Molecular Mechanisms of Action of Bacterial Exotoxins

Zbl. Bakt. 284,170-206 (1996) © Gustav Fischer Verlag, Stuttgart· Jena . New York

Molecular Mechanisms of Action of Bacterial Exotoxins* JOACHIM BALFANZ, PETER RAUTENBERG, and UWE ULLMANN Institut fur Medizinische Mikrobiologie und Virologie, Klinikum der Christian-AlbrechtsUniversitat, Kiel, Germany Received March 11, 1996 . Accepted March 25,1996

Summary Toxins are one of the inventive strategies that bacteria have developed in order to survive. As virulence factors, they playa major role in the pathogenesis of infectious diseases. Recent discoveries have once more highlighted the effectiveness of these precisely adjusted bacterial weapons. Furthermore, toxins have become an invaluable tool in the investigation of fundamental cell processes, including regulation of cellular functions by various G proteins, cytoskeletal dynamics and neural transmission. In this review, the bacterial toxins are presented in a rational classification based on the molecular mechanisms of action. Introduction

Pathogenic bacteria have evolved mechanisms for evading the defense system of their hosts and for destabilizing the cell's functional system. A common and effective way of achieving this is to produce toxins. Toxins are the principal pathogenic factors of microorganisms causing such infectious diseases as diphtheria, whooping cough, cholera, scarlet fever, listeriosis, botulism, tetanus, anthrax, gas gangrene, dysentery and gastroenteritis. Administration of a deactivated but still immunogenic variant of the toxin, i. e., vaccination with a toxoid, can often effectively protect the organism from the corresponding disease. In microbiology, the term "toxin" describes products of microbial metabolism that are lethal to cells or affect their function negatively even at very low concentrations. Often, this results in pathological changes in the entire body. In general, one should differentiate between endotoxins and exotoxins. Endotoxins are lipopolysaccharides that are part of the cell wall of gram-negative bacteria. Exotoxins are mostly proteins that are se<.:reted by microorganisms into the surrounding medium. The main mechanisms of action of the bacterial exotoxins will be described in this review. At the same time, this offers a chance to try to develop a useful classification of toxins. There is no generally accepted rational taxonomy for bacterial exotoxins like

* Dedicated to Prof. Dr. H. Brandis on the occasion of his 80th birthday.

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e. g. the enzyme nomenclature. A phenomenological description of the toxins according to their main effects (enterotoxic, neurotoxic, hemolytic, leukotoxic, cytotoxic, cytocidal, cytotonic, necrotic, emetic, paralytic, lethal) may be helpful and has often been the basis for the common name. For a logical classification, such names are not practical, because a toxin is frequently pleiotropic and can influence different types of cells and tissues. In this paper, we have tried to classify the bacterial exotoxins according to their molecular mechanisms of action, as already outlined in a previous review (168). A quite detailed overview of bacterial protein toxins was recently given by Menestrina et al. (130). Many of the approximately 240 known bacterial exotoxins (7) show remarkable similarities in their structure and their mechanisms of action. According to their cellular target, there are three main categories (Fig. 1): 1. Toxins that damage the cell membrane by direct action; 2. Toxins that possess receptor-modulating activities; and 3. Toxins that are internalized and have an intracellular enzymatic effect. Table 1 lists a number of toxins according to the bacterial species producing them. Bacterial toxins are not only a relevant topic of study for those interested in the pathogenesis and immunology of infectious diseases. They can also be used as an instrument for gaining insights into fundamental processes of cell biology. Toxins have been very helpful in detecting and clarifying the role of different G proteins (3.1.1 and 3.2). The recent analysis of the mode of action of the clostridial neurotoxins (3.4) has revealed the central role of VAMP/synaptobrevin and SNAP-25 in exocytosis and neurotransmitter release. This was a breakthrough not only in the understanding of botulism and tetanus but also of the molecular mechanism of neural transmission. Exciting progress is also being made in designing recombinant toxins targeted for specific cells so that such chimeric toxins may be used as therapeutic agents (145, 163). 1 Membrane-damaging toxins (cytolysins) The main effect of many toxins is to disintegrate the cell membrane. This is followed by a nonspecific increase in membrane permeability, which may culminate in cell death by lysis (9, 13, 65, 117). The mammalian membrane guarantees that the "interior milieu" of the cell is maintained. Because water passes freely through the lipid bilayer, the water influx caused by internal osmotically effective macromolecules, must be permanently compensated by actively pumping out ions, especially Na+. Cytolysins upset these vitally important ion gradients by disturbing the permeability of the membrane for ions. Subsequently, water flows into the cell heading to establish the Donnan equilibrium. The cell undergoes colloid osmotic swelling and eventually will even burst. Beside these effects on the water balance, the influx of such ions as Ca 2+ triggers pathophysiological events even at sublytic doses. The effects of sublethal concentrations of cytolysins, especially on leukocytes, are profound. They inhibit the chemotaxis of neutrophils and the proliferation and maturation of lymphocytes. Furthermore, they elicit the release of cytokines from monocytes and macrophages and of inflammatory mediators, especially leukotrienes and PAF (platelet activating factor).

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The membrane damage is caused by three different mechanisms: enzymatic degradation of the membrane lipids (1.1), formation of pores, i. e., well-defined membrane lesions (1.2), or surfactant activity of the toxins (1.3). The great majority of the cytoIysins belong to the group of pore-forming toxins.

Membrane damage

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.. enzymatic hydrolysis C. perfringens a-toxin .. pore formation cholesterol binding: streptolysin 0 S. aureus a·toxin RTX: E. coli Hly A, .. detergent like action S. aureus &-toxin

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ADP rlbosylatlon .. rho/rac proteins C. botulinum C3 toxin

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.. actin clostridial binary toxins: C. botulinum C2 toxin

hydrolysis of N-glycosidlc bonds

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.. 28S ribosomal RNA Shiga toxin Shiga-like toxins

.. elongatlonlactor EF-2 diphtheria toxin exotoxin A

Fig, 1. Cellular targets of bacterial toxins.

hemolysin (AshA) leukotoxin (AaltA) hemolysins (Apx lA, IIA, IlIA) aerolysin (AerA) cholera-like enterotoxin aero lysin (AerA) alveolysin (ALV) protective antigen (PA) edema factor (EF) and PA lethal factor (LF) and PA cereolysin 0 (CLO) phospholipase C exoenzyme (strain 2339) phospholipase A adenylate cyclase/hemolysin (AC/CyaA) pertussis toxin (PT) cholera-like enterotoxin cytotoxin heat-stable toxin (STa)

neurotoxin A (BoNt/A) neurotoxin B neurotoxin C neurotoxin D neurotoxin E neurotoxin F neurotoxin G C2 toxin C3 toxin botulinolysin (BLY)

Actinobacillus suis A. actinomycetemcomitans A. pleuropneumoniae Aeromonas hydrophila

Clostridium botulinum

Citrobacter freundii

Campylobacter jejuni

Bacillus subtilis Bordetella pertussis

Bacillus cereus

Aeromonas sobria Bacillus alvei Bacillus anthracis

Toxin

Bacterium species

adenylate cyclase (3.5) ? (3. pore-forming toxin/cholesterol (1.2.7) enzymatic cytolysin (1.1) ADP-ribosylation of G proteins (3.1.1) enzymatic cytolysin (1.1) pore-forming toxin/RTX (1.2.1) and adenylate cyclase (3.5) ADP-ribosylation of G proteins (3.1.1) ADP-ribosylation of G proteins? (3.1.1) internalized toxin/target unknown stimulation of the membranous intestinal guan yla te cyclase (2.1) metalloprotease/SNAP-25 (3.4) metalloprotease/synaptobrevin (3.4) metalloprotease/syntaxin (3.4) metalloprotease/syna ptobrevin (3.4) metalloprotease/SNAP-25 (3.4) metalloprotease/synaptobrevin (3.4) metalloprotease/synaptobrevin (3.4) ADP-ribosylation of actin (3.1.3) ADP-ribosylation of G proteins (3.1.1) pore-forming toxin/cholesterol (1.2.7)

pore-forming toxin/RTX (1.2.1) pore-forming toxinlRTX (1.2.1) pore-forming toxin/RTX (1.2.1) pore-forming toxin (1.2.3) ADP-ribosylation of G proteins? (3.1.1) pore-forming toxin (1.2.3) pore-forming toxin/cholesterol (1.2.7)

