Histotoxic Clostridial Infections MASAHIRO 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 or Although the histotoxic clostridia cause severe, gas gangrene involves an interruption to the blood supply to rapidly developing infections, these potent toxigenic the infected tissues, often via a traumatic wound, anaerobic bacteria still must be regarded as opportunistic patho- growth of the infecting clostridial cells, the production of gens. All of these infections require predisposing extracellular toxins, and toxin-mediated cell and tissue damage. This review focuses on host-pathogen interactions conditions to cause disease, often a traumatic wound in Clostridium perfringens-mediated and Clostridium that enables the entry of spores from the soil or gastro- septicum-mediated myonecrosis. The major toxins involved intestinal tract to gain access to ischemic internal organs are C. perfringens α-toxin, which has phospholipase C and or soft tissues of the body. Alternatively, in nontrau- sphingomyelinase activity, and C. septicum α-toxin, matic myonecrosis caused by C. septicum, gastrointes- β a -pore-forming toxin that belongs to the aerolysin family. tinal malignancy is commonly associated with the onset ff Although these toxins are cytotoxic, their e ects on host of disease, with tumor development leading to ulceration cells are quite complex, with a range of intracellular cell signaling pathways induced by their action on host cell membranes. and necrosis of the gastrointestinal mucosa and entry of the gastrointestinal microbiota into the circulation and subsequent infection at distal sites (3, 4). Clostridial GENERAL CHARACTERISTICS OF necrotizing soft tissue infections, especially those caused HISTOTOXIC CLOSTRIDIAL INFECTIONS by C. perfringens, Clostridium sordellii, and Clostri- Many pathogenic clostridial species cause potentially dium novyi, also have resulted from the subcutaneous – fatal soft tissue infections in humans and animals be- injection of contaminated black tar heroin (5 7). cause of their ability to produce extracellular protein toxins (1, 2). The ability of these pathogens to produce Received: 19 March 2018, Accepted: 23 March 2018, spores that are resistant to environmental stress is an Published: 26 July 2019 Editors: Vincent A. Fischetti, The Rockefeller University, New York, important factor in the epidemiology of these diseases. NY; Richard P. Novick, Skirball Institute for Molecular Medicine, Disease pathogenesis involves the growth of the clos- NYU Medical Center, New York, NY; Joseph J. Ferretti, Department tridial pathogen in the tissues and extensive tissue de- of Microbiology & Immunology, University of Oklahoma Health Science Center, Oklahoma City, OK; Daniel A. Portnoy, Department struction, which is the result of the action of extracellular of Molecular and Cellular Microbiology, University of California, toxins. Typical histotoxic clostridial diseases include Berkeley, Berkeley, CA; Miriam Braunstein, Department of Microbiology and Immunology, University of North Carolina-Chapel human gas gangrene or myonecrosis and blackleg in Hill, Chapel Hill, NC, and Julian I. Rood, Infection and Immunity cattle. Although there are several clostridial species that Program, Monash Biomedicine Discovery Institute, Monash are responsible for these syndromes (Table 1), this University, Melbourne, Australia review will focus on histotoxic infections caused by Citation: Nagahama M, Takehara M, Rood JI. 2019. Histotoxic clostridial infections. Microbiol Spectrum 6(4):GPP3-0024-2018. Clostridium perfringens and Clostridium septicum, doi:10.1128/microbiolspec.GPP3-0024-2018. primarily because these are the species that have been Correspondence: Masahiro Nagahama, [email protected] the subject of the most extensive molecular and func- -u.ac.jp or Julian I. Rood, [email protected] tional studies. © 2019 American Society for Microbiology. All rights reserved.

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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.

PATHOGENESIS OF CLOSTRIDIAL coli, and therefore devoid of any other C. perfringens MYONECROSIS CAUSED BY toxins, showed that the C-terminal domain of α-toxin C. PERFRINGENS AND C. SEPTICUM was immunoprotective (14). Second, mutation of the As previously described (8), the pathogenesis of these α-toxin structural gene (plc or cpa) abrogated the ability infections involves three distinct stages, irrespective of of the bacterium to cause clostridial myonecrosis in a whether the source of infection involves a traumatic murine model. Virulence was restored by complemen- wound. Stage 1 involves an interruption to the blood tation in trans with a recombinant plasmid containing supply such that the redox potential in the tissues drops the wild-type plc gene (15), thereby fulfilling molecular to a level that facilitates spore germination and/or the Koch’s postulates and providing proof of the essential growth of the infecting clostridial cells. Stage 2 involves role of this toxin in disease. bacterial growth and establishment of the conditions for The other major toxin produced by C. perfringens toxin production. In C. perfringens, regulation of toxin type A is perfringolysin O, or θ-toxin, a pore-forming production primarily utilizes the VirSR two-component toxin that is a member of the cholesterol-dependent cy- signal transduction system (9) and an accessory growth tolysin family (16, 17). Although mutation of the per- regulator-like quorum sensing system (10, 11), as well fringolysin O structural gene, pfoA, did not eliminate as other regulatory networks. Stage 3 encompasses the the ability to cause disease (18, 19) subsequent studies toxin-mediated cell and tissue damage that leads to ne- of plcpfoA double mutants (20) provided evidence that crosis, systemic toxicity, and clinical disease (8). perfringolysin O has a synergistic effect with α-toxin on C. perfringens type A is the major cause of traumatic disease pathogenesis (20, 21). gas gangrene, although other histotoxic clostridia may Recent studies have involved the concurrent analy- also be responsible for this sporadic, but fulminant sis of the transcriptomes of both the host and C. per- and often fatal, disease (Table 1)(1). The major toxin fringens in a murine myonecrosis infection (22). The produced by gas gangrene strains of C. perfringens is results showed that many host genes involved in the α-toxin, a zinc metallophospholipase that has both innate immune response to infection were upregulated phospholipase C and sphingomyelinase activity (12). in C. perfringens-infected muscle tissues. In the C. per- α-Toxin was the first bacterial toxin to be shown to have fringens cells, upregulated genes included those en- enzymatic activity (13). Two independent studies using coding potential adhesins and proteins associated with different experimental approaches have shown that the cell envelope. α-toxin is essential for C. perfringens-mediated myo- C. septicum is the major causative agent of nontrau- necrosis. First, immunization studies in mice using re- matic gas gangrene, often associated with a gastro- combinant α-toxin variants purified from Escherichia intestinal malignancy (4). The major toxin produced

