Histotoxic Clostridial Infections · Typical histotoxic clostridial diseases include human gas...

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Histotoxic Clostridial InfectionsMASAHIRO NAGAHAMA,1 MASAYA TAKEHARA,1 and JULIAN I. ROOD2

1Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho,Tokushima 770-8514, Japan; 2Infection and Immunity Program, Monash Biomedicine Discovery Institute

and Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia

ABSTRACT The pathogenesis of clostridial myonecrosis orgas gangrene involves an interruption to the blood supply tothe infected tissues, often via a traumatic wound, anaerobicgrowth of the infecting clostridial cells, the production ofextracellular toxins, and toxin-mediated cell and tissuedamage. This review focuses on host-pathogen interactionsin Clostridium perfringens-mediated and Clostridiumsepticum-mediated myonecrosis. The major toxins involvedare C. perfringens α-toxin, which has phospholipase C andsphingomyelinase activity, and C. septicum α-toxin,a β-pore-forming toxin that belongs to the aerolysin family.Although these toxins are cytotoxic, their effects on hostcells are quite complex, with a range of intracellular cell signalingpathways induced by their action on host cell membranes.

GENERAL CHARACTERISTICS OFHISTOTOXIC CLOSTRIDIAL INFECTIONSMany pathogenic clostridial species cause potentiallyfatal soft tissue infections in humans and animals be-cause of their ability to produce extracellular proteintoxins (1, 2). The ability of these pathogens to producespores that are resistant to environmental stress is animportant factor in the epidemiology of these diseases.Disease pathogenesis involves the growth of the clos-tridial pathogen in the tissues and extensive tissue de-struction, which is the result of the action of extracellulartoxins. Typical histotoxic clostridial diseases includehuman gas gangrene or myonecrosis and blackleg incattle. Although there are several clostridial species thatare responsible for these syndromes (Table 1), thisreview will focus on histotoxic infections caused byClostridium perfringens and Clostridium septicum,primarily because these are the species that have beenthe subject of the most extensive molecular and func-tional studies.

Although the histotoxic clostridia cause severe,rapidly developing infections, these potent toxigenicbacteria still must be regarded as opportunistic patho-gens. All of these infections require predisposingconditions to cause disease, often a traumatic woundthat enables the entry of spores from the soil or gastro-intestinal tract to gain access to ischemic internal organsor soft tissues of the body. Alternatively, in nontrau-matic myonecrosis caused by C. septicum, gastrointes-tinal malignancy is commonly associated with the onsetof disease, with tumor development leading to ulcerationand necrosis of the gastrointestinal mucosa and entry ofthe gastrointestinal microbiota into the circulation andsubsequent infection at distal sites (3, 4). Clostridialnecrotizing soft tissue infections, especially those causedby C. perfringens, Clostridium sordellii, and Clostri-dium novyi, also have resulted from the subcutaneousinjection of contaminated black tar heroin (5–7).

Received: 19 March 2018, Accepted: 23 March 2018,Published: 26 July 2019

Editors: Vincent A. Fischetti, The Rockefeller University, New York,NY; Richard P. Novick, Skirball Institute for Molecular Medicine,NYU Medical Center, New York, NY; Joseph J. Ferretti, Departmentof Microbiology & Immunology, University of Oklahoma HealthScience Center, Oklahoma City, OK; Daniel A. Portnoy, Departmentof Molecular and Cellular Microbiology, University of California,Berkeley, Berkeley, CA; Miriam Braunstein, Department ofMicrobiology and Immunology, University of North Carolina-ChapelHill, Chapel Hill, NC, and Julian I. Rood, Infection and ImmunityProgram, Monash Biomedicine Discovery Institute, MonashUniversity, Melbourne, Australia

Citation: Nagahama M, Takehara M, Rood JI. Histotoxic clostridial infections. Microbiol Spectrum 6(4):GPP3-0024-2018. doi:10.1128/microbiolspec.GPP3-0024-2018.

Correspondence: Masahiro Nagahama, [email protected] or Julian I. Rood, [email protected]

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© 2019 American Society for Microbiology. All rights reserved.

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PATHOGENESIS OF CLOSTRIDIALMYONECROSIS CAUSED BYC. PERFRINGENS AND C. SEPTICUMAs previously described (8), the pathogenesis of theseinfections involves three distinct stages, irrespective ofwhether the source of infection involves a traumaticwound. Stage 1 involves an interruption to the bloodsupply such that the redox potential in the tissues dropsto a level that facilitates spore germination and/or thegrowth of the infecting clostridial cells. Stage 2 involvesbacterial growth and establishment of the conditions fortoxin production. In C. perfringens, regulation of toxinproduction primarily utilizes the VirSR two-componentsignal transduction system (9) and an accessory growthregulator-like quorum sensing system (10, 11), as wellas other regulatory networks. Stage 3 encompasses thetoxin-mediated cell and tissue damage that leads to ne-crosis, systemic toxicity, and clinical disease (8).

C. perfringens type A is the major cause of traumaticgas gangrene, although other histotoxic clostridia mayalso be responsible for this sporadic, but fulminantand often fatal, disease (Table 1) (1). The major toxinproduced by gas gangrene strains of C. perfringens isα-toxin, a zinc metallophospholipase that has bothphospholipase C and sphingomyelinase activity (12).α-Toxin was the first bacterial toxin to be shown to haveenzymatic activity (13). Two independent studies usingdifferent experimental approaches have shown thatα-toxin is essential for C. perfringens-mediated myo-necrosis. First, immunization studies in mice using re-combinant α-toxin variants purified from Escherichia

coli, and therefore devoid of any other C. perfringenstoxins, showed that the C-terminal domain of α-toxinwas immunoprotective (14). Second, mutation of theα-toxin structural gene (plc or cpa) abrogated the abilityof the bacterium to cause clostridial myonecrosis in amurine model. Virulence was restored by complemen-tation in trans with a recombinant plasmid containingthe wild-type plc gene (15), thereby fulfilling molecularKoch’s postulates and providing proof of the essentialrole of this toxin in disease.