Mode of action

Table 1. Bacterial species and their toxins. Approximately half of the known bacterial exotoxins is listed.

botulism

opportunistic infections

enteritis

opportunistic infections whooping cough

food poisoning

6)

diarrhea, wound infections European foul brood of bees anthrax

porcine septicemia juvenile periodontitis porcine pleuropneumonia diarrhea, wound infections

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Corynebact. ulcerans Escherichia coli

Corynebacterium bovis Corynebacterium diphtheriae Corynebact. pseudotuberculosis

Clostridium spiroforme Clostridium tetani

Clostridium sordellii

Clostridium septicum

heat-stable toxin (STb phospholipase A hemolysin (Gvh)

= ST II)

toxin A toxin B histolyticolysin 0 exoenzyme perfringolysin 0 (PFO, theta tx) alpha toxin (phospholipase C) beta toxin (Cpb) iota toxin enterotoxin (CPE) alpha toxin septicolysin 0 (delta toxin) sordellilysin hemorrhagic toxin lethal toxin iota-like toxin tetanus toxin (TeTx) tetanolysin (TLY) phospholipase D diphtheria toxin phospholipase D diphtheria toxin phospholipase D Shiga-like toxins (SLT I, II) = verotoxins hemolysin HlyA heat-labile toxins (LT I, II) heat-stable toxin (STa = ST I)

Clostridium difficile

Clostridium histolyticum Clostridium limosum Clostridium perfringens

Toxin

Bacterium species

Table 1. Continued

pore-forming toxinIRTX (1.2.1) ADP-ribosylation of G proteins (3.1.1) stimulation of the membranous intestinal guanylate cyclase (2.1) ? (2.1) enzymatic cytolysin (1.1) pore-forming toxin (1.2.6)

glucosylation of Rho (3.2) glucosylation of Rho (3.2) pore-forming toxin/cholesterol (1.2.7) ADP-ribosylation of G proteins (3.1.1) pore-forming toxin/cholesterol (1.2.7) enzymatic cytolysin (1.1) pore-forming toxin (1.2.8) ADP-ribosylation of actin (3.1.3) pore-forming toxin (1.2.9) pore-froming toxin (1.2) pore-forming toxin/cholesterol (1.2.7) pore-forming toxin/cholesterol (1.2.7) similar to C. difficile toxins? (3.2) ADP-ribosylation of actin (3.1.3) metalloprotease/synaptobrevin (3.4) pore-forming toxin/cholesterol (1.2.7) enzymatic cytolysin (1.1) ADP-ribosylation of EF-2 (3.1.2) enzymatic cytolysin (1.1) ADP-ribosylation of EF-2 (3.1.2) enzymatic cytolysin (1.1) rRNA N-glycosidase (3.3)

Mode of action

bacterial vaginosis

infections of nasopharynx haemorrhagic colitis HUS (hemolytic uremic syndrome) extra-intestinal infections e.g., UTI traveller's diarrhea

bovine mastitis diphtheria lymphadenitis

tetanus

gas gangrene

food-poisoning; SID? gas gangrene

gas gangrene necrotic enteritis

pseudomembranous enterocolitis gas gangrene wound infections

Disease

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Streptococcus agalactiae Streptococcus mutans

Pseudomonas pseudomallei Salmonella spec. Serratia marcescens Shigella dysenteriae Staphylococcus au reus

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Proteus mirabilis Proteus vulgaris

Listeria seeligeri Morganella morganii Mycoplasma arthritidis Pasteurella haemolytica Al

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toxic shock syndrome tx (TSST) EDIN (epid.-diff.-inhib.) streptolysin S-similar toxin streptolysin S-similar toxin

pore-forming toxin (1.2.2) pore-forming toxin/RTX (1.2.1) pore-forming toxin (1.2.2) ADP-ribosylation of EF-2 (3.1.2) ADP-ribosylation of G proteins (3.1.1) pore-forming toxin (1.2.5) enzymatic cytolysin (1.1) ADP-ribosylation (of EF-2 ?, 3.1.2) ADP-ribosylation of G proteins? (3.1.1) pore-forming toxin (1.2.2) rRNA N-glycosidase (3.3) pore-forming toxin (1.2.8) enzymatic cytolysin (1.1) pore-forming toxin (1.2.8) detergent-like toxin (1.3) pore-froming toxin (1.2.8) superantigen (2.2)

? (3.6) pore-forming (1.2) pore-forming toxin/cholesterol (1.2.7) pore-forming toxin/cholesterol (1.2.7) enzymatic cytolysin (1.1) pore-forming toxin/cholesterol (1.2.7) pore-forming toxin/RTX (1.2.1) superantigen (2.2) pore-forming toxin/RTX (1.2.1)

hemolysin (HmpA) hemolysin (PvxA) hemolysin (HmpA) exotoxin A (ETA) exoenzyme S cytotoxin (leukocidin, PACT) phospholipase C (PLC-H) exotoxin cholera-like enterotoxin hemolysin (ShIA) Shiga toxin alpha toxin beta toxin (sphingomyelinase) gamma toxin delta toxin leukocidin S & F enterotoxins (SE) A,B,C1,C2,C3,D,E exfoliatin = exfoliative toxin

vacuolating cytotoxin (VacA) legiolysin iva no lysin 0 listeriolysin 0 (LLO) phospholipase C seelerigolysin hemolysin (MmxA) M. arthr. mitogen (MAM) leukotoxin (LktA)

broad spectrum of infections dental plaque

staph. scalded skin syndr. (SSSS, Ritter's Disease) toxic shock syndrome (TSS)

food poisoning

opportun. infect. e.g. UTI dysentery (shigellosis) pyogenic infections and abscesses of many organs septicemia

melioidosis

wound & nosocomial infections

rather apathogenic nosocomial infections arthritis in rodents bovine pneumonia ("shipping fever") nosocomial infections nosocomial infections

gastritis, peptic ulcer legionellosis facultative pathogenic listeriosis

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pneumolysin (PLY) streptolysin 0 (5LO) streptolysin 5 erythrogenic toxins A, C erythrogenic toxins B cholera toxin (CT) heat-stable hemolysin (TDH) heat-labile cytolysin damselysin heat-stable hemolysin (TDH) heat-labile cytolysin cholera-like enterotoxin heat-stable hemolysin (TDH) phospholipase A2 heat-labile cytolysin heat-stable toxin (5Ta)

Streptococcus pneumoniae Streptococcus pyogenes

Vibrio vulnificus Yersinia enterocolitica

Vibrio parahaemolyticus

Vibrio cholerae - non-Ol - el Tor Vibrio damsela Vibrio mimicus

Toxin

Bacterium species

Table 1. Continued

pore-forming toxin/cholesterol (1.2.7) pore-froming toxin/cholesterol (1.2.7) detergent-like toxin (1.3) superantigen (2.2) superantigen ? (2.2) ADP-ribosylation of G proteins (3.1.1) pore-forming toxin (1.2.4) pore-forming toxin (1.2.4) enzymatic cytolysin pore-forming toxin (1.2.4) pore-forming toxin (1.2.4) ADP-ribosylation of G proteins? (3.1.1) pore-forming toxin (1.2.4) enzymatic cytolysin (1.1) pore-forming toxin (1.2.4) stimulation of the membranous intestinal guanylate cyclase (2.1)

Mode of action

wound infections gastroenteritis

seafood poisoning

wound infections diarrhea

cholera acute gastroenteritis

pneumoniae, meningitis pharyngitis, scarlet fever, impetigo, cellulitis, erysipelas, late sequelae

Disease

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Exotoxin Molecular Mechanisms

177

1.1 Enzymatic cytolysins

A number of bacterial toxins attack the phospholipids of eukaryotic membranes via a specific hydrolytic cleavage (Fig.2A) (116, 136, 206). This results in a destabilization of the lipid bilayer and may eventually lead to the lysis of the cell. Often this occurs in cooperation with a second cytolysin, even one from another bacterial species (59). The dramatic effect of cytolysis may be an important mechanism in some cases (especially near the infection site), but the more subtle effects of phospholipase toxins in pathogenesis should not be underestimated. For example, bacterial phospholipase C may act by generating free diacylglycerol, a second messenger in eukaryotic cells, which stimulates the arachidonic acid cascade and activates protein kinase C, mimicking the effects of normal eukaryotic cell enzymes (131, 206). Increased Cal. influx

Membrane damage A.