2 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 130.194.145.38 On: Fri, 06 Sep 2019 02:16:21 Histotoxic Clostridial Infections by C. septicum is also called α-toxin, but it is not related to C. perfringens α-toxin. C. septicum α-toxin is an aerolysin-like pore-forming toxin that is secreted as an inactive prototoxin that is cleaved near the C terminus by membrane-bound furin or furin-like proteases to form the active toxin (23). The toxin then oligomerizes on the membrane and subsequently inserts to form a membrane pore, which ultimately leads to cell lysis (24). Genetic studies have shown that α-toxin is essential for C. septicum-mediated myonecrosis in mice (25). Muta- tion of the α-toxin structural gene, csa, leads to a loss of virulence, which is restored by complementation with the wild-type csa gene.

STRUCTURE AND FUNCTION OF C. PERFRINGENS α-TOXIN FIGURE 1 Structure of α-toxin. The structure of α-toxin was α obtained from the Protein Data Bank (code 1QM6) and was -Toxin is a 43-kDa single polypeptide that is a hemo- created using MacPyMOL. A ribbon representation is shown, lytic, cytotoxic, dermonecrotic, and lethal extracellu- colored from blue (N-terminal) to red (C-terminal). Two zinc lar toxin (26–29). Moreover, it induces contractions metal ions in the active site are shown as red spheres. of blood vessels and smooth muscle, aggregation of platelets, superoxide production, and cytokine release. Importantly, together with perfringolysin O, it is re- domain of pancreatic lipase, the N-terminal domain of sponsible for the hallmark histopathological feature of soybean lipoxygenase, and the C2 phospholipid-binding C. perfringens-mediated myonecrosis, the paucity of the domains of eukaryotic intracellular proteins, such as polymorphonuclear leukocyte (PMNL) influx into the synaptotagmin I, which are involved in signal trans- infected lesion (15, 19–21, 30). duction and vesicular transport (37, 38). Determination of the crystal structure of α-toxin The active site in the N-terminal domain is situated revealed that it has an α-helical N-terminal domain within a solvent-accessible cleft that contains two zinc (amino acids 1 to 246) that is responsible for its enzy- ions and an exchangeable divalent cation. His-148 and matic activity, a flexible linker (amino acids 247 to 255), Glu-152 dock with one zinc ion, which is critical for the and a C-terminal (amino acids 256 to 370) β-sandwich catalytic site of the toxin. His-11 and Asp-130 tightly membrane-binding domain (31)(Fig. 1). The two do- dock with the other zinc ion, which is needed for main- mains also are linked by hydrophobic surface interac- tenance of the structure. His-68, His-126, and His-136 tions between their neighboring faces. The N-terminal dock with an exchangeable divalent cation, which is domain has significant sequence similarity to phospho- required for binding to target cell membranes (39). lipase C enzymes from Bacillus cereus (32), Clostridium The external loop regions (residues 70 to 90, 135 to 150, bifermentans (33), and Listeria monocytogenes (34). It 205 to 215, 265 to 275, 290 to 300, and 330 to 340) comprises nine densely packed α-helices and resembles bind to phospholipids in the host cell membranes (31). the structure of B. cereus phospholipase C (31, 35), Thr-74 is present in one of these loops, and although which has enzymatic activity, but is neither toxic nor the substitution derivative T74I is still active and can hemolytic. There are three loops in the N-terminal do- cleave a water-soluble substrate, its membrane-damaging main: loop 1 (residues 70 to 90), loop 2 (residues 135 activity toward red blood cells and phosphatidylcholine- to 150), and loop 3 (residues 205 to 215). These loops cholesterol liposomes is decreased compared to wild-type serve a vital role in the direct interaction between the toxin (40, 41). However, the structure of the T74I variant toxin and the membrane-binding component. The C- is identical to that of the wild-type toxin (41). The T74I terminal domain comprises eight-stranded antiparallel substitution results in altered recognition of the glycerol β-sheets (31). This domain plays a role in membrane moiety in the phospholipid, without affecting the func- binding and is required for hemolytic and toxic activity tion of the active site, supporting the hypothesis that the (28, 36). It is absent from nontoxic phospholipase C en- amino acid 70 to 90 loop is important for phospholipid zymes (31). It has structural similarity to the C-terminal recognition (42, 43).

