SUPPLEMENT ARTICLE

The Role of Clostridial Toxins

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

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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 6–8 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 C. perfringens infections is supported by numerous

studies that have used a variety of approaches [1]. Both
Clostridium perfringens is a gram-positive, spore-form-
active and passive immunization of animals against
ing, nonmotile, rod-shaped organism commonly found
PLC or its C-terminal fragment are protective in ex-
in soil and in the intestines of humans and other an-
perimental wild-type infections. Similarly, experimental
imals. The species has been divided into 5 distinct types,
infections established with genetic mutants of C. per-
A–E. Of these subgroups, C. perfringens type A causes
fringens lacking PLC or with strains that produce less
the majority of human infections. Although classified
PLC were markedly less fulminant, and mortality was
as an anaerobe, C. perfringens is somewhat aerotolerant.
significantly reduced [2].
Under optimal conditions, its generation time can be
The importance of PFO in the pathogenesis of gas
as little as 8–10 min, and growth is accompanied by
gangrene has been largely controversial, despite the
abundant gas production.
early knowledge of its hemolytic nature and its sero-
Gas gangrene caused by C. perfringens is character-
logical and antigenic relationships to the cholesterol-
ized by extensive local destruction of muscle (myone-
binding cytolysins from Streptococcus pyogenes, Strep-
crosis), rapid destruction of viable tissue, shock, and,
tococcus pneumoniae, and Listeria monocytogenes.
ultimately, death. Of the many extracellular toxins pro-
Recently, the amino acid and nucleotide sequences of
duced by this organism, the a (phospholipase C, PLC)
pneumolysin, streptolysin O, and PFO have been de-
and v (perfringolysin O, PFO) toxins are its major vir-
termined [3–6]. Impressive homology exists among the
ulence factors. PLC’s role as the major lethal factor in
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
This material is based on work supported by the Office of Research and
Development, Medical Research Service, Department of Veterans Affairs (D.L.S.).
12 amino acids is identical for all 3 toxins. Some of
these toxins, including PFO, have been shown to fa-
Reprints or correspondence: Dr. Dennis L. Stevens, Infectious Diseases Section,
Veterans Affairs Medical Center, 500 West Fort St., Bldg. 45, Boise, ID 83702 cilitate the growth of their respective organisms within
(dlsteven@mindspring.com). mammalian phagocytic cells. Furthermore, experimen-
Clinical Infectious Diseases 2002; 35(Suppl 1):S93–100
 2002 by the Infectious Diseases Society of America. All rights reserved.
tal animal studies have demonstrated the protective ef-
1058-4838/2002/3505S1-0018$15.00 ficacy of several antibody preparations against these

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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) 40–50 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 thiol-
activated 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 permeabil-
ity—effects that are likely related to its ability to cleave sphin-
gomyelin 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 pro-
tein. Titball et al. [8] have determined that the protein is com-
posed of 2 functional domains: the N-terminal domain pos-
sesses 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 devel-
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 physi-
ological mechanisms could contribute to such a reduction, we
subsequently demonstrated a direct reduction in myocardial
contractility (dF/dt) in isolated atrial strips bathed with recom-
binant 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 mech-
anism for a brief period before hypotension ultimately oc-
curred. 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

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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) 40–50 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 antago-
nistic 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 oc-
curred. 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 IL-
6 [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 attribut-
able, 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 mark-
edly increases cardiac output [9, 10]. PFO-induced after-load
reduction occurs undoubtedly through the induction of en-
dogenous mediators that cause relaxation of blood vessel wall
tension [15]. Reduced vascular tone develops rapidly, and, to
maintain adequate tissue perfusion, a compensatory host re-
sponse 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. perfringens–induced shock be-
cause 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 al-
ternatives save the centuries-old practice of radical amputation.
Trauma introduces organisms into the deep tissues and pro-
duces an anaerobic niche with a sufficiently low redox potential
and acid pH for optimal clostridial growth. Histologically, clos-
tridial myonecrosis is remarkable for both the absence of acute
inflammatory cells in tissues and the accumulation of leuko-
cytes between fascial planes and within small vessels (leukos-
tasis) [1, 16, 17]. This picture is distinctly different from in-