The other major toxin produced by C. perfringenstype A is perfringolysin O, or θ-toxin, a pore-formingtoxin that is a member of the cholesterol-dependent cy-tolysin family (16, 17). Although mutation of the per-fringolysin O structural gene, pfoA, did not eliminatethe ability to cause disease (18, 19) subsequent studiesof plcpfoA double mutants (20) provided evidence thatperfringolysin O has a synergistic effect with α-toxin ondisease pathogenesis (20, 21).

Recent studies have involved the concurrent analy-sis of the transcriptomes of both the host and C. per-fringens in a murine myonecrosis infection (22). Theresults showed that many host genes involved in theinnate immune response to infection were upregulatedin C. perfringens-infected muscle tissues. In the C. per-fringens cells, upregulated genes included those en-coding potential adhesins and proteins associated withthe cell envelope.

C. septicum is the major causative agent of nontrau-matic gas gangrene, often associated with a gastro-intestinal malignancy (4). The major toxin produced

TABLE 1 Histotoxic clostridial infections

Syndromea Causative agent Host Major toxinsb References

Traumatic gas gangrene C. perfringens type A Humans α-toxin 12, 17

Perfringolysin O (θ-toxin)

Traumatic gas gangrene C. novyi Humans α-toxin 151

C. septicum α-toxin 129

C. histolyticum Lethal factor 152

C. sordellii Lethal toxin (TcsL) 153

Hemorrhagic toxin (TcsH)

Nontraumatic gas gangrene C. septicum Humans α-toxin 3, 4

C. fallax Humans and animals Not known 154

Necrotizing pancreatitis C. perfringens type A Humans α-toxin 155

Clostridial myonecrosis C. perfringens types A, B, C, and D Sheep, cattle, and other animals α-toxin 29

β-toxin

ε-toxin

Blackleg C. chauvoei Cattle, sheep, and other ruminants Cytolysin A (CctA) 156

aThe terms “gas gangrene” and “clostridial myonecrosis” are used interchangeably in this review.bNote that the different α-toxins referred to in this column are not necessarily structurally or functionally related.

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by C. septicum is also called α-toxin, but it is not relatedto C. perfringens α-toxin. C. septicum α-toxin is anaerolysin-like pore-forming toxin that is secreted as aninactive prototoxin that is cleaved near the C terminusby membrane-bound furin or furin-like proteases toform the active toxin (23). The toxin then oligomerizeson the membrane and subsequently inserts to form amembrane pore, which ultimately leads to cell lysis (24).Genetic studies have shown that α-toxin is essential forC. septicum-mediated myonecrosis in mice (25). Muta-tion of the α-toxin structural gene, csa, leads to a loss ofvirulence, which is restored by complementation withthe wild-type csa gene.

STRUCTURE AND FUNCTIONOF C. PERFRINGENS α-TOXINα-Toxin is a 43-kDa single polypeptide that is a hemo-lytic, cytotoxic, dermonecrotic, and lethal extracellu-lar toxin (26–29). Moreover, it induces contractionsof blood vessels and smooth muscle, aggregation ofplatelets, superoxide production, and cytokine release.Importantly, together with perfringolysin O, it is re-sponsible for the hallmark histopathological feature ofC. perfringens-mediated myonecrosis, the paucity of thepolymorphonuclear leukocyte (PMNL) influx into theinfected lesion (15, 19–21, 30).

Determination of the crystal structure of α-toxinrevealed that it has an α-helical N-terminal domain(amino acids 1 to 246) that is responsible for its enzy-matic activity, a flexible linker (amino acids 247 to 255),and a C-terminal (amino acids 256 to 370) β-sandwichmembrane-binding domain (31) (Fig. 1). The two do-mains also are linked by hydrophobic surface interac-tions between their neighboring faces. The N-terminaldomain has significant sequence similarity to phospho-lipase C enzymes from Bacillus cereus (32), Clostridiumbifermentans (33), and Listeria monocytogenes (34). Itcomprises nine densely packed α-helices and resemblesthe structure of B. cereus phospholipase C (31, 35),which has enzymatic activity, but is neither toxic norhemolytic. There are three loops in the N-terminal do-main: loop 1 (residues 70 to 90), loop 2 (residues 135to 150), and loop 3 (residues 205 to 215). These loopsserve a vital role in the direct interaction between thetoxin and the membrane-binding component. The C-terminal domain comprises eight-stranded antiparallelβ-sheets (31). This domain plays a role in membranebinding and is required for hemolytic and toxic activity(28, 36). It is absent from nontoxic phospholipase C en-zymes (31). It has structural similarity to the C-terminal

domain of pancreatic lipase, the N-terminal domain ofsoybean lipoxygenase, and the C2 phospholipid-bindingdomains of eukaryotic intracellular proteins, such assynaptotagmin I, which are involved in signal trans-duction and vesicular transport (37, 38).

The active site in the N-terminal domain is situatedwithin a solvent-accessible cleft that contains two zincions and an exchangeable divalent cation. His-148 andGlu-152 dock with one zinc ion, which is critical for thecatalytic site of the toxin. His-11 and Asp-130 tightlydock with the other zinc ion, which is needed for main-tenance of the structure. His-68, His-126, and His-136dock with an exchangeable divalent cation, which isrequired for binding to target cell membranes (39).The external loop regions (residues 70 to 90, 135 to 150,205 to 215, 265 to 275, 290 to 300, and 330 to 340)bind to phospholipids in the host cell membranes (31).Thr-74 is present in one of these loops, and althoughthe substitution derivative T74I is still active and cancleave a water-soluble substrate, its membrane-damagingactivity toward red blood cells and phosphatidylcholine-cholesterol liposomes is decreased compared to wild-typetoxin (40, 41). However, the structure of the T74I variantis identical to that of the wild-type toxin (41). The T74Isubstitution results in altered recognition of the glycerolmoiety in the phospholipid, without affecting the func-tion of the active site, supporting the hypothesis that theamino acid 70 to 90 loop is important for phospholipidrecognition (42, 43).