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Computational modeling of α-toxin and phosphati- to the membrane and that the amino acid 70 to 90 loop dylcholine revealed that Tyr-57 and Tyr-65, which exist and Tyr-57 and Tyr-65 serve an important role in the re- at an external site near the catalytic cleft and are in cognition of target membranes (Fig. 2). Other studies proximity with the 70 to 90 loop, are responsible for have shown that substitution of the C-terminal domain substrate interaction (44). The replacement of these residues Asp-269, Asp-336, Tyr-275, and Tyr-331, Tyr residues with Ala did not have any effect on phos- which were predicted to be involved in membrane bind- pholipase C and sphingomyelinase activity but had a ing (31), were required for hemolytic and cytotoxic ac- marked effect on the membrane-damaging activity (he- tivity (45). molysis of sheep red cells and disruption of liposomes). These residues exist in or near the entrance of the cata- INTERACTION OF C. PERFRINGENS lytic cleft and are essential for the membrane-damaging α-TOXIN WITH HOST CELLS effect of α-toxin, because they are required for entry of the catalytic cleft into the hydrophobic region of plasma Membrane-Damaging Activity on membranes (44). Phospholipid Bilayers Several studies have shown that the calcium-binding The effect of α-toxin on host tissues is caused by the C-terminal domain is responsible for membrane bind- phospholipase C- and sphingomyelinase-mediated cleav- ing (28, 31, 36, 37). Two acrylodan-labeled C-terminal age of phospholipids in the host cell membranes. The domain derivatives, S263C and S365C, bound to lipid resultant disruption of the phospholipid bilayer together bilayers and displayed a chromogenic shift, demonstrat- with the end products of these cleavage reactions, diacyl- ing entry of the C-domain of the toxin into the hydro- glycerol and ceramide, respectively, activates lipid me- phobic regions of the liposomes (36). The data provide tabolism in host cell membranes and plays a role in host evidence that the C-terminal domain of the toxin binds cell damage (27, 28).

FIGURE 2 Binding model of α-toxin and membrane phospholipids. The structure of α-toxin was obtained from the Protein Data Bank (code 1QM6) and was created using MacPyMOL. Phospholipids are shown as light gray spheres for head groups and light gray lines for tail groups. The head group of a phospholipid, from the outer membrane into the active 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- Treatment of rabbit neutrophils with α-toxin pro- lates the extent of the α-toxin-mediated membrane dis- motes the formation of diacylglycerol, which is fol- ruption (46). In another study that measured the release lowed by adhesion of the cells to extracellular matrix of carboxyfluorescein from various liposomes, α-toxin- proteins and the generation of superoxide ions (Fig. 3B) mediated release, or liposomal leakage, decreased with (51). α-Toxin induces diacylglycerol generation through increasing chain length of the hydrocarbon residues activation of intrinsic phospholipase C enzymes by a in the phospholipid bilayer (40). It was concluded that pertussis toxin-sensitive GTP-binding protein. More- membrane disruption was related to the fluidity of the over, it increases the phosphorylation of neurotrophic membrane and double bonds in the acyl chain. Sub- tyrosine kinase receptor type 1 (TrkA), leading to 3- sequently, several phosphatidylcholines (C18:0/C18:1) phosphoinositide-dependent protein kinase 1 activation with a double bond in the sn-2 fatty acyl chain were (52). Therefore, α-toxin independently causes stimula- prepared (47). The phase transition temperature value tion of intrinsic phospholipase C and protein kinase 1 was lowest when the cis-double bond was positioned at activity through TrkA receptor activation. Both phe- the center of the C18:1 sn-2 acyl chain and increased nomena synergistically augment the activity of protein continuously as the double bond was moved toward kinase C θ, leading to the formation of superoxide ions either end of the acyl chain. α-toxin-mediated carboxy- via activation of the mitogen-activated protein kinase fluorescein release from various liposomes was highest (MAPK) cascade (Fig. 3B)(52). In human lung carci- when the cis-double bond was positioned at the center of noma (A549) cells, α-toxin mediates the activation of the the sn-2 fatty acyl chain, resulting in a bell-shaped curve p38 MAPK, NF-κB, and extracellular signal-regulated (47). Accordingly, the membrane-disrupting activity of kinase 1/2 (ERK1/2) through TrkA activation, which the toxin is strongly associated with the membrane flu- leads to interleukin-8 (IL-8) release (53)(Fig. 3B). idity of target membranes. Treatment of rabbit erythrocytes with α-toxin in- There is evidence that α-toxin also promotes per- duces the biphasic formation of phosphatidic acid fringolysin O-induced membrane damage in phospholipid (54)(Fig. 3A). α-Toxin-induced formation of phospha- bilayers. Hydrolysis of phosphatidylcholine residues of tidic acid is activated in the earlier stage (30 s) by in- liposomes that contained cholesterol increased the amount trinsic phospholipase C and in the later stage (ca. 25 offreecholesterolinthelipidbilayerandenhanced min) by intrinsic phospholipase D. Both processes perfringolysin O-dependent cytolytic activity (48). These involve stimulation of the G-protein-signaling pathway data may at least partly explain the synergistic effects of α- by toxin-mediated membrane phospholipid cleavage toxin and perfringolysin O that were previously observed (55)(Fig. 3A). in C. perfringens infections of mice (20). Sheep erythrocytes have higher amounts of sphingo- myelin but do not contain phosphatidylcholine. Cleav- Activation of Phospholipid age of sphingomyelin by α-toxin leads to increased levels Metabolism and Hemolysis of ceramide and sphingosine 1-phosphate (56), which is α-toxin provokes contraction of isolated rat ileum and essential for toxin-induced hemolysis (57) and activates aorta tissues in a dose-dependent manner through stim- the sphingomyelin metabolism system (Fig. 3A)(56). ulation of the phospholipid metabolic pathway (49, 50). The resultant cleavage of N-nervonoic sphingomyelin In isolated rat organs, α-toxin activates the phospha- (C24:1-SM) occurs in a detergent-resistant membrane tidylinositol metabolic pathway and then the arachi- component (57)(Fig. 3A). Pertussis toxin blocks the donic acid cascade, thereby promoting thromboxane production of C24:1-ceramide caused by α-toxin and production. Contraction caused by the toxin was evoked blocks red cell hemolysis, which indicates that intrinsic by thromboxane A2 generation induced by arachidonic sphingomyelinase, which can cleave C24:1-SM to C24:1- acid (49, 50). The metabolism of arachidonic acid results ceramide, is regulated by a pertussis toxin-sensitive G- in the production of bioactive lipid mediators, including protein in plasma membranes. In addition, the activation leukotrienes, thromboxanes, and prostaglandins (27). of Rho protein by α-toxin increases sphingosine kinase These bioactive lipid mediators play key roles in en- activity. Subsequently, the ceramides are quickly me- hancing the local inflammatory reaction and lead to tabolized to sphingosine, indicating that the toxin also vasoconstriction, which is responsible for inducing an- activates ceramidases (57). These results indicate that oxia in the early stages of bacterial infections. These the cleavage of C24:1-SM to sphingosine 1-phosphate by effects presumably contribute to the capacity of C. per- α-toxin in lipid rafts is essential for toxin-induced he- fringens to proliferate in the host and cause disease. molysis of sheep red cells (Fig. 3A).

