Pathophysiological effects of Vibrio cholerae and enterotoxigenic Escherichia coli and their exotoxins on eucaryotic cells.

MICROBIOLOGICAL REVIEWs, Sept. 1978, p. 592-613 0146-0749/78/0042-0592$02.00/0 Copyright i 1978 American Society for Microbiology

Vol. 42, No.3

Printed in U.S.A.

Pathophysiological Effects of Vibrio cholerae and Enterotoxigenic Escherichia coli and Their Exotoxins on Eucaryotic Cells KAREN L. RICHARDS AND STEVEN D. DOUGLAS* Departments of Microbiology and Medicine, University of Minnesota Medical School, Minneapolis, Minnesota 55455

iNTRODUCTION .5.9.2...... 592 PATHOPHYSIOLOGY ...................................... 593 Etiology ..... ......3.... .............. ... ........3.... 593 Factors in Pathogenesis ..................................................... 593 Genetic and Physiological Basis for Toxin Production .59.......3. 53 THE TOXINS 594 Structure 594 Antigenic Relatedness of Toxins .594 Binding Site 606 Action of Toxins on Adenylate Cyclase .596 Solubilized Adenylate Cyclase and Toxins .597 Cofactors Required for Toxin Activity .597 Involvement of Guanosine 5'-Monophosphate (GMP) ........................ 597 Effect of Increased Intracellular Cyclic Adenosine 3',5'-Monophosphate . 598 Variability in Activation of Adenylate Cyclase .598 Similarities to Glycoprotein Hormones and Other Bacterial Toxins ......... 598 Vaccines and Immunity .. .....5...............................599 ENTEROTOXINS IN IN VIVO SYSTEMS ... 600 Whole-Animal Models ........... ................................. 600 Ileal Loop Assay ................. 600 Skin Permeability Assay ......... 601 ENTEROTOXINS IN IN VITRO SYSTEMS ...... 601 Erythrocyte Ghosts ............................................... 601 Adrenal Cells ....... .... ........ ... 602 Isolated Fat Cells .......................... 603 Lymphocyte and Lymphoid Cell Lines and the Immune Response ..... ...... 603 Fibroblasts ................................................................. 604 Other Cell Systems .................................... 604 DISCUSSION ........6..0.5.................. 605 CONCLUSIONS .60...................................606 LITERATURE CITED .606 .................................................................

........................

INTRODUCTION Vibrio cholerae and enterotoxigenic strains of Escherichia coli cause diarrheal disease in man and several animal species. E. coli strains can produce two enterotoxins; a low-molecularweight heat-stable toxin (ST) and a heat-labile toxin (LT) that has many properties similar to those of cholera toxin (CT). Although the mechanism of action of E. coli ST is not known, clinical symptoms due to cholera and E. coli LT are the result of action of the toxins on mucosal cells of the small intestine. The major action of V. cholerae and E. coli heat-labile enterotoxins is to activate adenylate cyclase followed by increases in intracellular levels of cyclic 3',5'-adenosine monophosphate (cAMP) and hypersecretion of salts and water into the intestinal lumen. There are a number of features distinctive to the exotoxins of V. cholerae and E. coli. These

differences constitute the basis for further studies on the structural and functional relatedness ofthe toxins and aspects of mechanisms of action (63, 79, 80, 128). This review will analyze factors that are requisite for localization of these organisms within the intestinal tract and toxin productibn. The physicochemical properties of the toxins and the effects of intestinal cells will be discussed as well as the response of other organ and cellular systems. In addition, analogies will be drawn between these toxins and other biologically active molecules. The role they may play in the growth of the organisms and their use as probes into the molecular biology of eucaryotic cells will be discussed. (Part of this review was presented at the 77th Annual Meeting of the American Society for Microbiology, 8-13 May 1977, New Orleans, La.).

