ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 18, No. 1 Copyright © 1988, Institute for Clinical Science, Inc.

Bacterial MICHAEL M. LUBRAN, M.D., Ph.D. Department of Pathology, Harbor-UCLA Medical Center, Torrance, CA 90509

ABSTRACT Many bacterial toxins are , encoded by the bacterial chromo­ somal genes, plasmids or phages. Lysogenic phages form part of the chro­ mosome. The toxins are usually liberated from the organism by lysis, but some are shed with outer membrane proteins in outer membrane vesicles. An important non- is or endotoxin, which is a constituent of the cell wall of gram negative bacteria. Toxins may dam­ age the eukaryotic by combining with some structural component, or otherwise alter its function. Many toxins combine with specific receptors on the surface membrane, frequently glycoproteins or gangliosides, and penetrate the cell to reach their intracellular target. A common mechanism of entry is absorptive endocytosis. Many protein toxins have an A-B structure, B being a polypeptide which binds to the and A being an enzyme. Many toxins are activated, either when produced by the bacterium or when bound to the membrane receptor, by proteases (nicking). An enzymatic process common to many toxins is adenosine diphosphate (ADP)-ribosylation of the adenylate cyclase regula­ tory proteins, leading to an increase in intracellular cyclic adenosine monophosphate (eAMP). This is the mechanism of action of . catalyzes the transfer of ADP-ribose to elongation factor- 2, inhibiting protein synthesis. Most toxins act on the target cells to which they bind, but tetanus toxin, and, to a lesser degree, , ascend axons and affect more distant structures. Although many toxin effects caused by bacteria have been described, only a few toxins have been identified, characterized, and their mode of action determined at the molecular level. The best known of these are discussed.

Introduction some intracellular target. Occasionally, they are the sole cause of disease; in Toxins produced by pathogenic bacte­ most cases, they act in concert with ria interfere with the physiological func­ other virulence factors which enable the tions of cells and are sometimes lethal. bacteria to establish themselves in the They act on the cell membrane28 (e.g., host and resist or evade its defensive hemolysins, lysins, phospholipases) or mechanisms. Although bacterial toxins 58 0091-7370/88/0100-0058 $02.00 © Institute for Clinical Science, Inc. BACTERIAL TOXINS 59 have been studied for many years, only a TABLE II few have been fully characterized and Principal Toxins Produced by Pathogenic Bacteria their mode of action determined at a molecular level. Many of the toxins are Organism Toxins proteins produced within the organisms. Mycobacteriurn Mycosides, , tuberculosis are exported through the bac­ Neisseria Endotoxin terial membranes or released by lysis of meningitides Pseudomonas A the organisms. Gram negative bacteria aeruginosa Salmonella , cytoxin have an outer membrane containing a (strains) lipopolysaccharide endotoxin. This toxin Shigella Cytotoxin (Shiga) , pyrogens, toxic escapes from the organism in vesicles aureus shock toxin (TST), , , a-, 3-, y-, 6-toxins shed from the outer membrane. Toxins Streptococcus , erythrogenic toxin (group A, are also classified by their target organs ß-hemolytic) or cells, for example, , S. pneumoniae Pneumolysin V. cholera Enterotoxin enterotoxins, and cytotoxins; however, V. mimicus Enterotoxin V. parahemolyticus Kanagawa hemolysin one toxin may act on different target V. vulnificus Enterotoxin cells. The effects of toxins have been Yersinia Enterotoxin studied in vivo in intact animals and in enterocolitica vitro on cells or cell fractions, using whole bacteria, bacterial fractions or cell-free extracts. The sensitivity of dif­ Protein Toxin Export in Bacteria42 ferent animals and cells to toxins varies considerably, and it is often uncertain The protein toxins are synthesized on which effects observed experimentally ribosomes as single polypeptide chains can be extrapolated to human disease. with a signal aminoacid sequence com­ Bacteria and their toxins are listed in prising about ten hydrophobic residues tables I and II. Many protein toxins have at its NH 2 terminal. In gram positive similar or related properties, which will organisms, which have a single mem­ be discussed in general and in more brane, the protein traverses the mem­ detail for the different toxins: these prop­ brane by diffusion or by creating a pore. erties are export, surface binding, inter­ The signal peptide is removed by a pro­ nalization (or translocation), action on tease synthesized concurrently with the target structures and genetic control. toxin, acting at the membrane. In many

T A B L E I

Principal Toxins Produced by Pathogenic Bacteria

Organism Toxins