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FIGURE 3 Relationship between phospholipid metabolism and the biological activities of α-toxin. (A) Signaling events involved in α-toxin-induced hemolysis of sheep or rabbit – erythrocytes. (B) Signaling events involved in α-toxin-activated generation of O2 in neutrophils 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, phosphatidylinositide dependent kinase 1; DRM, detergent-resistant membrane; TrkA, neurotrophic tyrosine kinase receptor type1; MAPKK, mitogen-activated protein kinase kinase; Erk1/2, extra- cellular signal-regulated kinase 1/2.

Interaction of α-Toxin with Gangliosides the ganglioside recognition site of botulinum and teta- Amino acid sequence analysis of botulinum toxin and nus toxins, significantly reduced binding to ganglioside tetanus toxin showed that the ganglioside-GT1b- and GM1a-containing liposomes. W84A and Y85A deriva- lactose-binding site is determined by the existence of the tives had markedly decreased capabilities to stimulate conserved peptide motif, H.....SXWY.....G (ca. 30 amino TrkA (59). Ganglioside GM1a assembles with a TrkA acids [aa]), with the Tyr and Trp residues being partic- molecule on the cell surface and stimulates TrkA and ularly critical (58). A similar motif (H.....SWY.....G, ERK1/2 (60). It has been reported that ganglioside GM1 amino acids 68 to 93) is present in α-toxin (53), in the clustering leads to the condensation of TrkA in lipid rafts region (amino acids 55 to 93) that is located at the in- and the stimulation of downstream signaling targets terface between the N-terminal and C-terminal domains (61). α-Toxin-mediated diacylglycerol production leads (42). It has been demonstrated that the 70 to 90 loop to diacylglycerol flip-flop movement that affects the in α-toxin plays a role in binding to the cell surface dynamics of cell membranes (Fig. 4). Therefore, it serves (43)(Fig. 2). Computational docking studies indicated in GM1a clustering and intrinsic phospholipase C-γ1 that Trp-84 binds to the sialic acid residue on ganglio- activation through TrkA, triggering various signal side GM1a via an aromatic stacking interaction and a transduction activities (62–64). hydrogen-bonding interaction and that Tyr-85 associ- α-toxin causes higher cytotoxicity in a ganglioside- ates with the galactosamine residue on GM1a via an deficient cell line (Don Q) (65) and can be internalized α-glycosidic bonding interaction (59). Replacement of by dynamin-mediated endocytosis (66, 67). The toxin Trp-84 and Tyr-85 in α-toxin, which correspond to binds to caveolin-1 both on the cell membranes and in