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Fig. 2. Bacterial toxins acting on membranes or receptors. E. coli ST is Escherichia coli heatstable toxin, APC is antigen presenting cell, MHC II is major histocompatibility complex class II, and TCR is T cell receptor. 12

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]. Balfanz, P. Rautenberg, and U. Ullmann

caused by the toxin-induced disturbance of permeability may be another activator of the protein kinase C cascade. A well-known example of an enzymatic cytolysin is the phospholipase C (a-toxin, 30 kDa, E.C. 3.1.4.3) of Clostridium perfringens. The main substrate of a-toxin is phosphatidylcholine (lecithin), which occurs ubiquitously in mammalian membranes. Toxins with the same specificity are produced by Bacillus cereus (PCP, for phosphatidylcholine-preferring phospholipase C, 23 kDa), Pseudomonas aeruginosa (PLCH, 78 kDa), and Listeria monocytogenes (28 kDa). Staphylococcus aureus sphingomyelinase C ~-toxin, 39 kDa, E.C. 3.1.4.3) also has phospholipase C activity, but with a specificity for sphingomyelin. Phospholipases D (E.C. 3.1.4.4) are elaborated by Vibrio damsela (damselysin, 69 kDa), Corynebacterium pseudotuberculosis (31 kDa) and Corynebacterium ulcerans (31 kDa). Phospholipases Ai (E.C. 3.1.1.32) and A2 (E.C. 3.1.1.4) are produced by Escherichia coli. 1.2 Pore-forming toxins

Pore-forming toxins achieve their harmful effects by inserting themselves into the host membrane and forming well defined, stable lesions (Fig.2B) (15). This requires that the hydrophilic protein toxins undergo a transition to an amphiphilic state to become firmly embedded within the apolar regions of the membranes. The insertion into the lipid film is often accompanied by oligomerization of several toxin molecules. The size of pores varies widely between functional diameters of 1-2 nm (e. g., Escherichia coli hemolysin and staphyloccocal a-toxin) and 25 nm (cholesterol-binding toxins, e. g., streptolysin 0). It should be pointed out that the cell has the capacity to repair membrane lesions to some extent by phagocytosis and regeneration of the membrane moieties affected. The functional diameter of pores can be assessed by the use of polysaccharides of various sizes (sugars, dextrans, glycols, polyglycols) in the extracellular medium. If the molecular diameter of such colloid exceeds the diameter of the membrane pores, they will increase the extracellular colloid osmotic pressure, thereby preventing lysis (18, 178). Another method for approximating the functional dimensions of membrane pores is to monitor the release of labelled marker molecules of various sizes from the intracellular space, as was done for streptolysin S and streptolysin 0 (30). Some pore-forming cytolysins, such as staphylococcal a-toxin, streptolysin 0 or Escherichia coli hemolysin, are used as cell-permeabilizing agents for the generation of controlled-size membrane pores (20). 1.2.1 RTX cytolysins (e.g. Escherichia coli hemolysin)

This large group of closely related cytolysins of gram-negative bacteria includes the prototype Escherichia coli hemolysin (HlyA, 107 kDa), the bifunctional adenylate cyclase/hemolysin (CyaA, 177 kDa) of Bordetella pertussis (see also 3.5), the leukotoxins of Pasteurella haemolytica Al (LktA) and Actinobacillus actinomycetemcomitans (AaLtA), and further the hemolysins of Actinobacillus pleuropneumoniae (Apx lA, IIA and IlIA), Actinobacillus suis (AshA), Proteus vulgaris (PvxA), and Morganella morganii (MmxA) (122). All these toxins have in common the structural motif of several repeats of a consensus sequence of nine amino acids (LeullleIPhe-X-Gly-GlylX-X-GlyAsn/Asp-Asp-X) (46). The number of repeated units varies from 6 (P. haemolytica leukotoxin) to 47 (B. pertussis adenylate cyclase/hemolysin). Based on this genetically conserved structure, the acronym RTX ("repeats in toxins") has been proposed for this

Exotoxin Molecular Mechanisms

179

family of toxins. The repetitive units of the RTX toxins are responsible for Ca 2 +dependent binding to membranes (123). The synthesis and secretion of the RTX protein require the interaction of at least five gene products (220). Recently, it has been shown that Escherichia coli hemolysin and Bordetella pertussis adenyl ate cyclase are activated by a posttranslational modification, identified as a fatty acylation of two lysine residues (79, 89,93,194). The target cell and host specificities of the RTX cytolysins vary widely. E. coli hemolysin, for example, is highly active against erythrocytes from various mammalian species, human leukocytes, epithelial, and endothelial cells. In contrast, P. hemolytica leukotoxin lyses only ruminant leukocytes (220). Sublytic doses of RTX cytolysins have numerous immunomodulatory effects, e. g., E. coli hemolysin (10-200 ng/ml) induces apoptosis of T lymphocytes (97) as well as the release of inflammatory lipid mediators like leukotrienes from human neutrophils (78) and of interleukin 1b from monocytes (19). 1.2.2 Serratia marcescens hemolysin

The hemolysins of Serratia marcescens (ShIA, 165 kDa), Proteus mirabilis and most Proteus vulgaris isolates (HpmA) are highly homologous, unique cytolysins of gramnegative bacteria (211). In contrast to the RTX toxins, the action of these cytolysins is Ca 2 +-independent. The secretion and activation of ShlA involves the cleavage of a signal peptide and a modification in the periplasmatic area, mediated by a second polypeptide of 65 kDa called ShlB (158). ShlA is firmly integrated into the erythrocyte membrane, probably as a monomer creating small pores. like E. coli hemolysin, sublytic doses of ShlA induce the release of leukotrienes C4 and B4 from polymorphonuclear cells and from mast cells (220). 1.2.3 Aeromonas aerolysin

Members of the genus Aeromonas, especially A. hydrophila and A. sobria, produce a cytolytic toxin called aerolysin. Proteolytically activated aerolysin (50 kDa) binds to glycophorin on mammalian membranes. Aggregates of five to seven toxin molecules form anion-selective transmembrane channels 1-1.5 nm in diameter (213). The membrane-traversing domain of aerolysin spans the lipid bilayer with three ~-strands per monomer of aerolysin, as determined by X-ray crystallography (162). The unusual transmembranous ~-barrel motif resembles the recently determined structure of bacterial porins (48,219). Clostridium septicum a-toxin resembles aerolysin with regard to activation and the mechanism of pore formation. Sequence data have revealed 27% identity and 72% similarity over a 387-residue region between the primary structure of a toxin and aerolysin (10). Such a high level of sequence similarity between toxins of a gram-positive and a gram-negative bacterium was previously unknown. Both toxins are encoded chromosomally. 1.2.4 Vibrio hemolysins

Pathogenic strains of Vibrio parahemolyticus, a cause of seafood poisoning, produce a 32-44 kDa thermostable direct hemolysin (TDH), which forms pores about 2 nm in diameter (87). Almost identical heat-stable cytolysins are produced by V. cholerae non01 and V. mimicus. The halophilic Vibrio vulnificus produces a heat-stable 51 kDa cy-