Bacillus anthracis Edema factor, lethal factor, protective antigen B. cereus Enterotoxins Bordetella pertussis Pertussigen, dermonecrotic toxin, tracheal toxin, lipopolysaccharide, adenylate cyclase toxin Campylobacter jejuni Enterotoxin Lipopolysaccharide, enterotoxins, cytotoxin, Shiga-like cytotoxin botulinum Neurotoxins C. perfringens Enterotoxin C. tetani C. dificile Enterotoxin, cytoxin Corynebacterium diphtheriae Diphtheria toxin Klebsiella (species) Enterotoxins Listeria monocytogenes Endotoxin 60 LUBRAN cases, further mild proteolysis occurs as cholera toxin, can affect a wide range outside the organism (nicking), resulting of cells in vitro. In human cholera, how­ in two polypeptide chains held together ever, the toxin acts mainly as an entero- by one or more disulfide bonds. toxin. Specificity is related to the pres­ Protein export is more complicated in ence on the cell surface of membrane gram negative bacteria. The cell enve­ receptors specific for a particular toxin. lope consists of three layers or compart­ The receptors may be glycoproteins, ments:6,46 an inner, cytoplasmic mem­ gangliosides, which may also act as brane, an outer membrane, and a receptors of neurotransmitters and other periplasmic space between them. The physiological substances, sterols, and outer membrane is about 75 A thick and other molecules of unknown composi­ is a bilayer consisting of two leaflets, an tion. The receptors are usually randomly outer one of lipopolysaccharide and an distributed on the surface membrane. In inner one made of phospholipids. The some cases, the ligand-receptor com­ periplasmic space contains a peptidogly- plexes of toxins acting intracellularly ean membrane which gives the organism cluster after binding into specialized its rigid shape. The outer membrane areas of the membrane termed “coated contains porous areas, filled with pro­ pits”, which are lined with clathrin, a fil­ teins (porins), which allow the passage of amentous membrane protein. Clustering small molecules but not proteins. Some into coated pits is aided by a cross-link- toxins are released by cell lysis; others ing enzyme, transglutaminase. This pro­ pass into the lipopolysaccharide of the cess results in the concentration of toxin outer membrane and reach the exterior molecules from a dilute solution and in outer membrane vesicles as a protein- their localization to specific regions of lipopolysaccharide complex. True secre­ the surface membrane. tion of proteins by gram negative bacte­ Some protein toxins of high molecular ria is unusual, although it may occur weight are made up of two protomers, A with some hemolysins. and B. The active part of the toxin mole­ The complexity of protein export is cule, A, is responsible for its . It well illustrated by the excretion of heat- is usually an enzyme, acting on an intra­ labile toxin (LT) by Escherichia coli.23 cellular target; B may be composed of This toxin, as detailed, is composed of A smaller subunits. It is responsible for and B protomers. Both LT-A and LT-B binding the toxin molecule to the recep­ are synthesized with signal peptides by tor and may aid in releasing A from com­ cytoplasmic membrane-bound ribo­ bination, so that it may enter the cell; B somes. The LT-B is elongated rapidly, remains on the surface. Some toxins, processed by a signal peptidase and such as cholera toxin, bind to specific immediately assembled into B5-pen- receptors and are translocated as the tamers and transferred to the periplas­ complete toxin, not as subunits. The mic space. The LT-A is formed more mode of binding of many toxins is not slowly and remains associated externally known. with the cytoplasmic membrane until it meets and combines with a B5-pentamer, forming the soluble toxin molecule, T ranslocation o f P r o te in T o x in s28,35 which is shed in outer membrane vesi­ cles. Small molecular weight toxins proba­ Cell Surface Binding of Protein Toxins28 bly enter the cell by simple pinocytosis. Most toxins have a high affinity for A small amount of surrounding fluid specific cells, although a few toxins, such containing the toxin is enclosed in a pino- BACTERIAL TOXINS 61 cytic vesicle and moves into the cyto­ inhibiting its action and disrupting pro­ plasm. This is an inefficient process, and tein synthesis and the cell dies. its success depends on the presence of a Adenylate cyclase activity is increased high concentration of toxin in the neigh­ by cholera toxin, EC-LT and pertussis borhood of the cell. High molecular toxin from Bordetella pertussis. The weight toxins are translocated by absorp­ enzyme is bound to the cytoplasmic side tive endocytosis. of the surface membrane. Adenylate In this receptor-mediated process cyclase consists of a hormone receptor, a (RME), the toxin-receptor complexes catalytic portion and two guanyl nucleo- undergo translational diffusion, are com­ tide-binding proteins, Gs and Gj, 4,16 bined enzymatically, are concentrated in which, respectively, regulate stimulation coated pits, as described previously, and and inhibition of the enzyme by surface- pass into the cytoplasm as endosomes. bound hormones. Cholera toxin and The toxins leave the endosomes by dif­ EC-LT ADP-ribosylate the a-subunit of ferent processes (which will be described Gs; ADP-ribosylates the later) and reach their intracellular target. a-subunit of G;. These are two different Surface-active toxins exert their effects proteins, with respective Mr’s of 45,000 on specific cells by binding to compo­ and 41,000. The (3-subunits of these pro­ nents of the surface membrane and teins are the same. The Aj subunit of the destabilizing