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FIGURE 4 Model of α-toxin-induced membrane dynamics and clustering of the GM1a and TrkA complex. α-Toxin binds to GM1a and, through the phospholipase C activity of the toxin itself, causes diacylglycerol (DG) production at the outer membrane. The flip-flop movement of DG affects membrane dynamics, facilitating clustering of GM1a and TrkA activation 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 enhanced activation of TrkA, triggering activation of signal transduction pathways, which induce neutrophil activation. Abbreviations: PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; DG, diacylglycerol; PC, phosphatidylcholine; TrkA, neurotrophic tyro- sine kinase receptor type 1. endosomal vesicles, and it also induces ceramide pro- is sustained in a steady state through granulopoiesis, duction in the cell membrane (28), which generates neg- which is accelerated to replenish neutrophils during bac- ative membrane curvature and facilitates endocytosis terial infection (71–73). Pattern recognition receptors, (68). Internalized α-toxin is transported to the early and such as Toll-like receptors (TLRs), are responsible for late endosomes and lysosomes. Lysosomes are damaged the recognition of structural components of microor- during progressive α-toxin internalization into Don Q ganisms (74, 75). TLR2 and TLR4 have been identified cells. In addition, the internalization of α-toxin is also as being pivotal for the recognition of Gram-positive or mediated through the stimulation of MEK/ERK signal- Gram-negative bacteria by recognizing cell wall com- ing (66, 67). It has been reported that the toxin causes ponents such as peptidoglycan and bacterial endotoxin reactive oxygen species generation via PKC, MEK/ERK, (lipopolysaccharide), respectively (76–79). During bac- and NF-κB signaling activities in Don Q cells (66, 67). terial infection, these bacterial components stimulate Therefore, α-toxin perturbs the host cell signaling cas- the production of granulocyte colony-stimulating factor cade and acts not only on the cell membrane, but also in (G-CSF), which is a glycoprotein that promotes the the cytoplasm. proliferation, survival, and differentiation of neutrophils and their progenitor cells (80) from endothelial cells and Effect of α-Toxin on Hematopoietic Cells monocytes, resulting in the acceleration of granulopoie- The first line of defense of the innate immune system sis (81). G-CSF-deficient and G-CSF receptor-deficient involves neutrophils, which play an important role in the mice exhibit chronic neutropenia and reduced infection- phagocytosis and subsequent elimination of pathogenic driven granulopoiesis, leading to impaired ability to bacteria (69, 70). Normally, the number of neutrophils eliminate infecting bacteria (82, 83). Thus, granulo-

ASMscience.org/MicrobiolSpectrum 7 Downloaded from www.asmscience.org by IP: 130.194.145.38 On: Fri, 06 Sep 2019 02:16:21 Nagahama et al. poiesis is precisely regulated to replenish neutrophils infected muscle, in an α-toxin-dependent manner (90). during bacterial infection. However, C. perfringens can α-Toxin inhibits neutrophil differentiation and impairs still cause life-threatening infections, and the mecha- the replenishment of differentiated mature neutrophils in nisms by which C. perfringens avoids these host defenses the peripheral circulation, resulting in reduced recruit- are still not completely understood. ment of neutrophils to the C. perfringens infection site. C. perfringens-mediated myonecrosis progresses so The half-life of peripheral neutrophils is less than 12 rapidly that death, which is associated with muscle de- hours in vivo (91), meaning that the number of mature struction, shock, and multiple organ failure, sometimes neutrophils required to counter a bacterial infection is precedes an accurate diagnosis (84). C. perfringens in- sustained through continuous granulopoiesis. Therefore, fection is characterized by a greatly reduced PMNL in- blockage of both granulopoiesis and PMNL diapedesis flux to the site of the infection (19, 85–87), suggesting into the tissues, as a result of platelet aggregation, leads that the bacteria disrupt neutrophil-based innate immu- to a reduction in cell numbers in a short period of time. nity to evade host defenses. α-Toxin mediates the forma- Thus, C. perfringens infection impairs the host immune tion of platelet-leukocyte aggregates, leading to vascular system, which could explain the both scarcity of PMNL occlusion, and induces a marked reduction in micro- at the site of infection and a propensity for polymicrobial vascular perfusion (45, 86, 88), which is thought to infections in many patients (Fig. 5). impede neutrophil extravasation (89). Recently, it was α-Toxin is essential for disease pathogenesis (18–21, shown using a mouse model of infection that C. per- 30), and blockage of neutrophil differentiation is de- fringens infection reduced mature neutrophils in bone pendent on its phospholipase C and sphingomyelinase marrow, in peripheral blood, and in C. perfringens- activities (90). In human lymphocytes, S. aureus sphin-