180

J. Balfanz, P. Rautenberg, and U. Ullmann

tolysin which binds to cholesterol as a low-affinity receptor and forms tetrameric membrane pores. Genetically similar toxins are secreted by V. cholerae el Tor and also V. mimicus (112). 1.2.5 Pseudomonas aeruginosa cytotoxin (formerly called leukocidin) Pseudomonas aeruginosa cytotoxin (PACT, 42.5 kDa), an opportunistic pathogen with very simple nutritional requirements that is responsible for many hospitalacquired infections, generates distinct membrane pores with an internal diameter of 2 nm. In vitro, it is active mainly against white blood cells (hence its old name). A remarkable discovery was the identification of a 28 kDa protein receptor for this cytotoxin, called CHIP28 (124), a member of an abundant membrane channel protein family. The receptor itself does not seem to be part of the pores. This discovery may be a hint that more cytolysins may exert their activity by stimulating a specific protein receptor after they have bound unspecifically to lipid moieties of the membranes. 1.2.6 Gardnerella vaginalis hemolysin Gardnerella vaginalis, which is held to be responsible for bacterial vaginosis, produces a pore-forming hemolysin (Gvh), which seems to bind to cholesterol and phospholipids in the membrane (35) without being structurally related to the thiolactivated, cholesterol-binding toxins (s. 1.2.7). This hemolysin has not yet been completely characterized. 1.2.7 Cholesterol-binding thiol-activated toxins (CBT) Streptococcus pyogenes streptolysin 0 (SLO) is the prototype of a group of at least 19 closely related cytolysins of gram-positive bacteria belonging to the genera Streptococcus, Bacillus, Clostridium and Listeria. Examples are pneumolysin (PLY), cereolysin 0 (CLO), perfringolysin 0 (PFO), tetanolysin (TLY) and listeriolysin 0 (LLO, of L. monocytogenes) (8, 192). They consist of a single polypeptide chain in the range of 50-80 kDa. Tetanolysin was the first to be observed, almost a century ago (54). The common property of these potent cytolysins is their binding to cholesterol in eukaryotic cell membranes, even of intracellular compartments like Iysosomes. Intoxication can be prevented by minute amounts of free cholesterol (or closely related sterols). Because of this trait, they were named cholesterol-binding toxins (CBT). A second characteristic feature of CBTs is their possession of an essential SH group. The toxic activity can be abolished by oxidation and by inhibitors of SH groups. Reactivation is possible by thiols and other reducing agents. Sequence data revealed a genetically conserved region of 15 amino acids with a single cysteine at the carboxy terminus. Within this cysteine motif, there is a cluster of three tryptophans (Trp-Ile-Trp-Trp), which is essential for toxicity, as shown by site-specific mutagenesis on pneumolysin (28). After binding, the CBT molecules aggregate with cholesterol, increasing the permeability of the membrane. Even at sublytic doses, the toxins interfere with defense mechanisms like phagocytosis by macrophages, and mobility and chemotaxis of neutrophils. At very high toxin doses (100-1000 times higher than the minimum lytic concentration), the lesions may reach an external diameter of 30-50 nm and a thickness of about 5 nm. They are formed by 50-70 toxin monomers and are visible under the electron microscope as ring or arc-like structures (17, 186). These pores have a size large enough to permit larger molecules like hemoglobin to escape directly without ly-

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sis of the cell. Listeriolysin 0 is directly responsible for the invasive capacity of L. monocytogenes and allows the pathogen to enter the host cytoplasm through the phagolysosome membrane. An experimental transfer of the chromosomally encoded gene for LLO provides the normally extracellular Bacillus subtilis with the ability to grow intracytoplasmically (21). Interestingly, the cytolysin of the common large sea anemone, Metridium senile, is similar in mechanism to the CBTs. Metridiolysin is also an SH-activated 80 kDa protein, which is inhibited by cholesterol and is very similar in its biological activity to the bacteriallysins of this group (12, 14). 1.2.8 Staphylococcal a-hemolysin and related toxins Staphylococcus aureus a-hemolysin (a-toxin, Hla) is the prototype of an oligomerizing, poreforming cytotoxin (16, 204). It is secreted as a water-soluble, single-chain polypeptide of 33 kDa. a-toxin is active against all mammalian cells tested, although the effectivity differs widely. Thus, rabbit erythrocytes are highly sensitive, whereas human erythrocytes are highly resistant. However, human platelets, monocytes, lymphocytes and endothelial cells are also sensitive to a-toxin. Recent binding studies indicate that highly sensitive cells possess specific binding sites which increase their susceptibility to a-toxin. In contrast, binding to cells of low susceptibility is adsorptive and unspecific (83). Six molecules aggregate to form a stable transmembrane pore with a diameter of 1-2 nm, which in electron micrographs appears like a hollow cylinder penetrating the membrane (66, 69). The pore formation will eventually cause cell death due to continued loss of ions and small vital molecules. In the meantime, the pores trigger a series of pathophysiological reactions (e. g., via Ca 2 + influx) such as stimulation of eicosanoid production, secretory processes, activation of endonucleases and release of cytokines (16). The leukocidin (Luk) and y-lysin (Hlg) of Staphylococcus aureus and the ~-toxin of Clostridium perfringens type Band C strains (Cpb) are related to staphylococcal atoxin (91, 151). Staphylococcal leukocidin is remarkable because of its bicomponent assembly, which is rare in pore-forming toxins. For pore formation in white blood cells, the two polypeptide chains of about 33 and 34.5 kDa (named Sand F for slow and fast eluting) have to cooperate: the S component binds first and facilitates the embedding of the F component in the membrane. The formation of a complete pore seems to require oligomerization of leukocidin molecules in the membrane (60). Staphylococcal y-lysin also consists of two components, designated Yl (32 kDa) and Y2 (36 kDa). They act synergistically to potentiate hemolysis on rabbit, sheep and human erythrocytes, although each component is weakly hemolytic by itself (40). Clostridial ~-toxin (Cpb) is one of the main toxins of type Band C strains of C. perfringens which are responsible for necrotic enteritis in humans and animals. Little is known about the biochemical or molecular properties of the toxin, though its nucleotide sequence has been determined (91). 1.2.9 Clostridium perfringens type A enterotoxin (CPE)

The enterotoxin of Clostridium perfringens type A (CPE), a major cause of food poisoning, inserts itself· into the membrane of sensitive cells, e.g., epithelial cells of the small intestine. Pores are formed by one 35 kDa CPE molecule and two host membrane proteins of 50 and 70 kDa, thus cre·ating a large complex, which disturbs the permeability and leads to leakage of K+ and increased Ca2+ influx (126, 172).

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1.3 Detergent-like cytolysins Staphylococcus au reus produces a helical 26-residue peptide with a highly amphipathic character called 6-lysin (6-toxin). It interacts nonspecifically with membrane lipids (67). At low concentrations, 6-lysin may bind as a monomer directly to the lipid phase without penetration, increasing the fluidity and permeability of the membrane. This is in accordance with the concept of membrane damage by "leaky patches" (11). At higher doses, several toxin molecules may insert themselves into the lipid film, aggregate and expose their hydrophilic moieties to the centre. This would open a channel, allowing Ca 2+ influx, and consequently trigger the reaction cascade resulting in the release of inflammatory factors. At very high doses, the effects of 6-lysin may be only detergent-like, creating lipid-toxin micelles of various size, which can totally disintegrate the membrane. There is a striking similarity in structure and mechanisms of action between (,)-toxin and mellitin, the cytolytic component of the venom of the honey bee, Apis mel/ifera, and also mastoparan, the 14-amino acid cytolysin of the wasp, Vespula lewsii ( 13). Streptococcus pyogenes streptolysin S (termed "s" because of its oxygen stability, in contrast to streptolysin 0, see 1.2.7) consists of 32 amino acids and is a very potent cytolysin with a hemolytic activity of about 3 X 10 6 HU/mg. This makes it 10 5 times more active than staphylococcal 6-lysin. The active toxin molecule seems to be a dimer, composed of two 32 amino acid peptide chains (13). Streptolysin S is thought to be responsible for the streptococcal leukotoxicity in which leukocytes are killed after phagocytosis of streptolysin S-producing strains. Streptolysin S is inhibited by minute amounts of phospholipids, damages the membrane and forms channels of up to 4.5 nm functional diameter, hut the mechanism of action of streptolysin S is largely unclear.

2 Receptor-targeting toxins Another molecular mechanism of toxins is the modulation of physiological receptors of cells, which have an effect on intracellular reaction cascades. Escherichia coli heat-stable toxin stimulates the transmembrane guanylate cyclase of the intestine, without being internalized (2.1). The interaction of superantigens with major histocompatibility complex (MHC) class II molecules and T cell receptors leads to an immunomodulatory and often generalized reaction (2.2). 2.1 Stimulation of membrane-bound guanylate cyclase: Escherichia coli heat-stable enterotoxin Escherichia coli heat-stable enterotoxin (STa or ST I) acts by stimulating the membrane-bound ("particulate") guanylate cyclase C, which is part of a signal transduction pathway normally exploited by endogenous neurohormonal regulators of intestinal salt and water transport (Fig.2C). An endogenous activator of intestinal guanylate cyclase, the 15-amino acid peptide, guanylin, was recently isolated from the rat jejunum (50). The heat-stahle enterotoxin is a plasmid-encoded peptide of 18-20 amino acids with three disulfide bridges. STa has a 50% homology with guanylin. The molecular mechanism of action by means of which STa causes the activation of guanylyl cyclase remains unclear. Based on the known mechanisms of internalized toxins, one could assume an enzymatic mode of action, but there are no data available to support this prediction.