FIGURE 1 Structure of α-toxin. The structure of α-toxin wasobtained from the Protein Data Bank (code 1QM6) and wascreated using MacPyMOL. A ribbon representation is shown,colored from blue (N-terminal) to red (C-terminal). Two zincmetal ions in the active site are shown as red spheres.


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Computational modeling of α-toxin and phosphati-dylcholine revealed that Tyr-57 and Tyr-65, which existat an external site near the catalytic cleft and are inproximity with the 70 to 90 loop, are responsible forsubstrate interaction (44). The replacement of theseTyr residues with Ala did not have any effect on phos-pholipase C and sphingomyelinase activity but had amarked effect on the membrane-damaging activity (he-molysis of sheep red cells and disruption of liposomes).These residues exist in or near the entrance of the cata-lytic cleft and are essential for the membrane-damagingeffect of α-toxin, because they are required for entry ofthe catalytic cleft into the hydrophobic region of plasmamembranes (44).

Several studies have shown that the calcium-bindingC-terminal domain is responsible for membrane bind-ing (28, 31, 36, 37). Two acrylodan-labeled C-terminaldomain derivatives, S263C and S365C, bound to lipidbilayers and displayed a chromogenic shift, demonstrat-ing entry of the C-domain of the toxin into the hydro-phobic regions of the liposomes (36). The data provideevidence that the C-terminal domain of the toxin binds

to the membrane and that the amino acid 70 to 90 loopand Tyr-57 and Tyr-65 serve an important role in the re-cognition of target membranes (Fig. 2). Other studieshave shown that substitution of the C-terminal domainresidues Asp-269, Asp-336, Tyr-275, and Tyr-331,which were predicted to be involved in membrane bind-ing (31), were required for hemolytic and cytotoxic ac-tivity (45).

INTERACTION OF C. PERFRINGENSα-TOXIN WITH HOST CELLSMembrane-Damaging Activity onPhospholipid BilayersThe effect of α-toxin on host tissues is caused by thephospholipase C- and sphingomyelinase-mediated cleav-age of phospholipids in the host cell membranes. Theresultant disruption of the phospholipid bilayer togetherwith the end products of these cleavage reactions, diacyl-glycerol and ceramide, respectively, activates lipid me-tabolism in host cell membranes and plays a role in hostcell damage (27, 28).

FIGURE 2 Binding model of α-toxin and membrane phospholipids. The structure ofα-toxin was obtained from the Protein Data Bank (code 1QM6) and was created usingMacPyMOL. Phospholipids are shown as light gray spheres for head groups and light graylines for tail groups. The head group of a phospholipid, from the outer membrane into theactive site of α-toxin, is displayed. Amino acids that may play a role in membrane inter-action are shown.


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The composition of the phospholipid bilayer modu-lates the extent of the α-toxin-mediated membrane dis-ruption (46). In another study that measured the releaseof carboxyfluorescein from various liposomes, α-toxin-mediated release, or liposomal leakage, decreased withincreasing chain length of the hydrocarbon residuesin the phospholipid bilayer (40). It was concluded thatmembrane disruption was related to the fluidity of themembrane and double bonds in the acyl chain. Sub-sequently, several phosphatidylcholines (C18:0/C18:1)with a double bond in the sn-2 fatty acyl chain wereprepared (47). The phase transition temperature valuewas lowest when the cis-double bond was positioned atthe center of the C18:1 sn-2 acyl chain and increasedcontinuously as the double bond was moved towardeither end of the acyl chain. α-toxin-mediated carboxy-fluorescein release from various liposomes was highestwhen the cis-double bond was positioned at the center ofthe sn-2 fatty acyl chain, resulting in a bell-shaped curve(47). Accordingly, the membrane-disrupting activity ofthe toxin is strongly associated with the membrane flu-idity of target membranes.

There is evidence that α-toxin also promotes per-fringolysin O-induced membrane damage in phospholipidbilayers. Hydrolysis of phosphatidylcholine residues ofliposomes that contained cholesterol increased the amountof free cholesterol in the lipid bilayer and enhancedperfringolysin O-dependent cytolytic activity (48). Thesedata may at least partly explain the synergistic effects of α-toxin and perfringolysin O that were previously observedin C. perfringens infections of mice (20).

Activation of PhospholipidMetabolism and Hemolysisα-toxin provokes contraction of isolated rat ileum andaorta tissues in a dose-dependent manner through stim-ulation of the phospholipid metabolic pathway (49, 50).In isolated rat organs, α-toxin activates the phospha-tidylinositol metabolic pathway and then the arachi-donic acid cascade, thereby promoting thromboxaneproduction. Contraction caused by the toxin was evokedby thromboxane A2 generation induced by arachidonicacid (49, 50). The metabolism of arachidonic acid resultsin the production of bioactive lipid mediators, includingleukotrienes, thromboxanes, and prostaglandins (27).These bioactive lipid mediators play key roles in en-hancing the local inflammatory reaction and lead tovasoconstriction, which is responsible for inducing an-oxia in the early stages of bacterial infections. Theseeffects presumably contribute to the capacity of C. per-fringens to proliferate in the host and cause disease.