it. Hemolysins bind to cho­ cholera toxin and EC-LT cleaves NAD + lesterol causing a leak of potassium ions and transfers the resulting ADP-ribose to and then hemolysis. Toxins acting on the a-subunit of Gs. The ADP-ribosyl- leukocytes (e.g. streptolysins O and S, ated a-subunit has less affinity for the (3- Str. pneumoniae pneumolysin14) com­ subunit (of Gs), which increases in con­ bine with surface-membrane sterols or centration and stimulates cAMP produc­ phospholipids resulting in release of tion. Pertussis toxin ADP-ribosylates the lysosomal hydrolytic enzymes, degranu­ inhibitory regulator protein G; and lation and cell death. Lesser effects are allows the stimulatory regulator to suppression of chemotaxis and phagocy­ increase cAMP concentration. An unreg­ tosis. Lysogenic streptococci (i.e., hav­ ulated increase in cAMP concentration is ing prophage desoxyribonucleic acid equivalent to continued overstimulation [DNA] incorporated in the bacterial of the cell by hormones such as nor­ DNA) produce an erythrogenic toxin, adrenaline and eventually leads to the which is also pyrogenic. It enhances sus­ death of the cell. ceptibility to endotoxic shock and cardio- toxicity. The (3-toxin of Staph, aureus is an enzyme which reacts with sphingo­ Genetic Control of Protein myelin to split off phosphorylcholine. Toxin Production3 Leukocidins act by stimulating mem­ brane acylphosphatase to dephosphory- Genes controlling the production of late membrane triphosphoinositide. protein toxins may reside in the bacterial Protein synthesis is affected by some chromosome, phage particles or plas­ toxins, for example, diphtheria toxin and mids. The DNA of temperate phages is exotoxin A produced by Pseudomonas incorporated into the bacterial chromo­ aeruginosa. The A subunit of these some. Phage reproduction is turned off toxins is activated by lysosomal enzymes by repression of early and late gene in the perinuclear region. The free A functions: the repressed phage genome subunits bind to a specific aminoacid res­ remains in the cell as prophage and rep­ idue, diphthamide (a histidine deriva­ licates synchronously with the cell (lysog- tive), in elongation factor 2 (EF-2), geny). Occasionally, repression of the 62 LUBRAN prophage fails and the cells are lysed. face membrane of the target cells. The B Lysis can also be induced by external chain has two a-helical amphipathic factors affecting DNA replication. Plas­ domains, reminiscent of the domains of mids and phages may be transferred apolipoproteins, which react with the from a toxin-producing strain to a non membrane phospholipids and core toxin-producing strain, making the bac­ lipids; there is a third hydrophobic teria toxin-producing. Genetic control is domain which anchors the B chain discussed with the individual toxins. through the thickness of the membrane. The nature of the surface membrane receptor is not known. Glycoproteins of Diphtheria Toxin16’28’30,33’35,49 Mr 150,000 have been isolated which can bind diphtheria toxin in vitro, but their Diphtheria toxin is a single polypep­ physiological role has not been estab­ tide of Mr 58,342 secreted by toxic lished. The receptor area probably occu­ strains of Corynebacterium diphtheriae, pies a broad region of the surface and carrying the tox structural gene found in may consist of several functional groups. the lysogenic corynebacteriophages (3 Internalization of the toxin occurs tox+, 7 tox+ and to tox+. Avirulent mainly by receptor-mediated endocy- strains become lysogenic when infected tosis. Some toxin molecules may enter with these phages. Highly toxic strains the cell by direct plasma membrane have two or three tox+ genes inserted transversal through non-specific pinocy- into the genome. Expression of this gene tosis, but very few molecules survive in is regulated by the bacterial host. The the cytoplasm to reach their target. In repressor gene is iron-dependent: its RME, the toxin-receptor complexes action is inhibited, and toxin production gather into coated pits lined with is enhanced in the presence of very low clathrin. The pits form vesicles which concentrations of iron. The toxin is syn­ migrate towards the Golgi apparatus and thesized as a single polypeptide chain of fuse with primary lysosomes. The B 535 residues, with a 25 residue leader chain, because of its hydrophobic sequence and two disulfide bridges. The domains, becomes embedded in the signal peptide is cleaved by a concomi­ lipid membrane of the endosomes and tantly produced protease as the toxin is creates a pore. The lysosomal proteases shed in the outer membrane vesicles. cleave the toxin molecule into its A and After export from the organism, the pro­ B chains, which become separated by tein is cleaved by a proteolytic enzyme reduction of the disulfide bridge. The into two subunits, A and B, held acid milieu produced by the lysosomes together by an interchain disulfide bond causes a conformational change in the A between cystine residues at 186 and 2 0 1 . chain, which can leave the endosome Reduction of the disulfide bond causes through the pore and pass to its target. separation of the A and B subunits, but The A chain is very stable and is not they remain closely associated by