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The initial step of intoxication is that the enterotoxin binds directly to the guanylate cyclase receptor (184) orland another kind of receptor (84). Toxin-receptor interaction is coupled to the activation of guanylate cyclase and to a dose-dependent increase in the intracellular level of cyclic GMP (90). It leads further to stimulation of a cGMP-dependent protein kinase of the intestinal mucosa (109). The physiological consequences of these processes are comparable to those of cholera toxin and result in secretorydiarrhea. Besides ST I, enterotoxigenic Escherichia coli strains produce a second kind of heatstable enterotoxin, a 48-amino acid peptide termed ST II (or STb) (31, 85). ST II is methanol-insoluble and active only in the jejunal loops of pigs, whereas ST I is methanol-soluble and active in the suckling mouse assay. ST II does not stimulate guanylate cyclase, but little more is known about its biological properties. 2.2 Interaction with MHC class II and T cell receptor: Bacterial superantigens

Superantigens are a class of structurally related bacterial protein toxins, 22-29 kDa in size, which are extremely potent mitogens of T lymphocytes (125). The designation "superantigen" was chosen because the mechanism of T lymphocyte stimulation closely mimics that of "classical" antigens. Established members of the superantigen family are the staphylococcal enterotoxins (SE), A, B, C1, C2, C3, D and E which are among the leading sources of food poisoning. The toxic-shock syndrome toxin (TSST-1) of Staphylococcus au reus induces an acute and sometimes lifethreatening disease often attributed to the use of vaginal tampons, but sometimes also to soft tissue wound infections. The pyrogenic (erythrogenic) toxins A and C of Streptococcus pyogenes are involved in scarlet fever. Mycoplasma arthriditis mitogen (MAM) causes an acute inflammatory infection and probably chronic arthritis in rodents (42). Both types of antigens must be presented to a T cell antigen receptor (TCR) by class II molecules of the major histocompatibility complex (MHC II) of an antigenpresenting cell (APC). Additionally, both types of antigens form ternary complexes of MHC II- antigen/superantigen - TCR that trigger cytokine production and T cell proliferation (Fig.2D). "Classical" antigens are proteolytically processed in APCs into short peptide fragments that bind to a cleft on the surface of MHC II molecules for presentation to the TCR. In contrast to classical antigens, superantigens do not require proteolytic processing, but act as intact proteins (34). The affinity of superantigens to MHC class II in humans depends on the subtype of the human leukocyte antigen (HLA). Staphylococcal enterotoxins bind to HLA-DR and HLA-DQ but not to HLA-DP (64). Classical antigenic peptides interact with variable components of both the a-chain and ~-chain of the TCR. In this way, a small fraction of T cells is stimulated in a controlled way, with physiological amounts of cytokines being released. In contrast, the superantigenMHC II complex contacts specifically a small conserved region of the variable ~-chain (V~) of the TCR (51, 165). Because the superantigen-MHC II complex interacts with these V~ elements, it can stimulate a whole battery of T cells, regardless of the other variable parts of the a-chain and ~-chain of the TCR. In any individual, there are about 30 different V~ elements which are targets for superantigens. For example, TSST-l has been shown to induce a dramatic proliferation of V~2-bearing T lymphocytes in vitro, and humans recovering from toxic shock syndrome have been shown to have elevated levels of V~2 T lymphocytes (38).

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Stimulation induces T cells to release interleukin-2 and interferon-y and subsequently tumour necrosis factor a, interleukin-1 and other monocyte mediators (61). The massive release of cytokines and monokines is responsible for the severe acute illness and shock. T cells expressing the appropriate V~ chain are first activated and subsequently deleted upon exposure to the super antigen (106,221). In addition to these acute conditions, there is evidence that superantigens may playa role in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis (159). Members of the superantigen family differ, to a varying degree, in their sequence homology, but they show a similar three-dimensional structure. One ~-pleated domain is formed by the N- and C-terminal regions which is involved in MHC and TCR binding. Diametrically opposed to this domain is a shallow cavity at the C-terminal region which most likely constitutes an additional TCR binding site (1, 166,200). Whether Staphylococcus au reus epidermolytic (exfoliative) toxins A and Band streptococcal pyrogenic (erythrogenic) toxin B should be classified as superantigens is under debate. The effect of these proteins is most likely due to contaminations, since recombinant forms are not mitogenic at all (63). 3 Internalized toxins Despite their different effects on the individual cell and the organism as whole, toxins with an intracellular target show similar mechanisms of action (215). Their toxicity includes three main steps: 1. Binding 2. Internalization 3. Intracellular effect The internalized toxins also have basic similarities in their structural aspects. They possess a bifunctional AB structure (70, 132). The A component contains the enzymatically active domain, whereas the B component mediates the binding to the membrane and the translocation to the cytosol. For toxicity, i. e., the effect of toxins on intact cells, the A and B components must cooperate. In celllysates, the A chain is enzymatically active without the B chain. The three-dimensional structure of five AB toxins has been analyzed so far. In diphtheria toxin (37) and Pseudomonas aeruginosa exotoxin A (5), the two functions of the B component can be assigned to two distinct domains of the Y-shaped toxin molecule (see 3.1.2 and Fig. 4). Cholera toxin (226), E. coli heatlabile toxin (188) and pertussis toxin (196) are composed of an A component and a B component, the latter with five subunits.

1.: Binding to cell surface receptors Binding of the B component to the receptor of the cell surface is the initial step of intoxication. The type of receptor additionally determines the specificity of the toxin for individual cell types (56,104). 2.: Internalization After binding to specific receptors, the toxin is internalized by endosomes. Endocytosis seems to occur either by clathrin-coated vesicles or by noncoated vesicles (140). Once it has been internalized, the toxin is no longer accessible to proteases or antibodies. The further processing of the toxin can require fusion of the endocytosis vesicles

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with lysosomes. The toxin enters the cytosol by translocation through the membrane of the endosome or the endolysosome. In all known cases, translocation is connected with the reduction of a single disulfide bond between the binding and the catalytic component. The internalization and translocation process has been investigated intensively for diphtheria toxin (see 3.1.2). 3.: Intracellular effects All internalized toxins are characterized by their highly specific effects on vitally important target molecules within the host cell. As far as we know, the interacellular effect of toxins is enzymatic in nature in all cases and can be based on five different molecular mechanisms of target modification (Fig. 3): - Modification of heterotrimeric or monomeric G proteins, elongation factor or actin by ADP-ribosyltransferases (3.1); - Modification of a monomeric G protein (Rho) by glucosyltransferases (3.2); - Hydrolysis of ribosomal RNA by N-glycosidases (3.3); - Hydrolysis of synaptic vesicle proteins by metalloendoproteases (3.4); and - Production of cyclic AMP by adenylate cyclases (3.5). Thus, the toxins that act intracellularly can be classified in three categories of enzymes: transferases, hydrolysases, and lyases (in this case, in particular nucleotide cyclases). The effects of these toxins on the physiology of the cell are severe, e. g., inhibition of protein biosynthesis (diphtheria toxin, Shiga toxins), increase in second messenger cAMP (cholera toxin and adenylate cyclases), disaggregation of the microfilament system (Clostridium difficile toxins A and B, C. botulinum C2 toxin), or inhibition of neurotransmitter release (clostridial neurotoxins). The lethal amounts of bacterial toxins (specified as LDso in mice per kg of body weight) differ widely (72): about 1 ng for the botulinum and tetanus neurotoxins, 15 !J,g for pertussis toxin, 30 !J,g for Clostridium difficile toxin A (enterotoxin) (169), 114 !J,g for anthrax lethal factor, and 250 !J,g for cholera toxin. 3.1 AD P-ribosyltransferases Bacterial ADP-ribosyltransferases catalyze the cleavage of nicotinamidedinucleotide (NAD+) to nicotinamide and ADP-ribose (27, 88, 167). Simultaneously, ADP-ribose is transferred to diphthamide (a modified histidine), arginine, asparagine or cysteine, according to the substrate specificity of the individual toxin (Fig. 3A). Acceptor molecules of ADP ribose are heterotrimeric or monomeric GTP-binding proteins (3.1.1), the GTP-binding elongation factor EF-2 (3.1.2), and cytoskeleton proteins (3.1.3). The ADP transfer results in a conformational change in these physiologically important proteins and an alteration of their vital activity, and this could often be linked directly to the clincial findings. The resemblance in the mode of action corresponds to similarities in the amino acid sequence of the toxins, especially in the region of the NAD+ binding site (53). 3.1.1 AD P-ribosylation of heterotrimeric or monomeric G proteins Several bacterial toxins achieve their effects by ADP ribosylation of a GTP-binding protein (G protein) (27, 143). Cholera toxin, E. coli heat-labile enterotoxin, and pertussis toxin interact with heterotrimeric G proteins, whereas the C3 toxin of Clostridium botulinum ADP ribosylates small monomeric G proteins.