Treatment of rabbit neutrophils with α-toxin pro-motes the formation of diacylglycerol, which is fol-lowed by adhesion of the cells to extracellular matrixproteins and the generation of superoxide ions (Fig. 3B)(51). α-Toxin induces diacylglycerol generation throughactivation of intrinsic phospholipase C enzymes by apertussis toxin-sensitive GTP-binding protein. More-over, it increases the phosphorylation of neurotrophictyrosine kinase receptor type 1 (TrkA), leading to 3-phosphoinositide-dependent protein kinase 1 activation(52). Therefore, α-toxin independently causes stimula-tion of intrinsic phospholipase C and protein kinase 1activity through TrkA receptor activation. Both phe-nomena synergistically augment the activity of proteinkinase C θ, leading to the formation of superoxide ionsvia activation of the mitogen-activated protein kinase(MAPK) cascade (Fig. 3B) (52). In human lung carci-noma (A549) cells, α-toxin mediates the activation of thep38 MAPK, NF-κB, and extracellular signal-regulatedkinase 1/2 (ERK1/2) through TrkA activation, whichleads to interleukin-8 (IL-8) release (53) (Fig. 3B).

Treatment of rabbit erythrocytes with α-toxin in-duces the biphasic formation of phosphatidic acid(54) (Fig. 3A). α-Toxin-induced formation of phospha-tidic acid is activated in the earlier stage (30 s) by in-trinsic phospholipase C and in the later stage (ca. 25min) by intrinsic phospholipase D. Both processesinvolve stimulation of the G-protein-signaling pathwayby toxin-mediated membrane phospholipid cleavage(55) (Fig. 3A).

Sheep erythrocytes have higher amounts of sphingo-myelin but do not contain phosphatidylcholine. Cleav-age of sphingomyelin by α-toxin leads to increased levelsof ceramide and sphingosine 1-phosphate (56), which isessential for toxin-induced hemolysis (57) and activatesthe sphingomyelin metabolism system (Fig. 3A) (56).The resultant cleavage of N-nervonoic sphingomyelin(C24:1-SM) occurs in a detergent-resistant membranecomponent (57) (Fig. 3A). Pertussis toxin blocks theproduction of C24:1-ceramide caused by α-toxin andblocks red cell hemolysis, which indicates that intrinsicsphingomyelinase, which can cleave C24:1-SM to C24:1-ceramide, is regulated by a pertussis toxin-sensitive G-protein in plasmamembranes. In addition, the activationof Rho protein by α-toxin increases sphingosine kinaseactivity. Subsequently, the ceramides are quickly me-tabolized to sphingosine, indicating that the toxin alsoactivates ceramidases (57). These results indicate thatthe cleavage of C24:1-SM to sphingosine 1-phosphate byα-toxin in lipid rafts is essential for toxin-induced he-molysis of sheep red cells (Fig. 3A).




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Interaction of α-Toxin with GangliosidesAmino acid sequence analysis of botulinum toxin andtetanus toxin showed that the ganglioside-GT1b- andlactose-binding site is determined by the existence of theconserved peptide motif, H.....SXWY.....G (ca. 30 aminoacids [aa]), with the Tyr and Trp residues being partic-ularly critical (58). A similar motif (H.....SWY.....G,amino acids 68 to 93) is present in α-toxin (53), in theregion (amino acids 55 to 93) that is located at the in-terface between the N-terminal and C-terminal domains(42). It has been demonstrated that the 70 to 90 loopin α-toxin plays a role in binding to the cell surface(43) (Fig. 2). Computational docking studies indicatedthat Trp-84 binds to the sialic acid residue on ganglio-side GM1a via an aromatic stacking interaction and ahydrogen-bonding interaction and that Tyr-85 associ-ates with the galactosamine residue on GM1a via anα-glycosidic bonding interaction (59). Replacement ofTrp-84 and Tyr-85 in α-toxin, which correspond to

the ganglioside recognition site of botulinum and teta-nus toxins, significantly reduced binding to gangliosideGM1a-containing liposomes. W84A and Y85A deriva-tives had markedly decreased capabilities to stimulateTrkA (59). Ganglioside GM1a assembles with a TrkAmolecule on the cell surface and stimulates TrkA andERK1/2 (60). It has been reported that ganglioside GM1clustering leads to the condensation of TrkA in lipid raftsand the stimulation of downstream signaling targets(61). α-Toxin-mediated diacylglycerol production leadsto diacylglycerol flip-flop movement that affects thedynamics of cell membranes (Fig. 4). Therefore, it servesin GM1a clustering and intrinsic phospholipase C-γ1activation through TrkA, triggering various signaltransduction activities (62–64).

α-toxin causes higher cytotoxicity in a ganglioside-deficient cell line (Don Q) (65) and can be internalizedby dynamin-mediated endocytosis (66, 67). The toxinbinds to caveolin-1 both on the cell membranes and in

FIGURE 3 Relationship between phospholipid metabolism and the biological activitiesof α-toxin. (A) Signaling events involved in α-toxin-induced hemolysis of sheep or rabbiterythrocytes. (B) Signaling events involved in α-toxin-activated generation of O2

– inneutrophils and in α-toxin-mediated release of IL-8 from A549 cells. Abbreviations: SM,sphingomyelin; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; DG,diacylglycerol; PC, phosphatidylcholine; SMase, sphingomyelinase; PLD, phospholipase D;PA, phosphatidic acid; PKCθ, protein kinase Cθ; PI3K, phosphatidylinositol 3-kinase;PIP3, phosphatidylinositol 3,4,5-trisphosphate; CDase, ceramidase; CER, ceramide; SPH,sphingosine; S1P, sphingosine 1-phosphate; DGK, DG kinase; PDK1, phosphatidylinositidedependent kinase 1; DRM, detergent-resistant membrane; TrkA, neurotrophic tyrosinekinase receptor type1; MAPKK, mitogen-activated protein kinase kinase; Erk1/2, extra-cellular signal-regulated kinase 1/2.