non- attacked by the lysosomal enzymes; the covalent forces. The A subunit (which B chain, however, is readily destroyed. has 193 residues and Mr 21,150) is an Interaction of the A chain with EF-2 enzyme, which catalyzes the ADP-ribo- involves binding to NAD+ at a single sylation of elongation factor-2 , which is site near glu-148. The complex then involved in the translocation process of binds to EF-2 and the ADP-ribose protein synthesis. The B subunit is moiety is covalently bound to a single responsible for binding the intact toxin receptor in the EF-2 molecule, diphtha- molecule to specific receptors in the sur­ mide, at Nl, by an a-glycosidic linkage. BACTERIAL TOXINS 63 The reaction goes almost to completion. ribose from NAD+ to EF-2, inhibiting Diphthamide, 2-[3-carboxyamide-2-(tri- its action in protein synthesis. The ADP- methylammonia) propyl] histidine, ribose is covalently bound to diphtha­ located near the NH2-terminal of the A mide, as in the case of diphtheria toxin. chain, is a unique histidine derivative However, the two toxins are not the found only in EF-2 of eukaryotic cells same and there is no immunological and archaebacteria. The EF-2 is a cross-reaction between them. GTPase; ADP-ribosylation of diphtha­ Exotoxin S is also an ADP-ribosyl mide prevents this activity, without transferase, of Mr 49,000. Unlike exo­ altering the ability of EF-2 to bind GTP. toxin A, it does not act on EF-2. The Protein synthesis is arrested. About half probable substrate is EF-1. Protein syn­ of the diphtheria toxin molecules are thesis is also inhibited. Both toxins bind associated non-covalently with a nucleo­ to surface receptors and are internal­ tide, ApUp, similar in structure to NAD. ized by endocytosis. The nature of the ApUp blocks the nucleotide binding site receptor and the details of internal­ on the A chain. Diphtheria toxoid, pro­ ization are not known. The toxins are duced by treatment with formaldehyde encoded by the bacterial chromosome; or glutaraldehyde, has no enzymatic plasmids or bacteriophages are not activity, although it binds to cells. It is involved. internally crosslinked and does not sepa­ rate into A and B subunits. Enterotoxins Little is known about the effects of diphtheria toxin on cells at the molecular These toxins, produced by several dif­ level. There is apparently no specific tar­ ferent organisms, act on the mucosal epi­ get organ; arrest of protein synthesis thelium of the small intestine and pro­ eventually leads to cell death. Clinically, duce a profuse watery diarrhea. the organisms remain at the site of infec­ tion. The toxin produces necrosis of the Cholera Toxin3,7,12,15> !9,20,21,28,33,44 local , which later becomes the characteristic diphtheritic mem­ Cholera toxin, secreted by Vibrio cho- brane. Systemic effects are produced by lerae, is a protein composed of five iden­ entry of the exotoxin into the circulation. tical B protomers (Mr 11,677) and one A The myocardium and peripheral nerves protomer (Mr 27,215), held together are most affected. noncovalently. The B units form a ring, with the A protomer in the center. The A Exotoxin A12,16,24 protomer has a signal peptide at its N- terminal, composed of 18 hydrophobic Pseudomonas aeruginosa, a Gram- aminoacid residues; the B protomers negative opportunistic organism, pro­ have signal peptides of 21 aminoacid res­ duces exotoxin A and exotoxin S. Exo­ idues. The A and B protomers are toxin A is secreted as a single, assembled into the complete toxin in the polypeptide chain of Mr 62,583, with a outer membrane of the organism (in con­ signal peptide sequence. It has four trast to EC-LT, which is assembled in intrachain disulfide bonds, none in the the periplasmic space). The A protomer enzymatic region. The secreted protein is cleaved by a protease into an Ax ami- is a proenzyme: activation involves split­ noterminal peptide and a smaller A2 ting it into an A (enzymatic) chain (Mr peptide (Mr about 5,000), which remains 26,000) and B (binding) chain. The A closely associated with the B protomer. chain catalyzes the transfer of ADP- Ax and A2 are joined by a disulfide link­ 64 LUBRAN age. Both A and B protomers are kiihn, in this case stimulating a flux of encoded by chromosomal genes ctx A chloride and bicarbonate ions (accom­ and ctx B, regulated by a gene tox R and panied by sodium ions) into the lumen, possibly others. Protomer B is always brought about by an increase in anion produced in excess of A. Note, A1 is an conductance of the luminal membrane. ADP-ribosyl transferase; B is responsible The toxin also ADP-ribosylates other for binding the toxin to its surface-mem- substrates, but the consequences for brane receptor, A2 assisting in this pro­ human disease are not known. cess. The surface-membrane receptor on Escherichia coli eukaryotic cells is the monosialoganglio- Enterotoxins3,4,12,15’16’21,27’31 side GMl, which is widely distributed among different cell types.44 However, These enterotoxins are responsible for in the clinical disease, the organism is “travelers’ diarrhea”. Some strains of E. confined to the gastro-intestinal tract. coli colonize the mucosal surface of the The mode of internalization of the toxin small intestine and produce exotoxins