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G proteins act as genetically conserved molecular "switches" for very different cellular processes (22, 29, 39). They are "turned on" by exchanging GDP for GTP and "turned off" by hydrolyzing GTP to GDP by intrinsic GTPase activity. The regulatory status (active or inactive) is based on different conformations of the G proteins:

Hydrolases

Transferases A.

c.

ADP ribosylation

NN N=c :

0-

<

0-

N

N)

II~ II

-0 - P -0 -P -0-CH:z

II

o

0

HO

protein·

B.

OH

ADP-ribose

Glucosylation

E~\

H~H

Conserved region of 288 RNA

OH

glucose

nucleotidyl-cyclases E.

cAMP synthesis

Fig. 3. Mechanisms of bacterial toxins acting at the intracellular level.

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1. In the inactive regulatory conformation, G proteins bind GDP and can be activated by ligand/receptor interaction. 2. Receptor activation is coupled to an exchange of GDP for GTP. Subsequently, the heterotrimeric G proteins dissociate into a Ga-GTP subunit and a ~/y-dimeric protein. In this GTP-linked form, the G proteins can regulate the activity of subsequent effector systems. 3. The G proteins are inactivated by GTP hydrolysis catalyzed by the intrinsic GTPase activity. In the case of heterotrimeric G proteins, the Ga-GDP subunit associates with the ~/y-dimer again to form the Ga~y complex, which is ready to be stimulated anew by the receptor. ADP ribosylation of heterotrimeric G-proteins: - Cholera toxin and E. coli heat-labile enterotoxin Cholera toxin (CT) and E. coli heat-labile enterotoxin (LT) are able to transfer ADP ribose to the Gsa subunit of heterotrimeric G proteins (62, 73, 141,214). Both toxins are almost identical in function and structure, having an 80% sequence homology (129, 193,225). Cholera toxin is chromosomally encoded, whereas E. coli heat-labile toxin is encoded on a plasmid. They are composed of an enzymatically active A subunit and five receptor-binding B subunits (ABs) (71). The subunits of the B protomer are formed by five identical 103 amino acid polypeptide chains. Analysis of the threedimensional structure has confirmed that the A subunit is embedded in the center of the ring-shaped, "doughnut"-like arrangement of the five B subunits (171, 188, 190, 226). The B protomer binds to the ganglioside receptor GMl on the membrane of target intestinal epithelial cells (49, 86, 108, 189). A prerequisite for the enzymatic activity of the toxins is the proteolytic cleavage of the A subunit and the reduction of the disulfide bond to yield a catalytically active Al fragment (192 amino acids) and an A2 fragment (47 amino acids). A2 mediates the binding of Al to the B pentamer (71, 128). After translocation of the enzymatically active Al subunit into the cytosol, ADP ribose is transferred to arginine-174 of the Gsa subunit of the G protein (62, 142). ADP ribosylation results in inhibition of the GTPase activity of the Gsa protein. The Gs protein and the Gs dependent adenylate cyclase are fixed in their active statuses and cAMP is permanently produced. This inhibits the electro-neutral sodium/chloride transport systems of the jejunum and the ileum and decreases water absorption. The simultaneous opening of the electrogenic chloride channels of the colon increases the intraluminalliquid inflow. In summary, these alterations of the liquid flows result in the characteristic severe diarrheas. It should be noted that cholera toxin may also induce water and electrolyte secretion by stimulating prostaglandins, in addition to the elevated cAMP level (164). Cholera-like enterotoxins are produced by various other bacteria including Vibrio mimieus, Salmonella, Pseudomonas, Campylobaeter jejuni, and Aeromonas hydrophila (167). - Pertussis toxin Like cholera and the heat-labile toxin, pertussis toxin (PT) acts by transferring ADPribose to a heterotrimeric G protein (144). However, in the case of pertussis toxin, the main substrate is the a subunit of Gj, the inhibitory GTP-binding protein of the adenylate cyclase system. As a result of the modification of Gia, the exchange of guanine nucleotides is blocked. Consequently, Gi is fixed in its inactive conformation and cannot

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exert its inhibitory effect on cAMP production. 50me of the symptoms of whooping cough, such as the release of histamine, lymphocytosis and hyperinsulinemia, can be attributed to the effects of pertussis toxin (105,218). Pertussis toxin (formerly known as islet-activating protein) is composed of a single catalytic subunit, S1 (21 kDa), and a B protomer consisting of five heterogeneous subunits (52, 53, two copies of 54, and 55) (201). The 51 subunit contains two regions homologous to the A subunit of both cholera toxin and E. coli heat-labile toxin (119). The pentameric organization of the B oligomer is very similar to that of the members of the cholera and the Shiga toxin families, although no sequence homologies exist between the B subunits of these three toxin groups. The five subunits of the B protomer are arranged like a ring with a central pore. Each B subunit is folded into six antiparallel ~-strands with an ex-helix in the center lining the central pore (196), as was demonstrated for the cholera-like heat-labile toxin and Shiga-like toxin (191). This folding has now been found in several proteins that bind an oligosaccharide or oligonucleotide and was named the oligomer-binding fold (146). The cellular glycoreceptor of pertussis toxin has not yet been exactly determined and the mechanism of membrane translocation also remains unclear. The Sl subunit has an intrachain disulfide bond, which has to be reduced, probably to make it hydrophobic, and pass through the pore of the B protomer into the lipid bilayer. In the cytosol, S1 exerts its ADP-ribosylrransferase activity and modifies the carboxy-terminal cysteine (residue 352 \ of C;, and a number of other G proteins, e. g., transducin Gt protein. In fact, PT is widely used as a tool in research on G proteins (210). 50me biological activities of pertussis toxin, e. g., T-cell proliferation, do not seem to be caused by the catalytic 51 subunit, but are directly attributed to the B proto mer binding (14S). ADP-ribosylation of small monomeric G-proteins: Clostridium botulinum C3 and related toxins Beside the heterotrimeric G proteins, a number of smaller (21 kDa) monomeric G proteins are known. They are involved in controlling many different cellular functions, including growth, differentiation, cytoskeletal organization, and intracellular vesicle transport and secretion (SO). Several bacterial toxins interfere with this regulatory system by transferring either ADP ribose or glucose (see 3.2) to small G proteins (27,102). Certain strains of Clostridium botulinum produce the ADP-ribosyltransferring C3 toxin (syn. exoenzyme C3), in addition to the neurotoxins C 1 and D (3.3) and the C2 toxin (1.3.3). The substrates of C3 toxin are Rho and Rho-like G proteins, which are ADP-ribosylated at arginine-41 (185). Rho is involved in the regulation of actin polymerization; thus, C3 may ultimately have the same physiological effect as C2 toxin which directly ADP-ribosylates actin. Recently, it has been shown that several other bacterial species transfer ADP-ribose to monomeric G proteins. For example, an exoenzyme of Clostridium limosum and one of Bacillus cereus ADP-ribosylate the same substrate as the C3 toxin (99, 100). Pseudomonas aeruginosa exoenzyme 5 transfers ADP ribose to several G proteins, including H-Ras (41).