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endosomal vesicles, and it also induces ceramide pro-duction in the cell membrane (28), which generates neg-ative membrane curvature and facilitates endocytosis(68). Internalized α-toxin is transported to the early andlate endosomes and lysosomes. Lysosomes are damagedduring progressive α-toxin internalization into Don Qcells. In addition, the internalization of α-toxin is alsomediated through the stimulation of MEK/ERK signal-ing (66, 67). It has been reported that the toxin causesreactive oxygen species generation via PKC, MEK/ERK,and NF-κB signaling activities in Don Q cells (66, 67).Therefore, α-toxin perturbs the host cell signaling cas-cade and acts not only on the cell membrane, but also inthe cytoplasm.

Effect of α-Toxin on Hematopoietic CellsThe first line of defense of the innate immune systeminvolves neutrophils, which play an important role in thephagocytosis and subsequent elimination of pathogenicbacteria (69, 70). Normally, the number of neutrophils

is sustained in a steady state through granulopoiesis,which is accelerated to replenish neutrophils during bac-terial infection (71–73). Pattern recognition receptors,such as Toll-like receptors (TLRs), are responsible forthe recognition of structural components of microor-ganisms (74, 75). TLR2 and TLR4 have been identifiedas being pivotal for the recognition of Gram-positive orGram-negative bacteria by recognizing cell wall com-ponents such as peptidoglycan and bacterial endotoxin(lipopolysaccharide), respectively (76–79). During bac-terial infection, these bacterial components stimulatethe production of granulocyte colony-stimulating factor(G-CSF), which is a glycoprotein that promotes theproliferation, survival, and differentiation of neutrophilsand their progenitor cells (80) from endothelial cells andmonocytes, resulting in the acceleration of granulopoie-sis (81). G-CSF-deficient and G-CSF receptor-deficientmice exhibit chronic neutropenia and reduced infection-driven granulopoiesis, leading to impaired ability toeliminate infecting bacteria (82, 83). Thus, granulo-

FIGURE 4 Model of α-toxin-induced membrane dynamics and clustering of the GM1aand TrkA complex. α-Toxin binds to GM1a and, through the phospholipase C activity of thetoxin itself, causes diacylglycerol (DG) production at the outer membrane. The flip-flopmovement of DG affects membrane dynamics, facilitating clustering of GM1a and TrkAactivation by phosphorylation. Activation of TrkA results in the activation of PLCγ-1,leading to the increased production of DG. Finally, DG formation results in the enhancedactivation of TrkA, triggering activation of signal transduction pathways, which induceneutrophil activation. Abbreviations: PIP2, phosphatidylinositol 4,5-bisphosphate; PLC,phospholipase C; DG, diacylglycerol; PC, phosphatidylcholine; TrkA, neurotrophic tyro-sine kinase receptor type 1.




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poiesis is precisely regulated to replenish neutrophilsduring bacterial infection. However, C. perfringens canstill cause life-threatening infections, and the mecha-nisms by which C. perfringens avoids these host defensesare still not completely understood.

C. perfringens-mediated myonecrosis progresses sorapidly that death, which is associated with muscle de-struction, shock, and multiple organ failure, sometimesprecedes an accurate diagnosis (84). C. perfringens in-fection is characterized by a greatly reduced PMNL in-flux to the site of the infection (19, 85–87), suggestingthat the bacteria disrupt neutrophil-based innate immu-nity to evade host defenses. α-Toxin mediates the forma-tion of platelet-leukocyte aggregates, leading to vascularocclusion, and induces a marked reduction in micro-vascular perfusion (45, 86, 88), which is thought toimpede neutrophil extravasation (89). Recently, it wasshown using a mouse model of infection that C. per-fringens infection reduced mature neutrophils in bonemarrow, in peripheral blood, and in C. perfringens-

infected muscle, in an α-toxin-dependent manner (90).α-Toxin inhibits neutrophil differentiation and impairsthe replenishment of differentiated mature neutrophils inthe peripheral circulation, resulting in reduced recruit-ment of neutrophils to the C. perfringens infection site.The half-life of peripheral neutrophils is less than 12hours in vivo (91), meaning that the number of matureneutrophils required to counter a bacterial infection issustained through continuous granulopoiesis. Therefore,blockage of both granulopoiesis and PMNL diapedesisinto the tissues, as a result of platelet aggregation, leadsto a reduction in cell numbers in a short period of time.Thus, C. perfringens infection impairs the host immunesystem, which could explain the both scarcity of PMNLat the site of infection and a propensity for polymicrobialinfections in many patients (Fig. 5).

α-Toxin is essential for disease pathogenesis (18–21,30), and blockage of neutrophil differentiation is de-pendent on its phospholipase C and sphingomyelinaseactivities (90). In human lymphocytes, S. aureus sphin-

FIGURE 5 Inhibition of granulopoiesis and erythropoiesis in a C. perfringens-infectedhost. Infection with C. perfringens diminishes mature hematopoietic cells in the bonemarrow, leading to impairment of replenishment of differentiated neutrophils and eryth-rocytes in the peripheral circulation and resulting in host innate immune deficiency. Themajor virulence factor of C. perfringens, α-toxin, plays an important role in this phe-nomenon by interfering with cell differentiation of myeloblasts and erythroblasts.


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gomyelinase disrupts lipid rafts, which are cholesterol-rich plasma membrane microdomains (92). Lipid raftsfunction as platforms of signaling molecules involved inthe regulation of cell differentiation in many cell types(93, 94). Ceramide, the hydrolysis product of sphingo-myelinase, also is known to be a lipid messenger impli-cated in various cellular responses, including apoptosis,differentiation, and cell growth (95). Recently, C. per-fringens α-toxin was reported to perturb the integrityof lipid rafts, leading to the blockage of neutrophil dif-ferentiation (90). The detailed molecular mechanismbehind this blockage has not been elucidated, but it maybe important to focus on the relationship between therole of sphingomyelinase and cell differentiation to shedfurther light on host-pathogen interactions in clostridialmyonecrosis.