has not been fully elucidated. It is EC-LT and EC-ST; a second group is believed that when bound to its recep­ enteroinvasive, like Shigella organisms, tor, the B protomers and A2 bring A1 and multiply intracellularly, producing close to the plasma membrane and, at the same exotoxins. A third group, the same time, create an opening for it to enteropathogenic E. coli (EPEC), pro­ enter the cytoplasm. Ax is then sepa­ duces a cytotoxin, similar to that pro­ rated from the rest of the toxin by reduc­ duced by Shigella dysenteriae 1. tion of the disulfide bond. The target for EC-LT11 is a heat-labile protein toxin, A1 is adenylate cyclase, which is mem- very similar to cholera toxin in its com­ brane-bound on the cytoplasmic side of position and properties. Like that toxin, the membrane. Internalization of the it consists of an A subunit (Mr 25,500 to toxin is not effected by RME. The 29,000) and five identical B pentamers of enzyme transfers ADP-ribose from Mr 11,800). The subunits are synthe­ NAD+ to the regulatory protein Gs, as sized with signal peptides, assembled described previously, and produces an into the complete toxin in the periplas- uncontrolled increase in cAMP. Cholera mic space and exported with outer mem­ toxin acts like a hormone in its produc­ brane proteins in vesicles, as has been tion of a second messenger, cAMP; how­ described previously. The A subunit ever, unlike hormonal action, the pro­ consists of an Ax chain (Mr 21,000), duction of the messenger is irreversible. which has enzymatic activity, and an A2 Cholera toxin attaches itself to recep­ chain, which fixes the A subunit to the tors on the luminal surface of the intes­ ring of B pentamers and assists the B’s to tinal epithelium, the effects of increased bind to a specific receptor. There is con­ cAMP production depending on the cell. siderable homology between these In villus cells, it has an antiabsorptive chains and the corresponding ones in effect, that is, the inward flux of sodium cholera toxin; there is also antigenic and chloride ions from lumen to mucosa cross-reactivity. The toxin is coded for by is inhibited; water absorption is there­ plasmid genes. fore also prevented. The cause is EC-LT has the same enzymatic action believed to be phosphorylation of brush as cholera toxin. It is an ADP-ribosyl border components by a cAMP-depen- transferase, transferring ADP-ribose dent protein kinase. The cAMP also from NAD+ to the Gs regulatory pro­ acts on the cells of the crypts of Lieber- tein of surface-membrane bound adenyl­ BACTERIAL TOXINS 65 ate cyclase, leading to an unregulated targets and mode of action of these toxins increase in cAMP production, and the are not known. same effects on salt and water absorption described for cholera toxin. EC-LT Other Enterotoxins binds to the same GMi receptors on the brush border membrane of the small Some strains of Klebsiella, Entero- intestinal mucosa that bind cholera toxin; bacter cloacae, Yersinia enterocolitica, in addition, it binds to other receptors, and Shigella flexneri produce entero­ probably galactoproteins.21 toxins similar to STA; Pseudomonas aeru­ EC-ST is a heterogeneous group of ginosa, ,i5 and Klebsiella heat-stable, low molecular weight, pep­ produce an LT-like toxin. Some Salmo­ tides. There are two varieties, STA and nella organisms produce a cholera-like STb, differing in methanol solubility. STB enterotoxin, which is heat-labile, of Mr is not an enterotoxin.27 STA peptides about 90,000 and have A and B pro- have an Mr of about 2,000 and consist of tomers. Aeromonas organisms produce 18 to 19 aminoacid residues, of which six an enterotoxin different from LT, ST, are cysteine. The toxin is tissue specific and cholera toxin. Clostridium perfrin- to the small intestinal brush border cells, gens produces an enterotoxin similar to where it binds to a high molecular EC-LT, while C. difficile produces an weight protein, and not a ganglioside. It enterotoxin stimulating guanylate has no known enzymatic activity, but it cyclase activity. Vibrio vulnificus, V. flu- produces an increase in cGMP in the cell vialis, and V. mimicus produce a chol­ by its stimulating effect on guanylate era-like enterotoxin. Campylobacter cyclase. This results in inhibition of the jejuni produces enterotoxins resembling sodium coupled uptake of chloride by those of cholera and EC-LT. the villus cells, as occurs with cholera toxin. The mechanisms of internalization Shigella dysenteriae Type 1 Toxin3,12,32 of the toxin and activation of guanylate cyclase are not known. This toxin () has cytotoxic, neurotoxic and enterotoxic actions, although its principal effect is cytotoxic. E nter otoxins36,40 The toxin (Mr 65,000) is composed of an A protomer (Mr 32,000) and six or seven Some strains of this organism produce B protomers (Mr of each about 5,000). heat-stable enterotoxins, which cause The A protomer can be cleaved into Ax severe diarrhea and vomiting. Seven dif­ and A2 chains, linked by a disulfide ferent toxins have been described, A, B, bond. Aj is an enzyme. The toxin is inac­ C1; C2, C3, D, and E. They are all single tive when formed, but is activated in the chain polypeptides, with Mr’s in the target cell by proteolysis and reduction range