3.1.2 ADP-ribosylation of the elongation factor 2: Diphtheria toxin and Pseudomonas aeruginosa exotoxin A Diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A (ETA) catalyze the ADP ribosylation of the GTP-binding eukaryotic and archae bacterial elongation fac-

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tor 2 (EF-2) at a diphthamide residue. In the process, these toxins interrupt the biosynthesis of proteins and can lead to cell death (74, 88, 92, 107, 161, 198). EF-2 is responsible for the translocation of the growing polypeptide chain from the aminoacyltRNA position to the peptidyl-tRNA position at the ribosome. Energy for this reaction is created by hydrolysis of GTP. The ADP-ribosylation of EF-2 at diphthamide (a posttranslationally modified histidine) inhibits protein synthesis by blocking the GTPdependent translocation (43, 209, 222). Diphtheria toxin is produced only by lysogenized Corynebacterium diphtheriae, which has been infected with p-corynebacteriophages carrying the DT gene. The virusencoded toxin gene is controlled by an Fe 2 +-dependent repressor protein of the bacterium and toxin synthesis is inhibited by iron in the medium. Next to the clostridial neurotoxins, diphtheria toxin is one of the most effective bacterial toxins. The lethal dosage for humans is 0.13 Ilg/kg, according to the data from the Kyoto catastrophe of 1948 (450 children died after injection of an insufficiently detoxified vaccine) (6). A single internalized diphtheria toxin molecule may be sufficient to kill a sensitive cell (224). Structure of diphtheria toxin. After splitting off a secretory signal (leader) peptide of 25 amino acids, the diphtheria toxin is secreted as a single polypeptide chain with 535 amino acids and a molecular weight of 58 342 (77). The toxin is cleaved into the A and B fragments in vivo by furin and probably by additional proteases (75,207) and in vitro, by several proteases. The nicked toxin remains connected by a single disulfide bond. The carboxy-terminal B chain (342 residues, 37 kDa) is responsible for the binding, whereas the amino-terminal A chain (193 residues, 21 kDa) is enzymatically active after separation from the B chain by reduction of the disulfide bond. Recently, Xray crystallography has shown that the B chain has two structural domains (37). The receptor-binding R domain has a p-pleated structure and mediates the binding of the toxin to receptor structures on the target cell. The translocation-mediating T domain is rich in a-helices and is responsible for the translocation of the toxin through the membrane. Together with the catalytic A chain (C domain), the three domains of the holotoxin form a Y-shaped structure (FigA). Structure of exotoxin A. Like diphtheria toxin, Pseudomonas aeruginosa exotoxin A is produced in a single polypeptide chain (66 kDa, 613 amino acids) (76), which has to be activated by proteolytic cleavage into an N-terminal (28 kDa) and a C-terminal (37 kDa) fragment, the latter representing the enzymatically active domain (153). Although the amino acid sequences of ETA and DT show little homology, there is a remarkable similarity in the tertiary structure of the catalytic centres and the assembly in three domains (5, 37). There is, however, a difference in the arrangement of the A and B components. In exotoxin A, the catalytic A domain is localized at the C-terminus, whereas in diphtheria toxin the A domain is an N-terminal one (36). Since its three-dimensional structure was determined, efforts have been made to construct novel forms of the toxin so that ETA may be used as a therapeutic agent (163). Cell binding. The first step in the binding of diphtheria toxin seems to involve interaction with acidic phospholipids (S2, 157). This low-affinity receptor mediates the binding to a high-affinity receptor occurring in lower concentrations. It has been suggested that such multiple site binding might be a general model of how enterotoxins and clostridial neurotoxins bind to cells (138). The high affinity receptor of the diphtheria toxin is a membrane protein with a relative molecular mass of 14500 (diphtheria toxin receptor, DTR 14.5) (127). DTR 14.5 is probably identical to the transmembrane precursor molecule of a heparin-binding growth factor (147). This receptor is

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Diphtheria toxin eOOH leader peptide 25 amino acids

catalytic

receptorbindlng

domain

domain

amino acid 1 - 193

amino acid 386 - 535 186 S

\

201 S translocation domain amino acid 205 - 378 ACTIVE-

BINDINGCHAIN

1. Receptor binding

2. Endocytosis & nicking

3. H+ influx & translocation

4. Reduction of A chain & ADP ribosylation

SH

.• . + S", ,S. . . • . . . . . . . . . . . . . .,. . . . . . •. f

;'

NAD

ADP·ribosyl-

diphttlamlcle

EF·2

Fig. 4. Structure and internalization of diphtheria toxin. EF-2 is elongation factor 2.

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associated with another membrane protein of 27 kDa (diphtheria toxin receptorassociated protein, DRAP 27), which is identical to CD9, and mediates between the binding and the toxic action (94, 133). CD9 occurs in human monocytes, B-cell precursors and thrombocytes. Its physiological role is still unclear. Pseudomonas aeruginosa exotoxin A binds to the receptor for a2-macroglobulin (111). The identification of these binding sites of diphtheria toxin and exotoxin A illustrates that cells do not have specific "toxin receptors", but that bacterial toxins use physiological membrane proteins to enter the target cell. Internalization and membrane translocation. The translocation of diphtheria toxin across the cell membrane following receptor-mediated endocytosis is initiated by the influx of protons into the endosome (174) (Fig. 4). The decrease in pH to below 5.5 changes the conformation of the toxin's T domain; hydrophobic regions are exposed and inserted into the lipid double-layer of the endosome membrane. The T domain forms a hydrophilic region in the membrane, which allows the A chain to penetrate through the endosome membrane and to reach the cytosol. The disulfide bond between the A chain and the B chain is cleaved by thioreductases in the cytosol and the A fragment achieves its catalytically active conformation (121, 149, 175). Diphtheria toxin was the first bacterial toxin to be isolated (173), four years after Loeffler (120) had suggested in Berlin in 1884 that a diffusible poison was the likely explanation for the observed tissue damage. This was the beginning of the era of bacterial toxinology. 3.1.3 ADP-ribosylation of actin: The clostridial binary toxins (e. g., Clostridum botulinum C2 toxin) Actin is part of the microfilament system of the cell and interacts with components of the intermediate filament system and the microtubuli. The various elements of the cytoskeleton system contribute to cell motility, endocytosis, mitosis and cytokinesis. Monomeric globular G-actin is in a state of dynamic balance with the polymerized filamentous F-actin (33, 217). In vertebrates, actin occurs in several isoforms: skeletal a-actin, cardiac a-actin, smooth muscle a- and y-actin, and non-muscle cell ~- and yactin (4). The actin-modifying toxins of several Clostridium species are binary molecules: they consist of two separate polypeptide chains, which are functionally equivalent to the AB model of other toxins (45, 155). The first step in intoxication is the binding of the B component (100 kDa) to a receptor. The binding induces selective proteolysis of the B component into an 88 kDa fragment and subsequent association with the enzymatically active A component (55 kDa), followed by endocytosis of the AB receptor complex. The clostridial binary toxins ADP-ribosylate G actin at arginine177. The prototype of these toxins is Clostridium botulinum C2 toxin (3, 154,216). Only the y-actins and the non-muscle cell ~-actin are modified by C2 toxin. In contrast, Clostridium perfringens iota-toxin transfers ADP ribose to all actin isoforms (4).

The ADP ribosylation of actin by the binary toxins inhibits the polymerization of globular G actin to filamentous F actin, leading to disorganization of the microfilament system of the cell. Probably as a sequel, C2 toxin influences functions of the unspecific immune response, such as the production of superoxide and the migration and endocytosis of neutrophilic granulocytes. These findings reflect the participation of actin in such immune processes.

