Clin Infect Dis. 2002 Stevens S93 S100

7/30/2019 Clin Infect Dis. 2002 Stevens S93 S100

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Clostridial Myonecrosis CID 2002:35 (Suppl 1) S93

S U P P L E M E N T A R T I C L E

The Role of Clostridial Toxinsin the Pathogenesis of Gas Gangrene

Dennis L. Stevens1,3 and Amy E. Bryant1,2

1Veterans Affairs Medical Center, Boise, Idaho; and 2University of Idaho, Moscow; and 3University of Washington School of Medicine, Seattle

Clostridium perfringensgas 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-form-

ing, nonmotile, rod-shaped organism commonly found

in soil and in the intestines of humans and other an-

imals. 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. perfringensis 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 character-

ized by extensive local destruction of muscle (myone-

crosis), rapid destruction of viable tissue, shock, and,

ultimately, death. Of the many extracellular toxins pro-

duced by this organism, the a (phospholipase C, PLC)

and v (perfringolysin O, PFO) toxins are its major vir-

ulence 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

([email protected]).

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 ex-

perimental wild-type infections. Similarly, experimental

infections established with genetic mutants of C. per-

fringens 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 sero-

logical and antigenic relationships to the cholesterol-

binding cytolysins from Streptococcus pyogenes, Strep-

tococcus pneumoniae, and Listeria monocytogenes.

Recently, the amino acid and nucleotide sequences of

pneumolysin, streptolysin O, and PFO have been de-

termined [36]. Impressive homology exists among the

amino acid sequences of these toxins, particularly in

the region that contains the cysteine residue near theamino 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 fa-

cilitate the growth of their respective organisms within

mammalian phagocytic cells. Furthermore, experimen-

tal animal studies have demonstrated the protective ef-

ficacy of several antibody preparations against these


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S94 CID 2002:35 (Suppl 1) Stevens and Bryant

Figure 1. Effects of clostridial exotoxins on cardiac index (CI). Awake rabbits ( per group) were slowly infused (0.33 mL/min) via catheternp 6in 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 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 cytotoxicto platelets and leukocytes, and increases capillary permeabil-

ityeffects 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 ( per group) were slowly infused (0.33 mL/min) via catheternp 6in 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 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 rapidlyexpand 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 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 supernatantfluid from 106 human mononuclear cells stimulated with recombinant PLC. Samples were collected at 24 h and assayed in duplicate by commercialenzyme-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

(2040 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

human platelet/neutrophil responses and reduced perfusiondef-

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 [2022]. These studies have shown that this interaction

is fibrinogen-dependent [21, 22], that CD11b/CD18 serves asthe 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/neutrophilag-

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


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Figure 4. Phospholipase C (PLC) induces a rapid and irreversible decrease in skeletal muscle blood flow. Rat abdominal muscles were injectedwith 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 toxinelaboration 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 gangrenethe paucity of inflammatory cells in the tis-

suesbut 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

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 cascadeof 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 vessels luminal surfaceprocesses 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 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 neu-

trophil chemoattractant/activator, IL-8, to increase ICAM-1 ex-

pression, and to transiently express endothelial leukocyte ad-

hesion molecule1 (E-selectin). Strongly adherent, activated

leukocytes move to EC junctions and emigrate between en-

dothelial cells, aided by platelet-endothelial cell adhesion

molecule1.

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 isothiocynateconjugated 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 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 themfor enhanced respiratory burst activity. Alternatively, high con-

centrations of IL-8 attenuate transmigration of leukocytes

through an endothelial cell monolayer in response to che-

moattractantsa 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 pretreatedwith 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 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 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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