergo profound shape changes similar to those
described after prolonged TNF or IFN-g exposure. In vivo,
conversion of EC to this fibroblastoid morphology could con-

Figure 6. Phospholipase C (PLC)induced platelet/granulocyte complex formation is mediated by gpIIbIIIa. Heparinized whole blood was pretreated
with a neutralizing antibody against gpIIbIIIa (anti-CD41a) or isotype-matched control IgG or peptides or proteins that mimic fibrinogen, including (1)
Arg-Gly-Asp-Ser (RGDS) or Arg-Gly-Glu-Ser (RGES; an analogous but inactive peptide), (2) echistatin, an RGD-containing polypeptide and potent anticoagulant from the venom of the viper Echis carinatus, (3) fibrinogen fragment 400411, or (4) the P-selectin inhibitor, fucoidan. Blood was stimulated
with recombinant PLC and processed for dual color-flow cytometry. Data represent the mean (SD) fluorescence intensity of granulocyte-associated
CD62P expression of 3 experiments done in duplicate. From Bryant et al. [19].

tribute to the localized vascular leakage and massive swelling

observed clinically with this infection. Similarly, the direct cytotoxicity of PFO could disrupt endothelial integrity and contribute to progressive edema both locally and systemically.
Thus, via the mechanisms outlined above, both PLC and
PFO may cause local, regional, and systemic vascular dysfunction. For instance, the local absorption of exotoxins within the
capillary beds could affect the physiological function of the
endothelium lining the postcapillary venules, resulting in impairment of phagocyte delivery at the site of infection. Toxininduced endothelial dysfunction and microvascular injury
could also cause loss of albumin, electrolytes, and water into
the interstitial space, resulting in marked localized edema. These
events, combined with intravascular platelet aggregation and
leukostasis, would increase venous pressures and favor further
loss of fluid and protein in the distal capillary bed. Ultimately,
a reduced arteriolar flow would impair oxygen delivery, thereby
attenuating phagocyte oxidative killing and facilitating anaerobic glycolysis of muscle tissue. The resultant drop in tissue
pH, together with reduced oxygen tension, might further decrease the redox potential of viable tissues to a point suitable
for growth of this anaerobic bacillus. As infection progresses
and additional toxin is absorbed, larger venous channels would

become affected, causing regional vascular compromise, increased compartment pressures, and rapid anoxic necrosis of
large muscle groups. When toxins, or the cytokines they induce,
reach arterial circulation, systemic shock and multiorgan failure
rapidly ensue, and death is common.

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