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

8 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 130.194.145.38 On: Fri, 06 Sep 2019 02:16:21 Histotoxic Clostridial Infections gomyelinase disrupts lipid rafts, which are cholesterol- raft integrity in erythroid progenitors, leading to the rich plasma membrane microdomains (92). Lipid rafts impairment of erythroid differentiation. During eryth- function as platforms of signaling molecules involved in roblast enucleation, the clustering of lipid rafts at the the regulation of cell differentiation in many cell types furrow between incipient reticulocytes and pyrenocytes (93, 94). Ceramide, the hydrolysis product of sphingo- is observed, and an inhibitor of lipid rafts impedes myelinase, also is known to be a lipid messenger impli- enucleation (100). Therefore, lipid rafts play an impor- cated in various cellular responses, including apoptosis, tant role in the regulation of erythropoiesis, but the differentiation, and cell growth (95). Recently, C. per- detailed mechanism by which the disturbance in lipid fringens α-toxin was reported to perturb the integrity raft integrity by α-toxin impairs erythroid differentiation of lipid rafts, leading to the blockage of neutrophil dif- remains unclear. ferentiation (90). The detailed molecular mechanism behind this blockage has not been elucidated, but it may be important to focus on the relationship between the STRUCTURE AND HOST CELL role of sphingomyelinase and cell differentiation to shed INTERACTIONS OF C. PERFRINGENS further light on host-pathogen interactions in clostridial PERFRINGOLYSIN O myonecrosis. Perfringolysin O (θ-toxin) is a pore-forming hemolytic toxin that belongs to the cholesterol-dependent cyto- Effect of α-Toxin on Erythropoiesis lysin (CDC) family (16, 101), which includes streptoly- α-Toxin has been reported to lyse erythrocytes from sin O from Streptococcus pyogenes, listeriolysin O from various species. It activates T-type Ca2+ channels and in- L. monocytogenes, pneumolysin from Streptococcus creases intracellular Ca2+, which plays an important role pneumoniae (17), and related cytolysins produced in the hemolysis of horse erythrocytes (96). The toxin by several other clostridia (102). The perfringolysin O activates the sphingomyelin metabolic system, leading structural gene, pfoA, is located on the chromosome to the hemolysis of sheep erythrocytes (56, 57), and the (103, 104). Perfringolysin O is elaborated by most, but lysis of rabbit erythrocytes involves α-toxin-mediated not all, strains of C. perfringens (104–107), and mature activation of endogenous phospholipase C activity (54, perfringolysin O (53 kDa, 473 aa) (108) is secreted 55). Recently, Bacillus anthracis lethal toxin was re- as a water-soluble monomer and binds to host cell ported not only to induce hemolysis, but also to inhibit membranes in a cholesterol-dependent manner (102). erythroid differentiation of cord blood-derived CD34+ Binding to the cell membrane initiates oligomerization hematopoietic stem cells, leading to the suppression and the formation of a pore complex that can contain of erythropoiesis (97). The toxin-induced suppression up to 50 monomeric subunits and is responsible for of erythropoiesis is potentially part of a lethal toxin- cell lysis. mediated pathophysiology in anemia and hypoxia. Sim- Perfringolysin O monomers have a hydrophilic, ilarly, it was reported that mature erythroblasts were β-sheet-rich structure that can be divided into four dis- preferentially decreased when isolated mouse bone tinct domains (109, 110)(Fig. 6). Domain 1 contains a marrow cells were treated with α-toxin, and subsequent seven-stranded antiparallel β-sheet flanked by helices experiments revealed that the blockage of erythroid and loops. Domain 2 forms an elongated β-sheet struc- differentiation was involved in the reduction in mature ture. Domain 3 comprises α-helices and β-sheets, and erythroblasts by α-toxin (98). Massive intravascular domain 4 (the C-terminal region) is divided into two hemolysis and severe anemia have been reported in distinct β-sandwich regions. Domain 4 also contains a C. perfringens-infected patients (99), although it is a highly conserved 11-aa Trp-rich motif (ECTGLAWEW relatively uncommon event. The detailed mechanisms WR) or undecapeptide loop that is near the C-terminus. by which C. perfringens infection causes severe anemia This structure subsequently was shown to be similar to have not been elucidated, but blockage of erythropoiesis that of members of the eukaryotic membrane attack by α-toxin may be important (Fig. 5). complex/perforin family (111). Experiments carried out using an inactive α-toxin Perfringolysin O and most of the other cholesterol- H148G variant revealed that its enzymatic activity was dependent cytolysins bind directly to cholesterol in the required for the α-toxin-mediated reduction of erythro- host cell membrane. Contrary to what was thought blast levels (98). Furthermore, the cell surface expression for many years, the undecapeptide is not the receptor- of a lipid raft marker, GM1, decreased in association binding motif (112). Instead, there is a Thr-Ile pair with erythroid differentiation, and α-toxin affected lipid located in a nearby loop (L1) of domain 4 that is re-

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PMNL and macrophages in the blood vessels around the site of infection, which leads to vascular leukostasis and contributes to the paucity of leukocyte infiltration to the site of infection (20, 21). Similarly, intramuscular injection of a crude C. perfringens toxin preparation causes and promotes the formation of intravascular platelet aggregates (87). Leukocytes and fibrin accumu- late rapidly in the aggregate, leading to obstruction of the blood vessels and the production of thrombi that reduce 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-activating factor in endothelial cells (19, 118, 119, 121). The toxin triggers platelet/leukocyte and platelet/platelet aggrega- tion by activating the platelet fibrinogen receptor gpIIb/ IIIa synergistically with α-toxin (86, 122). Moreover, it has been reported to impair the polymerization of actin filaments in leukocytes and the migration of neutrophils FIGURE 6 Structure of perfringolysin O. The structure of per- in response to chemoattractants (118, 123). These events fringolysin O was obtained from the Protein Data Bank (code 1PFO) and was created using MacPyMOL. A ribbon repre- lead to the formation of intravascular platelet/leucocyte/ fi sentation is shown, colored from blue (N-terminal) to red brin aggregates, and the adherence of the aggregates (C-terminal). to the vascular endothelial cells results in vascular in- jury, vessel obstruction, hypoxia, and tissue destruction (1, 30, 124). In addition, a direct cytotoxic effect of sponsible for binding to cholesterol (113). The role of perfringolysin O on endothelial cells has been demon- the undecapeptide is to assist in anchoring the toxin strated, which also impairs leukocyte transmigration monomer to the membrane and to couple membrane (30, 88). Disruption of the endothelium contributes to insertion with oligomerization (112). The next step in progressive edema (30). α-Toxin impairs granulopoiesis, the oligomerization process is a conformational change leading to the reduction of neutrophils, and a direct of two α-helical bundles in domain 3 to form two longer cytolytic effect of perfringolysin O on leukocytes has amphipathic β-hairpins; this extended β-hairpin struc- been reported (123). Therefore, perfringolysin O works ture inserts into the host cell membrane, initially to form synergistically with α-toxin to reduce the level of leuko- toxin dimers and then to form a large oligomeric β-barrel cytes. Finally, C. perfringens cells can escape from the pore that contains 35 to 45 monomers. The biological phagosome of macrophages and survive, and perfringo- effect of large pore formation is to induce cell lysis by lysin O is an important factor in this escape process osmotic stress (114). In addition, perfringolysin O can (125, 126). These data are in agreement with genetic interfere with cell signaling events by activating TLR4 studies that showed that α-toxin and perfringolysin O and inducing inducible nitric oxide synthase expres- act synergistically in the disease process (19, 20). sion and tumor necrosis factor-α (TNFα) secretion in In the latter stage of clostridial gas gangrene, cardio- bone marrow-derived macrophages (115) and by stim- vascular collapse, tachycardia, and multiorgan failure ulating the degradation of Ubc9, thereby reducing the are observed in patients (1). Toxins such as perfringolysin posttranslational SUMOylation of host proteins (116). Oandα-toxin are released into the blood and can act at Therefore, perfringolysin O has effects on both mem- a distance from the site of infection. Perfringolysin O re- brane integrity and internal cell signaling. duces systemic vascular resistance, increases cardiac out- Perfringolysin O works synergistically with α-toxin to put, and decreases the heart rate without a drop in mean cause gas gangrene by promoting leukostasis and in- arterial pressure (117, 127). In addition, it promotes the travascular coagulation (19–21, 117). The result is de- release of inflammatory cytokines, such as TNFα,IL-1, creased blood flow (87, 88) and the accumulation of IL-6, platelet-activating factor, and prostaglandin I2, by