of 26,000 to 30,000. While A is of the disulfide bond, liberating the free immunologically distinct, the others A2 chain. The protomers are synthesized show considerable homology. Cx has with N-terminal hydrophobic signal been sequenced. It has an Mr of 27,500 sequences and the toxin molecule is and has 239 aminoacid residues. It is assembled in the periplasmic space of encoded by a plasmid. C3 has 236 resi­ the organism. Toxin synthesis is phage dues and an Mr of 27,111. It is also encoded. encoded by a plasmid. The trio of Cl5 C2, The toxin binds with high affinity to and C3 cross-react, but are not identical. glycoprotein receptors on the surface A is encoded by a lysogenic phage. The membrane and probably enters the cell 6 6 LUBRAN by RME; however, the details are not GQ-type gangliosides on neuronal mem­ known. Because the organism is intracel­ branes. These gangliosides are mainly lular in the intestinal mucosal cells, neuronal in distribution and determine binding and internalization apply to its the specificity of the toxin. The nerve other target cells. The A2 chain inhibits terminals have many binding sites, protein synthesis in the target cells by accounting for the high toxicity of teta­ catalyzing the inactivation of the 60 S nus toxin. The bound toxin is internal­ ribosomal subunit, by an unknown ized, possibly by adsorptive pinocytosis, mechanism. and undergoes retrograde axonal trans­ Enteropathogenic E. coli (EPEC ) 10 port of motor, sensory and adrenergic produce a toxin, similar to but not iden­ fibers in smooth vesicles, cisternae and tical with Shiga toxin. The molecular tubules, slowly reaching the post-synap­ weight is less. The toxin is composed of tic dendrites. The toxin is selectively A and B protomers; A is nicked by pro­ released from these and is taken up by teases to give Aj and A2 chains, the the presynaptic nerve terminals. Some former enzymatically inactivating the 60 toxin is found in the lysosomes of the S ribosome. Toxin production is encoded ganglion cells. It acts by preventing by a plasmid, which is easily transmitted the release of acetylcholine, thus block­ to other E. coli. ing inhibitory synapses in the spinal cord and causing a spastic paralysis. Neurotoxins Botulinum Toxin12,22,28,43 The principal neurotoxins are teta­ nus and botulinus toxins. Although they There are eight antigenically different are similar in many respects, their toxins, each produced by a different target organs are different and thus their strain of . The clinical effects. organisms and the toxins are named A, B, Cl, C2, D, E, F, and G. All except Tetanus Toxin12,22,28’41 C2 are neurotoxins. C2 is a eytotoxin which induces hypotension, an increase This is a protein exotoxin of Mr in intestinal secretions, pulmonary hem­ 150.000 produced by , orrhage and increased vascular perme­ encoded by a plasmid. The toxin mole­ ability. The toxin is an ADP-ribosyl cule consists of a light a-chain, Mr transferase, its substrate being p/7 -actin, 50.000 and a heavy (3-chain, Mr 100,000, preventing actin polymerization. Skele­ which consists of two fragments, (3X and tal muscle actin is not affected. 1 C2 is |32. A disulfide linkage connects a and secreted as two separate proteins, C2-I there is an intrachain sulfide linkage and C2-II, of respective Mr’s 50,000 and in Pj. The toxin remains in the bacte­ 1 0 0 ,0 0 0 , which must be combined to rium until its release by autolysis. It is produce a toxic effect. C2-I is the activated by nicking by an intracellular enzyme; C2-II binds the complete toxin protease before release, the (3X fragment to the cell and aids the translocation of being separated. However, both chains the enzyme. are required for toxicity. Neither chain Toxins of C. botulinum are phage nor the whole molecule has demonstra­ encoded, except for C2. Both Cl and D ble enzymatic activity. have a lysogenic phage, while the others The target structures for tetanus toxin have either a phage or a plasmid. The are the peripheral nerve endings. The toxin is synthesized as a single chain heavy chain binds to GDlb, GTlb and polypeptide of Mr 150,000 to 160,000 BACTERIAL TOXINS 67 and is nicked extracellularly by an the O-chain through one or more keto- endogenous protease after release by deoxysaccharidic acids (KDO), forming autolysis, to give a heavy (H) and a light the inner core, to residues, often (L) chain, similarly to tetanus toxin. The substituted by phosphate and phosphor- toxins act on cholinergic nerve endings, ylethanolamine. Rough (R) mutants lack initially at the peripheral neuromuscular the O-polysaccharide and part of the junctions and, after retrograde axonal inner core. ascent, to nerve endings for which ace­ is covalently bound to the tylcholine is the transmitter. Botulinum polysaccharide portion of LPS, through a toxin can reach any area of the central KDO. Lipid A s of various endotoxins nervous system neuronally connected resemble one another in their general with the peripheral nerve endings. The structure. They have a phosphorylated blood brain-barrier is impermeable to glucosamine disaccharide backbone, the toxin. The heavy chain at its COOH with medium chain fatty acids attached end binds to gangliosides, probably Gxla to the 