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3.2 Glucosyltransferases: Clostridium difficile toxins A and B Clostridium difficile toxins A and B are the causative factors of antibiotic-associated pseudomembranous colitis (110). Their potent cytotoxicity in cultured cells is caused by disaggregation of the microfilament cytoskeleton (205). Recently it was shown that toxins A and B glucosylate the low molecular weight GTP-binding proteins of the Rho family at threonine-37 (101, 102, 103) (Fig. 3B), which is involved in the regulation of the actin cytoskeleton. This glucosylation is a novel mechanism of action of intracellular toxins and resembles the ADP-ribosylation of Rho by Clostridium botulinum C3 toxin (3.1.1). Toxins A and B of Clostridium difficile are the largest known bacterial protein toxins. Both consist of one single polypeptide chain, that of toxin A being 308 kDa and that of toxin B, 270 kDa, with 63% amino acid homology between the two (55). Both toxins are presumed to have a dimeric organization, because the native toxins have a molecular size in the range of 400000 to 600000 (toxin A) and 360000 to 500000 (toxin B). Toxin A binds to three carbohydrate antigens, designated I, X and Y, which occur in the intestinal epithelium of humans (208). The C-terminal of both toxins contains repetitive units, which appear to mediate the interaction between toxin and receptor (223). 3.3 RNA N-glycosidases: Shiga and Shiga-like toxins The group of Shiga toxins causes inhibition of protein biosynthesis (2, 152). The molecular mechanism differs, however, from that of diphtheria toxin and Pseudomonas aeruginosa exotoxin A. Shiga toxin and the Shiga-like toxins (SLT-I and SLT-II, formerly named verotoxins) of Escherichia coli inactivate the eukaryotic 60S-ribosome subunit by hydrolysis of a single N-glycosidic binding between adenine and ribose of a certain nucleotide (adenosine-4324) in the highly conserved sequence of the 28S-ribosome RNA (57, 170) (Fig. 3C). Consequently, the interaction of the elongation factor EF-2 with the 28S RNA is disturbed and the translation is blocked. In a remarkable analogy, the plant toxins ricin, abrin, and other ribosome-inactivating proteins (RIPs) hydrolyze the ribosomal RNA at the same adenosine position as the bacterial toxins (197). Shiga and Shiga-like toxins (mol. wt. 70000) are composed of one A component (mol. wt. 32 000) and a B component of five identical 69 amino acid subunits (mol. wt. 7700), a quaternary structure, which is analogous to that of cholera toxin, E. coli heat-labile enterotoxin and pertussis toxin (190, 191, 195, 196). The A subunit has to be cleaved by proteolysis and reduction of a disulfide bond to release the active Al fragment (mol. wt. 27000) (156). Shiga toxin and Shiga-like toxin SLT-I are identical except for a single amino acid (32, 199). Binding of Shiga toxins occurs via a glycolipid receptor identified as globosid Gb3 (95, 115). Translocation seems to be localized in the endoplasmic reticulum following retrograde transport of endocytosed material (176). Shiga-like toxins are able to induce the expression of proinflammatory cytokines (TNF-a, IL-l and IL-6) and may thus exacerbate colonic ulceration and bloody diarrhea (203). 3.4 Metalloendoproteases: the clostridial neurotoxins The clostridial neurotoxins, i. e., tetanus toxin (TeTx) and the seven different botulinum neurotoxin serotypes (BoNT/A, B, C, D, E, F and G), are the most effective toxins of all (150, 187). Both TeTx and BoNTs exert their effects by blocking neuroexocytosis. Tetanus toxin inhibits the release of neurotransmitters like y-aminobutyric ac-

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id (GABA) at interneural synapses, whereas botulinum toxin blocks the release of acetylcholine at neuromuscular synapses. Until 1991, neither the target molecule nor the mechanism of action of these toxins was known. Recently, it has been detected that the clostridial neurotoxins have enzymatic activity. Both tetanus toxin and the botulinum neurotoxins are zinc-dependent endoproteases (139, 180, 181). The substrates of the clostridial neurotoxins are several membrane proteins involved in neuroexocytosis (Fig. 3D). TeTx and BoNTIB, D, F and G specifically hydrolyze VAMP/synaptobrevin in the membrane of synaptic vesicles. BoNT/A and E cleave SNAP-25, whereas BoNT/ C hydrolyzes syntaxin, both being proteins of the presynaptic membrane (24,25,118, 179,182,183). The clostridial neurotoxins are produced as a single polypeptide chain (150 kDa). For activation, the prototoxin is cleaved into two chains, which remain connected by a disulfide bond. The heavy fragment (H chain, 100 kDa) is responsible for the binding to neuronal cells and the translocation of the light chain (L chain, 50 kDa) into the neuron. The L chain is the catalytic part of the neurotoxins (23,137) and contains the zinc-binding motif His-Glu-X-X-His (26, 98). The receptors for the clostridial neurotoxins are localized on the presynaptic membrane of neurons, but have not yet been precisely identified. High affinity binding seems to be achieved by the cooperative interaction of the toxin, first with membrane lipids, particularly polysialogangliosides, followed by concomitant binding to a protein receptor, similar to that assumed for diphtheria toxin (138). In contrast, the oligomeric toxins like cholera, pertussis and Shiga toxin accomplish strong cell binding by multiple binding subunits. Botulinum neurotoxins are used as therapeutic agents for various types of dystonia (96,177). 3.5 Invasive adenylate cyclases of Bordetella pertussis and Bacillus anthracis Bordetella pertussis and Bacillus anthracis produce adenylate cyclase toxins (ACTs), which are internalized by the target cell and cause a transient increase in the intracellular levels of cAMP of up to 100-1000 times the normal status (44, 82, 113, 135). The enzymatic activity of the bacterial invasive adenylate cyclases is stimulated by calmodulin, a Ca 2+-binding protein of the eukaryotic cytosol (Fig.3E). It has been suggested that the high cAMP levels are responsible for the disturbance of the unspecific immune response. B. pertussis adenylate cyclase, e. g., reduces the reactivity of polymorphonuclear granulocytes and macrophages (218). B. pertussis adenylate cyclase toxin is an integral part of the bifunctional adenylate cyclase/hemolysin, a single polypeptide of 177 kDa that possesses both the binding and the toxic functions. The C-terminal domain of 1300 amino acid residues constitutes the membrane-damaging hemolysin with its typical RTX motif (see 1.2.1), whereas the N-terminal domain of 400 amino acid residues is the adenylate cyclase that acts in the cytosol. Thus, B. pertussis has evolved two mechanisms for elevating cAMP in the host, the immediately but transiently effective adenylate cyclase toxin, on the one hand, and the much slower but persistently active pertussis toxin, on the other (see 3.1.1). Bacillus anthracis adenylate cyclase (edema factor) is one of the three distinct proteins of the anthrax toxin complex (Fig. 5). This plasmid-encoded toxin complex is composed of the edema factor (EF, 89 kDa), the lethal factor (LF, 90 kDa), and the protective antigen (PA, 83 kDa) (113, 114). Each component is secreted separately. The protective antigen acts as a B protomer and mediates the binding to a specific, as yet 13

Zhl. Bakt. 284/2-3

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..

edema factor (adenylate cyclase) 89kDa

protective antigen 83kDa

lethal factor 90kDa Lethal toxin

Fig. 5. The anthrax toxin complex. unidentified protein receptor. After binding, a cell surface protease identified as furin (137) cuts off a 20 kDa fragment from the N-terminus of PA. The remaining receptorbound PA of 63 kDa (PA63) is now able to bind either EF or LF, which compete for the binding, to form either adenylate cyclase toxin (PA-EF or ACT) or lethal toxin (PALF). The toxin-receptor complex is subsequently internalized by receptor-mediated endocytosis and released into the cytosol. The binding to calmodulin than activates AC, and the cAMP level of the host cell becomes elevated. However, the major pathogenic effects of anthrax are not caused by the invasive adenylate cyclase, but by the lethal factor (see 3.4). The adenylate cyclases of Bordetella pertussis and Bacillus anthracis show a homologous amino acid sequence, especially in the calmodulin-binding regions and the ATPbinding motif (58). 3.6 Toxins with unknown mechanisms of action Helicobacter pylori vacuolating cytotoxin In addition to its urease, Helicobacter pylori, a pathogen responsible for gastritis and peptic ulcer, produces a vacuolating cytotoxin (VacA, 95 kDa) (47,202), which

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provokes the formation of large vacuoles in epithelial cells. The vacuoles are coated with large quantities of Rab7, a small GTP-binding protein (160). The molecular mechanism is largely unknown. Anthrax lethal toxin The lethal toxin of the anthrax toxin complex (see 3.5 and Fig. 5) induces lysis of macrophages but shows little or no effect on other cells (68). Recently it was discovered that sublytic doses of lethal toxin induce macrophages to express interleukin 1 and tumour necrosis factor. Systemic shock and death from anthrax are presumed to be the result of this overexpression of cytokines (81). The lethal factor contains the zinc-binding motif His-Glu-X-X -His (212). This indicates that anthrax lethal toxin has metalloendopeptidase activity like the clostridial neurotoxins (3.4). However, no enzymatic activity has been detected so far, and the intracellular target of the lethal factor is not known. Acknowledgement. We are very grateful to Annegret Stuhr for her support and help in preparing this review and to Katherine Dege for thorough proofreading.

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