10 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 130.194.145.38 On: Fri, 06 Sep 2019 02:16:21 Histotoxic Clostridial Infections acting synergistically with α-toxin, which contributes to formation (23, 102, 133). The resultant cleaved pro- the symptoms of toxic shock, including hypotension, peptide prevents premature oligomerization (134). The hypoxia, and reduced cardiac output (30, 128). 46-kDa prototoxin binds to glycophosphatidylinositol- anchored protein receptors located in lipid rafts in the host cell membrane (135). After protease cleavage on FUNCTION AND HOST CELL INTERACTIONS the cell membrane, the 43-kDa mature toxin undergoes OF C. SEPTICUM α-TOXIN a conformational change and oligomerizes to form a C. septicum is the primary causative agent of nontrau- heptameric prepore complex that is required for pore matic clostridial myonecrosis and enumerates an α-toxin formation (24). Cleavage of the C-terminal extension en- that is essential for disease and is responsible for reduced ables the formation of a transmembrane β-barrel struc- PMNL influx into the infected lesions (25). C. septicum ture, with each monomer contributing one extended α-toxin is a member of the aerolysin family of pore- β-hairpin loop to the β-barrel that forms the trans- forming toxins (129, 130). These toxins include aero- membrane pore (130, 132). lysin from Aeromonas hydrophila, ε-toxin and C. per- The analysis of substitution derivatives of α-toxin fringens enterotoxin (CPE) from C. perfringens, and has yielded insights into the functional domains of parasporin toxins produced by Bacillus thuringiensis. the toxin (132, 136–138). Y155F and S189C α-toxin These toxins all form small, often heptameric, β-barrel derivatives had reduced toxicity for CHO cells but pores in host cell membranes (130). Although the crystal were still cleaved by host proteases and could bind to structures of several members of the aerolysin family glycophosphatidylinositol-anchored receptors and form have been determined, there is no such structure available oligomers. A S189C/S238C derivative retained its ability for α-toxin. However, molecular modelling of α-toxin to bind, act as a substrate for furin cleavage, and form shows that it does not have the small-lobe lectin-binding oligomers but was not toxic (136). Like aerolysin, domain (D1) of aerolysin, known as the aerolysin per- α-toxin has a Trp-rich motif (amino acids 302 to 312) tussis toxin domain (131), but has homologous domains in its C-terminal domain (138). Several of the residues to the other three aerolysin domains (Fig. 7)(132). in this motif, including three Trp residues, are essential α-Toxin is synthesized as a preprototoxin, with a 31- for binding and/or cytotoxic activity (137, 138). Fluo- aa leader sequence that is cleaved upon secretion from rimetric studies using Cys-substituted derivatives led to the bacterial cell and a 45-aa C-terminal extension that is the identification of the amphipathic transmembrane cleaved by host proteases such as furin prior to pore β-hairpin (Lys-203 to Gln-232) that spans the host cell membrane. Deletion of this region eliminated the toxin’s FIGURE 7 Crystal structure of aerolysin and molecular model ability to form pores and kill cells, but the resultant of C. septicum α-toxin. Domains are indicated. Note that protein could still bind, undergo cleavage, and form domain 1 (D1) of aerolysin is not present in α-toxin. AT, oligomers (132). Subsequently, saturation Ala or Cys C. septicum α-toxin. Reprinted with permission from refer- substitutions were made across the remaining residues in ence 137. Copyright (2006) American Chemical Society. the α-toxin molecule, and the resultant proteins were purified and analyzed for their biological and biophysi- cal properties (137). The results showed that the only α-toxin Cys residue, Cys-86 in domain 1, was required for receptor binding. In summary, derivatives altered in receptor binding all had substitutions in domain 1, but proteins with altered oligomerization properties had changes in domains 1 and 3. Several of these mutants have been examined in the mouse myonecrosis model (139). The results showed that both the amphipathic β-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-mediated myonecrosis (129, 140), but pore-mediated osmotic cell lysis is not the only consequence of intoxication by α-toxin. Treatment of C2C12 mouse myoblast cells