Effect of α-Toxin on Erythropoiesisα-Toxin has been reported to lyse erythrocytes fromvarious species. It activates T-type Ca2+ channels and in-creases intracellular Ca2+, which plays an important rolein the hemolysis of horse erythrocytes (96). The toxinactivates the sphingomyelin metabolic system, leadingto the hemolysis of sheep erythrocytes (56, 57), and thelysis of rabbit erythrocytes involves α-toxin-mediatedactivation of endogenous phospholipase C activity (54,55). Recently, Bacillus anthracis lethal toxin was re-ported not only to induce hemolysis, but also to inhibiterythroid differentiation of cord blood-derived CD34+

hematopoietic stem cells, leading to the suppressionof erythropoiesis (97). The toxin-induced suppressionof erythropoiesis is potentially part of a lethal toxin-mediated pathophysiology in anemia and hypoxia. Sim-ilarly, it was reported that mature erythroblasts werepreferentially decreased when isolated mouse bonemarrow cells were treated with α-toxin, and subsequentexperiments revealed that the blockage of erythroiddifferentiation was involved in the reduction in matureerythroblasts by α-toxin (98). Massive intravascularhemolysis and severe anemia have been reported inC. perfringens-infected patients (99), although it is arelatively uncommon event. The detailed mechanismsby which C. perfringens infection causes severe anemiahave not been elucidated, but blockage of erythropoiesisby α-toxin may be important (Fig. 5).

Experiments carried out using an inactive α-toxinH148G variant revealed that its enzymatic activity wasrequired for the α-toxin-mediated reduction of erythro-blast levels (98). Furthermore, the cell surface expressionof a lipid raft marker, GM1, decreased in associationwith erythroid differentiation, and α-toxin affected lipid

raft integrity in erythroid progenitors, leading to theimpairment of erythroid differentiation. During eryth-roblast enucleation, the clustering of lipid rafts at thefurrow between incipient reticulocytes and pyrenocytesis observed, and an inhibitor of lipid rafts impedesenucleation (100). Therefore, lipid rafts play an impor-tant role in the regulation of erythropoiesis, but thedetailed mechanism by which the disturbance in lipidraft integrity by α-toxin impairs erythroid differentiationremains unclear.

STRUCTURE AND HOST CELLINTERACTIONS OF C. PERFRINGENSPERFRINGOLYSIN OPerfringolysin O (θ-toxin) is a pore-forming hemolytictoxin that belongs to the cholesterol-dependent cyto-lysin (CDC) family (16, 101), which includes streptoly-sin O from Streptococcus pyogenes, listeriolysin O fromL. monocytogenes, pneumolysin from Streptococcuspneumoniae (17), and related cytolysins producedby several other clostridia (102). The perfringolysin Ostructural gene, pfoA, is located on the chromosome(103, 104). Perfringolysin O is elaborated by most, butnot all, strains of C. perfringens (104–107), and matureperfringolysin O (53 kDa, 473 aa) (108) is secretedas a water-soluble monomer and binds to host cellmembranes in a cholesterol-dependent manner (102).Binding to the cell membrane initiates oligomerizationand the formation of a pore complex that can containup to 50 monomeric subunits and is responsible forcell lysis.

Perfringolysin O monomers have a hydrophilic,β-sheet-rich structure that can be divided into four dis-tinct domains (109, 110) (Fig. 6). Domain 1 contains aseven-stranded antiparallel β-sheet flanked by helicesand loops. Domain 2 forms an elongated β-sheet struc-ture. Domain 3 comprises α-helices and β-sheets, anddomain 4 (the C-terminal region) is divided into twodistinct β-sandwich regions. Domain 4 also contains ahighly conserved 11-aa Trp-rich motif (ECTGLAWEWWR) or undecapeptide loop that is near the C-terminus.This structure subsequently was shown to be similar tothat of members of the eukaryotic membrane attackcomplex/perforin family (111).

Perfringolysin O and most of the other cholesterol-dependent cytolysins bind directly to cholesterol in thehost cell membrane. Contrary to what was thoughtfor many years, the undecapeptide is not the receptor-binding motif (112). Instead, there is a Thr-Ile pairlocated in a nearby loop (L1) of domain 4 that is re-




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sponsible for binding to cholesterol (113). The role ofthe undecapeptide is to assist in anchoring the toxinmonomer to the membrane and to couple membraneinsertion with oligomerization (112). The next step inthe oligomerization process is a conformational changeof two α-helical bundles in domain 3 to form two longeramphipathic β-hairpins; this extended β-hairpin struc-ture inserts into the host cell membrane, initially to formtoxin dimers and then to form a large oligomeric β-barrelpore that contains 35 to 45 monomers. The biologicaleffect of large pore formation is to induce cell lysis byosmotic stress (114). In addition, perfringolysin O caninterfere with cell signaling events by activating TLR4and inducing inducible nitric oxide synthase expres-sion and tumor necrosis factor-α (TNFα) secretion inbone marrow-derived macrophages (115) and by stim-ulating the degradation of Ubc9, thereby reducing theposttranslational SUMOylation of host proteins (116).Therefore, perfringolysin O has effects on both mem-brane integrity and internal cell signaling.