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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 charac-
terized by minimal tissue destruction and a luxuriant leukocytic
response at the site of infection. The rapid progression of in-
fection and tissue necrosis associated with clostridial gas gan-
grene is related to the absence of an acute tissue inflammatory
response, to tissue perfusion deficits resulting from toxin-me-
diated 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 toxin-
mediated 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, dose-
dependent, and irreversible decrease in blood flow (figure 4)
that was not due to vasoconstriction but did parallel the for-
mation of freely moving intravascular aggregates initially in
venules (!2 min) and, later (8 min), in arterioles [18]. Im-
munohistochemistry demonstrated that these early aggregates
consisted of activated (i.e., P-selectin positive) platelets. Later
(20–40 min), aggregates enlarged and consisted of platelets,
fibrin, and neutrophils [18]. These heterotypic aggregates be-
came lodged within vessels, completely obstructing local and
regional blood flow. Flow cytometry of human whole blood
confirmed that PLC induced formation of both activated plate-
let/platelet (not shown) and platelet/neutrophil aggregates (fig-
ure 5) [19]. Neutralization of PLC activity completely abrogated
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human platelet/neutrophil responses and reduced perfusion def-
icits in the rat model [18].
Although P-selectin binding of polymorphonuclear leuko-
cyte (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 [20–22]. 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 inter-
action is further enhanced when the functionally active con-
formation 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 ag-
gregation 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 gp-
IIbIIIa is responsible for PLC-induced platelet/platelet and
platelet/neutrophil aggregation in vivo.
Mechanisms of toxin-induced suppression of the acute in-
flammatory 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 ex-
tracellular 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 con-
centrations [26], and the destruction of any infiltrating phag-
ocytes at the site of active bacterial proliferation and toxin
elaboration likely contributes to the marked reduction of in-
flammatory 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 pro-
portions. Thus, cytotoxicity alone could account for what is
classically observed in both human and experimental cases of
gas gangrene—the paucity of inflammatory cells in the tis-
sues—but 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 ac-
cumulation, adherence, and extravasation (see below). Toxin-
induced dysregulation of these events, which orchestrate the
pyogenic responses with other infections, could, in part, explain
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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 ad-
herence and activational events. Initially, the circulating, in-
activated leukocyte is tethered to the activated EC and rolls
along the vessel’s luminal surface—processes mediated by se-
lectins. Tethering results in juxtacrine activation of the leuko-
cyte by PAF, functional up-regulation of leukocyte CD11b/
CD18, and firm adhesion to intercellular adhesion molecule–1
(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 neu-
trophil chemoattractant/activator, IL-8, to increase ICAM-1 ex-
pression, and to transiently express endothelial leukocyte ad-
hesion molecule–1 (E-selectin). Strongly adherent, activated
leukocytes move to EC junctions and emigrate between en-
dothelial cells, aided by platelet-endothelial cell adhesion
molecule–1.
Our work has shown that PLC strongly induces the expres-
sion of E-selectin and ICAM-1 on cultured human umbilical
vein endothelial cells [28]. The magnitude and duration of these
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Figure 5. Platelet/granulocyte complex formation is induced by phospholipase C (PLC). Heparinized whole blood was activated by (1) PBS, (2) n-
formyl-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 isothiocynate–conjugated anti-human CD11b (granulocyte marker) and phycoerythrin-conjugated anti-
human 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 gram-
negative organisms. In addition, PLC also stimulated produc-
tion of endothelial cell–derived 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 con-
centrations of IL-8 attenuate transmigration of leukocytes
through an endothelial cell monolayer in response to che-
moattractants—a process termed “heterologous desensitiza-
tion” [29]. This desensitization results from inhibition of neu-
trophil 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 intra-
muscular 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,
function. Indeed, sublytic concentrations of PFO dose-de-
pendently stimulated random migration of neutrophils but de-
creased directed migration toward n-formyl-methionyl-leucyl-
phenylalanine or a complement-derived chemoattractant [26]
and prevented F-actin polymerization by leukocytes in response
to chemotactic factor stimulation [17]. Thus, in vivo, desen-
sitization of neutrophils could contribute to the lack of phag-
ocyte 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 mol-
ecules and initiation of platelet aggregation. Toxin-induced hy-
peradhesion of leukocytes with enhanced respiratory burst ac-
tivity 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 pro-
longed 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-

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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 anti-
coagulant from the venom of the viper Echis carinatus, (3) fibrinogen fragment 400–411, 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 cy-
totoxicity of PFO could disrupt endothelial integrity and con-
tribute to progressive edema both locally and systemically.
Thus, via the mechanisms outlined above, both PLC and
PFO may cause local, regional, and systemic vascular dysfunc-
tion. 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 im-
pairment of phagocyte delivery at the site of infection. Toxin-
induced 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 anaer-
obic glycolysis of muscle tissue. The resultant drop in tissue
pH, together with reduced oxygen tension, might further de-
crease 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, in-
creased 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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