ASMscience.org/MicrobiolSpectrum 11 Downloaded from www.asmscience.org by IP: 130.194.145.38 On: Fri, 06 Sep 2019 02:16:21 Nagahama et al. with α-toxin leads to a rapid Ca2+ influx into the cell but demonstrating that toxin-induced TNF-α release is re- does not induce apoptosis. Instead, α-toxin induces pro- lated to stimulation of the ERK/MAPK signaling path- grammed cellular necrosis (140), which is consistent way through TrkA. Anti-TNF-α antibodies, but not with the fact that it is more active against nucleated cells anti-IL-1β or anti-IL-6 antibodies, blocked the death (141, 142). α-Toxin-mediated Ca2+ influx resulted in the of mice and intravascular hemolysis by α-toxin. Com- activation of proteolytic calpain activity, a reduction in pounds that block the release of TNF-α therefore may lysosomal integrity, leading to cathepsin release, mito- be valuable in treating patients with C. perfringens in- chondrial dysfunction and ATP depletion, and release fection. Recently, experiments with C. perfringens in- from the nucleus of the histone-binding protein HMGB1 fections in mice provided evidence that treatment with (140). Induction of this programmed necrotic pathway opioids such as buprenorphine stop the development was dependent on the ability to form pores; treatment of disease (148), but this treatment option has not been with a substituted α-toxin lacking the β-hairpin loop did tested in clinical settings. not induce a Ca2+ influx or programmed necrosis. All of Several studies have investigated the use of recombi- these changes are consistent with α-toxin-mediated pore nant α-toxin variants to vaccinate against C. perfringens- formation causing the induction of the programmed cel- mediated gas gangrene, primarily from a military or lular necrosis pathway (140). Other studies have shown biosecurity perspective or for elderly patients that are that α-toxin treatment of Madin-Darby Canine Kidney at risk. Initial studies showed that the isolated nontoxic (MCDK) cells causes a rapid K+ efflux, ATP depletion, C-terminal C2 lipid-binding domain of α-toxin (amino and caspase-3-independent necrosis and nonapoptotic acids 247 to 370) was highly immunoprotective in mice cell death (129). Subsequently, it was shown that pore (14). Mice immunized with this formaldehyde-treated formation by α-toxin led to ERK, c-Jun N-terminal pro- recombinant antigen were protected from the effects tein kinase, and p38 phosphorylation and activation of of α-toxin and from challenge with a 10× LD50 dose of their resultant MAPK pathways and the resultant dose- C. perfringens. By contrast, mice immunized with the dependent release of TNFα (143). C. septicum α-toxin equivalent N-terminal enzymatic domain (amino acids 1 also leads to a significant reduction in vascular perfusion to 249) produced immunoreactive antibodies, but these of muscle tissues, as shown by comparative intravital antibodies did not protect against the toxin or disease. microscopy studies using culture supernatants from an These results were confirmed in a subsequent, more de- isogenic series of C. septicum strains (88). tailed, vaccination study that also showed that mice vaccinated with the C-terminal domain and then chal- lenged in a murine infection model had reduced throm- CONTROL AND TREATMENT OF bosis and an increased PMNL influx into the site of HISTOTOXIC CLOSTRIDIAL INFECTIONS infection (149). To further define the immunoprotective The treatment of myonecrotic infections caused by region of the C-terminal domain, the potency of several C. perfringens and C. septicum is complicated by the recombinant fragments (amino acids 251 to 370, 281 to rapid onset and progression of disease. Death can result 370, and 311 to 370) fused to glutathione S-transferase within 24 hours of initial symptoms. Standard treatment (GST) were examined in an active vaccination model regimens involve surgical debridement of the infected (150). Vaccination with the 251 to 370 and 281 to 370 tissues, which needs to occur as early as possible (2), GST-fusions protected against the toxicity of α-toxin and treatment with penicillin and clindamycin (144). and against C. perfringens challenge, whereas the amino The effectiveness of the penicillin on its own has been acid 311 to 370 derivative yielded only partial protec- questioned (145), and there is experimental evidence tion. Despite these three promising studies, no commer- that clindamycin and metronidazole, alone or in com- cial C. perfringens vaccine is currently available. bination with penicillin are more effective in mice (145, 146). Often, surgical removal of an infected limb is ACKNOWLEDGMENTS the only option available in life-threatening infections. Research in Julian Rood’s laboratory was supported by project In other studies, mice lacking TNF-α were protected grant GNT1082401 from the Australian National Health and from C. perfringens α-toxin-induced lethality (147), Medical Research Council. demonstrating that TNF-α release is essential for the lethal effect of the toxin. Treatment with erythromycin REFERENCES α α 1. Stevens DL, Aldape MJ, Bryant AE. 2012. Life-threatening clostridial blocked -toxin-induced release of TNF- from periph- infections. Anaerobe 18:254–259 http://dx.doi.org/10.1016/j.anaerobe eral neutrophils and the activation of TrkA and ERK1/2, .2011.11.001.

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