3 and 3'-hydroxyl and the amine and others. The toxin is internalized at radicals. The fatty acids have 12, 14, or the neuromuscular junction, possibly by 16 carbon atoms, are mostly saturated RME, but the true mechanism is not and some have hydroxyl groups. Lipid A known. At the cholinergic nerve end­ is antigenic, antisera protecting against ings, it antagonizes events triggered by some of the effects of endotoxin, but not calcium ions that culminate in the all. Endotoxicity resides in the complete release of acetylcholine, possibly by LPS molecules, but the lipid A compo­ blocking calcium ion channels. The nent plays an important part. The role of mechanism of axonal ascent is not clear. various structures in the molecule in producing toxic effects is under intense Endotoxins study, using newly available synthetic analogues. The outer membrane of gram negative The LPS is released from the bacteria, bacteria contains lipopolysaccharide combined with outer membrane pro­ (LPS) as an integral part. The amphi- teins, and other compounds, in outer pathic macromolecule carries the anti­ membrane vesicles. Thus, although an genic determinants of O specificity. It is endotoxin, it is found in the environment also an endotoxin, responsible for symp­ of the organisms and can be dissemi­ toms of a wide variety of septic and non- nated throughout the body. In the septic conditions.25 plasma, it is bound to high density lipo­ consist of a hydrophilic polysaccharide proteins17 and is slowly removed from portion, which is responsible for its O- the circulation by phagocytes. Most is and R-antigenic properties and a cova­ eventually removed by the liver; some is lently bound hydrophobic lipid compo­ degraded intracellularly in tissues. nent called lipid A, which is responsible Endotoxin is also released within phago­ for the endotoxic properties of cytic cells as they process ingested bacte­ LPS.18,26’29,37,38 The polysaccharide can ria and it may be produced locally by be divided into an O-specific polysac­ lysis of organisms. charide chain joined to a core heterooli­ The mechanism of endotoxin toxicity gosaccharide. The O-chain is made up of is not clear. Some cells are particularly repeating oligosaccharide units, the sensitive to endotoxin, namely, mono­ diversity and arrangement of which cyte, granulocytes, and endothelial cells. differ in different bacteria and provide Endotoxin is also a potent activator of the O-specificity. The core is linked to the complement system; activated com­ 6 8 LUBRAN plement could be responsible for cell walls of all bacteria, can in large amounts membrane damage and activation of the produce endotoxin-like effects.9 Group A coagulation system.34 The entry of LPS Streptococci, some Staphylococci and into target cells5 is believed to involve Mycobacterium tuberculosis produce binding to surface receptors, endocytosis endotoxin-like effects. and translocation of LPS through the cytoplasm to a receptor on the mito­ Toxic Shock Syndrome2,8,39 chondrial membrane. The mitochondrial proton gradient is destroyed, ADP and This condition (TSS) is associated with NADH accumulate in the cytosol leading strains of Staphylococcus aureus infected to enhanced glycolysis and lactic acid with group I bacteriophages, especially production. The permeability of the cell phage 29. On lysis, these bacteria pro­ membrane to calcium ions is increased; duce an exotoxin, TST. This is a single mitochondrial injury permits the accu­ chain polypeptide of Mr 24,000. Other mulation of peroxides and superoxides protein toxins, including staphylococcal and lipids are oxygenated producing enterotoxin B, have been found asso­ prostaglandins. End results are release ciated with TST. The role of these other of lysosomal enzymes, active degrada­ toxins in producing TSS is not clear. tion of intracellular proteins and the new However, it is known that endotoxin synthesis of replacements. Endogenous enhances the severity of TSS, probably pyrogen is formed. The cellular events by suppressing the immune system. account for the observed effects of endo­ Toxic shock syndrome is pyrogenic, par­ toxin toxicity: disturbed temperature ticularly when following small amounts regulation, changes in and cal­ of endotoxin. cium concentrations, increased lactic Toxic shock syndrome occurs mainly in acid production and lysosomal enzyme menstruating women using tampons, but activities, disseminated intravascular it also occurs in other women and to a coagulation, and circulatory hypoten­ lesser degree in males. The severe, often sion. lethal, form of the disease is marked by Endotoxin is always being produced in multi-system organ damage and massive the host by resident bacteria. Under capillary vasodilation with intravascular normal conditions, the host defense fluid loss. Clinically, there are fever, a mechanisms, many induced by the toxin rash, desquamation of the skin of the in small amounts, clear it from the sys­ palms and soles after one to two weeks, tem in the liver or metabolically degrade severe hypotension, and vomiting or it. Breakdown of the defense mecha­ diarrhea, myalgia, renal damage, liver nisms and resulting endotoxemia may damage, bone marrow depression and occur with