Perfringolysin O works synergistically with α-toxin tocause gas gangrene by promoting leukostasis and in-travascular coagulation (19–21, 117). The result is de-creased blood flow (87, 88) and the accumulation of

PMNL and macrophages in the blood vessels around thesite of infection, which leads to vascular leukostasis andcontributes to the paucity of leukocyte infiltration tothe site of infection (20, 21). Similarly, intramuscularinjection of a crude C. perfringens toxin preparationcauses and promotes the formation of intravascularplatelet aggregates (87). Leukocytes and fibrin accumu-late rapidly in the aggregate, leading to obstruction ofthe blood vessels and the production of thrombi thatreduce local blood flow (87, 88).

Perfringolysin O upregulates the expression of ad-herence molecules, including platelet-activating factor,CD11b, and endothelial adherence molecules (118–120). In addition, it stimulates the expression of inter-cellular adherence molecule 1 and platelet-activatingfactor in endothelial cells (19, 118, 119, 121). The toxintriggers platelet/leukocyte and platelet/platelet aggrega-tion by activating the platelet fibrinogen receptor gpIIb/IIIa synergistically with α-toxin (86, 122). Moreover, ithas been reported to impair the polymerization of actinfilaments in leukocytes and the migration of neutrophilsin response to chemoattractants (118, 123). These eventslead to the formation of intravascular platelet/leucocyte/fibrin aggregates, and the adherence of the aggregatesto the vascular endothelial cells results in vascular in-jury, vessel obstruction, hypoxia, and tissue destruction(1, 30, 124). In addition, a direct cytotoxic effect ofperfringolysin O on endothelial cells has been demon-strated, which also impairs leukocyte transmigration(30, 88). Disruption of the endothelium contributes toprogressive edema (30). α-Toxin impairs granulopoiesis,leading to the reduction of neutrophils, and a directcytolytic effect of perfringolysin O on leukocytes hasbeen reported (123). Therefore, perfringolysin O workssynergistically with α-toxin to reduce the level of leuko-cytes. Finally, C. perfringens cells can escape from thephagosome of macrophages and survive, and perfringo-lysin O is an important factor in this escape process(125, 126). These data are in agreement with geneticstudies that showed that α-toxin and perfringolysin Oact synergistically in the disease process (19, 20).

In the latter stage of clostridial gas gangrene, cardio-vascular collapse, tachycardia, and multiorgan failureare observed in patients (1). Toxins such as perfringolysinO and α-toxin are released into the blood and can act ata distance from the site of infection. Perfringolysin O re-duces systemic vascular resistance, increases cardiac out-put, and decreases the heart rate without a drop in meanarterial pressure (117, 127). In addition, it promotes therelease of inflammatory cytokines, such as TNFα, IL-1,IL-6, platelet-activating factor, and prostaglandin I2, by

FIGURE 6 Structure of perfringolysin O. The structure of per-fringolysin O was obtained from the Protein Data Bank (code1PFO) and was created using MacPyMOL. A ribbon repre-sentation is shown, colored from blue (N-terminal) to red(C-terminal).


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acting synergistically with α-toxin, which contributes tothe symptoms of toxic shock, including hypotension,hypoxia, and reduced cardiac output (30, 128).

FUNCTION AND HOST CELL INTERACTIONSOF C. SEPTICUM α-TOXINC. septicum is the primary causative agent of nontrau-matic clostridial myonecrosis and enumerates an α-toxinthat is essential for disease and is responsible for reducedPMNL influx into the infected lesions (25). C. septicumα-toxin is a member of the aerolysin family of pore-forming toxins (129, 130). These toxins include aero-lysin from Aeromonas hydrophila, ε-toxin and C. per-fringens enterotoxin (CPE) from C. perfringens, andparasporin toxins produced by Bacillus thuringiensis.These toxins all form small, often heptameric, β-barrelpores in host cell membranes (130). Although the crystalstructures of several members of the aerolysin familyhave been determined, there is no such structure availablefor α-toxin. However, molecular modelling of α-toxinshows that it does not have the small-lobe lectin-bindingdomain (D1) of aerolysin, known as the aerolysin per-tussis toxin domain (131), but has homologous domainsto the other three aerolysin domains (Fig. 7) (132).

α-Toxin is synthesized as a preprototoxin, with a 31-aa leader sequence that is cleaved upon secretion fromthe bacterial cell and a 45-aa C-terminal extension that iscleaved by host proteases such as furin prior to pore

formation (23, 102, 133). The resultant cleaved pro-peptide prevents premature oligomerization (134). The46-kDa prototoxin binds to glycophosphatidylinositol-anchored protein receptors located in lipid rafts in thehost cell membrane (135). After protease cleavage onthe cell membrane, the 43-kDa mature toxin undergoesa conformational change and oligomerizes to form aheptameric prepore complex that is required for poreformation (24). Cleavage of the C-terminal extension en-ables the formation of a transmembrane β-barrel struc-ture, with each monomer contributing one extendedβ-hairpin loop to the β-barrel that forms the trans-membrane pore (130, 132).

The analysis of substitution derivatives of α-toxinhas yielded insights into the functional domains ofthe toxin (132, 136–138). Y155F and S189C α-toxinderivatives had reduced toxicity for CHO cells butwere still cleaved by host proteases and could bind toglycophosphatidylinositol-anchored receptors and formoligomers. A S189C/S238C derivative retained its abilityto bind, act as a substrate for furin cleavage, and formoligomers but was not toxic (136). Like aerolysin,α-toxin has a Trp-rich motif (amino acids 302 to 312)in its C-terminal domain (138). Several of the residuesin this motif, including three Trp residues, are essentialfor binding and/or cytotoxic activity (137, 138). Fluo-rimetric studies using Cys-substituted derivatives led tothe identification of the amphipathic transmembraneβ-hairpin (Lys-203 to Gln-232) that spans the host cellmembrane. Deletion of this region eliminated the toxin’sability to form pores and kill cells, but the resultantprotein could still bind, undergo cleavage, and formoligomers (132). Subsequently, saturation Ala or Cyssubstitutions were made across the remaining residues inthe α-toxin molecule, and the resultant proteins werepurified and analyzed for their biological and biophysi-cal properties (137). The results showed that the onlyα-toxin Cys residue, Cys-86 in domain 1, was requiredfor receptor binding. In summary, derivatives alteredin receptor binding all had substitutions in domain 1,but proteins with altered oligomerization properties hadchanges in domains 1 and 3. Several of these mutantshave been examined in the mouse myonecrosis model(139). The results showed that both the amphipathicβ-hairpin and Cys-86 were essential for disease, pro-viding evidence that receptor binding and pore forma-tion are important in pathogenesis.