septic lesions, liver dysfunc­ CNS involvement. Some of these find­ tion, severe hemorrhagic shock, adrenal ings are directly due to the toxic insufficiency and other conditions.25 action on target cells, others are secondary Endotoxin can be detected in the blood to them. Mild forms of the disease occur. and other body fluids by use of the Limulus amebocyte lysate test (LAL). Bordetella pertussis toxins4,12,28,47,49 The lysate is clotted by endotoxin. A sensitive colorimetric modification of the These have been studied intensively LAL test is now available.13 in recent years because of the high Although endotoxin is a product of degree of reactogenicity of whole cell gram negative bacteria, peptidoglycan, a pertussis vaccines and the severity of basic structural component of the cell some of the reactions. The major toxin is BACTERIAL TOXINS 69 pertussis toxin or pertussigen. In the amounts of this toxin may produce no past, this toxin was given many different overt effects, unless the subject is names, each associated with one of its stressed and homeostatic mechanisms toxic effects in experimental animals. It fail. is no?, known that these diverse effects Adenylate cyclase toxin is secreted by are caused by one molecule, probably by the growing organism and its activity is one mechanism. Other toxins are ade­ increased a thousand times by eukaryotic nylate cyclase toxin, dermonecrotic calmodulin. In some way, this toxic toxin, tracheal cytotoxin and lipopolysac- enzyme enters eukaryotic cells and cata­ charide. Filamentous hemagglutinin, lyzes the conversion of endogenous ATP although not a toxin, assists the attach­ to cAMP. The toxic enzyme has an Mr of ment of the organism to its target cells 190,000. Non-toxic adenylate cyclase, and is therefore a . also produced by the organism, has an Pertussigen is a hexameric protein of Mr of 70,000. Mr 117,000, the subunits being asso­ Dermonecrotic toxin is heat-labile. It ciated into an A-B dimer. The A pro- has an Mr of 102,000 and has subunits of tomer, Mr 28,000, is an ADP-ribosylase. 24,000 and 30,000 Mr. It produces vaso­ The heavier B-protomer is composed of constriction of small blood vessels and four dissimilar subunits held together local ischemia, when injected subcutane- non-covalently and united by a second ously in mice. The mechanism of action smallest subunit, which connects two is not clear, but the toxin does inhibit pairs of dimers. The whole structure is Na+-K+ ATPase activity. reminiscent of cholera toxin. The B pro- Tracheal cytotoxin is a small peptide, tomer binds the toxin to surface-mem­ probably derived from peptidoglycan. It brane receptors on the target cell, possi­ binds specifically to ciliated cells and bly a ganglioside. The mechanism of inhibits DNA synthesis. Its molecular internalization is not known. The A pro- mechanism of action is unknown. tomer transfers ADP-ribose from NAD + Lipopolysaccharide differs from the to the membrane bound 41,000 Mr sub­ LPS of gram negative bacteria. There are unit of the inhibitory protein, G;, of the two immunologically distinct lipopoly- guanine nucleotide-binding regulatory saccharides, containing lipid A and lipid component of adenylate cyclase. The X, respectively. There are also two differ­ binding is enhanced by Mg2+ and the ent oligosaccharide chains. The lipid X 35,000 Mr component of G,. As a result, fraction has the characteristic endotoxic the response of the regulatory compo­ effects, but the lipid A fraction has little nent to signals from cell surface recep­ toxicity and is not pyrogenic. However, tors is attenuated or abolished and there it has potent adjuvant and antiviral activ­ is an unregulated increase in cAMP pro­ ities. Polypeptides associated with the duction when the cells are stimulated. lipopolysaccharides also have immuno- Insulin secretion is potentiated through modulating properties. loss of a-adrenergic inhibition. Other non-cAM P coupled receptor pathways Bacillus anthracis28 are also blocked by pertussigen, e.g., the stimulation of phosphatidylinositol This organism produces three factors hydrolysis, arachidonate release and cal­ which combined are toxic: protective cium mobilization. Immune effector factor, edema factor and lethal factor. cells are particularly affected. Similar Protective factor plus edema factor form mechanisms produce heightened sensi­ an A-B type toxin, which is responsible tivity to histamine stimulation. Small for the dermonecrotic effect of the toxin. 70 LUBRAN Edema factor is an inactive form of ade­ 10. C leary, T. G ., M a thew son, J. J., F aris, E., and Pickering, L. K.: Shiga-like cytotoxin pro­ nylate cyclase, which is activated after duction by enteropathogenic Escherichia coli entry into the target cell by calmodulin, serogroups. Infect. Immun. 47:335—337, 1985. elevating cellular cAMP. Protective fac­ 11. C le m e n ts , J. D., Y ancey, R. J., and F in k le - STEIN, R. A.: Properties of homogeneous heat- tor promotes entry of the enzyme into labile enterotoxin from Escherichia coli. Infect. the cell. Lethal factor is lethal to animals Immun. 29:91-97, 1980. when combined with protective factor. 12. E id e l s , L., P roia, P. 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