Pore formation is essential for C. septicum-mediatedmyonecrosis (129, 140), but pore-mediated osmoticcell lysis is not the only consequence of intoxication byα-toxin. Treatment of C2C12 mouse myoblast cells

FIGURE 7 Crystal structure of aerolysin and molecular modelof C. septicum α-toxin. Domains are indicated. Note thatdomain 1 (D1) of aerolysin is not present in α-toxin. AT,C. septicum α-toxin. Reprinted with permission from refer-ence 137. Copyright (2006) American Chemical Society.




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with α-toxin leads to a rapid Ca2+ influx into the cell butdoes not induce apoptosis. Instead, α-toxin induces pro-grammed cellular necrosis (140), which is consistentwith the fact that it is more active against nucleated cells(141, 142). α-Toxin-mediated Ca2+ influx resulted in theactivation of proteolytic calpain activity, a reduction inlysosomal integrity, leading to cathepsin release, mito-chondrial dysfunction and ATP depletion, and releasefrom the nucleus of the histone-binding protein HMGB1(140). Induction of this programmed necrotic pathwaywas dependent on the ability to form pores; treatmentwith a substituted α-toxin lacking the β-hairpin loop didnot induce a Ca2+ influx or programmed necrosis. All ofthese changes are consistent with α-toxin-mediated poreformation causing the induction of the programmed cel-lular necrosis pathway (140). Other studies have shownthat α-toxin treatment of Madin-Darby Canine Kidney(MCDK) cells causes a rapid K+ efflux, ATP depletion,and caspase-3-independent necrosis and nonapoptoticcell death (129). Subsequently, it was shown that poreformation by α-toxin led to ERK, c-Jun N-terminal pro-tein kinase, and p38 phosphorylation and activation oftheir resultant MAPK pathways and the resultant dose-dependent release of TNFα (143). C. septicum α-toxinalso leads to a significant reduction in vascular perfusionof muscle tissues, as shown by comparative intravitalmicroscopy studies using culture supernatants from anisogenic series of C. septicum strains (88).

CONTROL AND TREATMENT OFHISTOTOXIC CLOSTRIDIAL INFECTIONSThe treatment of myonecrotic infections caused byC. perfringens and C. septicum is complicated by therapid onset and progression of disease. Death can resultwithin 24 hours of initial symptoms. Standard treatmentregimens involve surgical debridement of the infectedtissues, which needs to occur as early as possible (2),and treatment with penicillin and clindamycin (144).The effectiveness of the penicillin on its own has beenquestioned (145), and there is experimental evidencethat clindamycin and metronidazole, alone or in com-bination with penicillin are more effective in mice (145,146). Often, surgical removal of an infected limb isthe only option available in life-threatening infections.

In other studies, mice lacking TNF-α were protectedfrom C. perfringens α-toxin-induced lethality (147),demonstrating that TNF-α release is essential for thelethal effect of the toxin. Treatment with erythromycinblocked α-toxin-induced release of TNF-α from periph-eral neutrophils and the activation of TrkA and ERK1/2,

demonstrating that toxin-induced TNF-α release is re-lated to stimulation of the ERK/MAPK signaling path-way through TrkA. Anti-TNF-α antibodies, but notanti-IL-1β or anti-IL-6 antibodies, blocked the deathof mice and intravascular hemolysis by α-toxin. Com-pounds that block the release of TNF-α therefore maybe valuable in treating patients with C. perfringens in-fection. Recently, experiments with C. perfringens in-fections in mice provided evidence that treatment withopioids such as buprenorphine stop the developmentof disease (148), but this treatment option has not beentested in clinical settings.

Several studies have investigated the use of recombi-nant α-toxin variants to vaccinate againstC. perfringens-mediated gas gangrene, primarily from a military orbiosecurity perspective or for elderly patients that areat risk. Initial studies showed that the isolated nontoxicC-terminal C2 lipid-binding domain of α-toxin (aminoacids 247 to 370) was highly immunoprotective in mice(14). Mice immunized with this formaldehyde-treatedrecombinant antigen were protected from the effectsof α-toxin and from challenge with a 10× LD50 dose ofC. perfringens. By contrast, mice immunized with theequivalent N-terminal enzymatic domain (amino acids 1to 249) produced immunoreactive antibodies, but theseantibodies did not protect against the toxin or disease.These results were confirmed in a subsequent, more de-tailed, vaccination study that also showed that micevaccinated with the C-terminal domain and then chal-lenged in a murine infection model had reduced throm-bosis and an increased PMNL influx into the site ofinfection (149). To further define the immunoprotectiveregion of the C-terminal domain, the potency of severalrecombinant fragments (amino acids 251 to 370, 281 to370, and 311 to 370) fused to glutathione S-transferase(GST) were examined in an active vaccination model(150). Vaccination with the 251 to 370 and 281 to 370GST-fusions protected against the toxicity of α-toxinand against C. perfringens challenge, whereas the aminoacid 311 to 370 derivative yielded only partial protec-tion. Despite these three promising studies, no commer-cial C. perfringens vaccine is currently available.

ACKNOWLEDGMENTSResearch in Julian Rood’s laboratory was supported by projectgrant GNT1082401 from the Australian National Health andMedical Research Council.

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