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EFFECTS OF E. COLI AND V. CHOLERAE TOXINS

PATHOPHYSIOLOGY Etiology Cholera is characterized clinically by an acute enteritis after bacterial colonization of the small intestine. Massive fluid loss frequently leads to severe dehydration and shock. V. cholerae is a gram-negative, motile, curved rod that grows on simple nutrient media at alkaline pH (5, 181). The organism has been classified into the classical and El Tor biotypes on the basis of geographical distribution and the ability to lyse sheep erythrocytes (61, 62). Two serotypes, Inaba and Ogawa, are distinguishable by heat-stable somatic antigens. Certain strains of E. coli, commonly found in great numbers in the large intestine, are capable of colonizing the small intestine and have been identified as causative agents of a severe choleralike disease in man (6, 82, 175) and domestic animals (116). Although several studies have attempted to relate toxigenic strains to particular somatic antigens (84, 88), clinical disease correlates more closely with the presence of distinct protein capsular (K) antigens (109). In addition, certain antigenic types are often associated with a particular host species; those isolated from pigs possess antigens different from those isolated from calves, lambs, (117, 161).

or

humans

Factors in Pathogenesis Adherence to intestinal surfaces and toxin production are two factors necessary for pathogenesis by these two microorganisms. Motility is also a virulence factor for V. cholerae (72, 115). Motile vibrios are more virulent than nonmotile organisms of the same strain (72, 85, 115) and are able to penetrate the intestinal mucus and enter villous crypts (85, 86). Adherence is thought to be important to enable the organisms both to resist removal by peristalsis and to multiply near the site of action of the toxin (86). Studies of V. cholerae adherence to intestinal epithelium by Freter and Jones (72), Jones et al. (114), and Nelson et al. (155) have demonstrated that the organisms penetrate the intestinal muand attach to microvilli at the brush border of the epithelial cells. Several factors are important determinants of adhesion of V. cholerae and include bacterial motility (72), temperature, and cations (71, 114, 115). The exact nature of the adhesive antigen (adhesin) is not known; however, it may be associated with the bacterial flagellum. Nonmotile vibrios are unable to adhere to intestinal brush border membranes and intestinal tissue slices (72); nonmotile mutants are unable to adhere to brush border membranes cus

593

after centrifugation. Motile vibrios show little or no movement after adherence (116). Vibrio species also possess pili (14, 197) which may be involved in adhesion. Adhesion to brush borders occurs at 37 and 220C but not at 40C and is optimum at calcium concentrations of 1 to 10 mM (114), although strontium is also effective. Cholera vibrios also agglutinate human group O erythrocytes (115). This hemagglutination, as well as intestinal adherence, is inhibited by Lfucose and D-mannose. Nelson et al. (155) observed more rapid and uniform adhesion of V. cholerae to infant rabbit ileal loops than to adult rabbit ileal loops. Colonization and clearance of the organisms were similar in the infant and adult rabbit ileum. Enterotoxigenic strains of E. coli adhere to intestinal epithelium and agglutinate guinea pig and human type 0 erythrocytes (15, 50, 94). Certain protein K antigens on the surface of animal isolates are important for adhesion (77, 116, 117, 146, 162). A fimbrial antigen demonstrated in certain diarrheagenic human strains acts as a colonization factor (50, 56, 84). In contrast to V. cholerae, adhesion is not temperature dependent (77) and is mannose resistant (50). K88, the adhesin of E. coli strains pathogenic for pigs, and K99, found on isolates from calves and lambs, have been isolated and purified (88, 117, 146, 161). Agglutination of guinea pig erythrocytes by K8 is inhibited by mucous glycoproteins with terminal beta-D-galactosyl residues (77). Certain protein K antigens of pathogenic animal strains and colonization factor antigens of human isolates are thin, flexible pili that are coded for by transmissible plasmids (50, 56, 88, 116, 146). These are distinct from type I pili of E. coli, which are also involved in adhesion and are receptors for specific phages (14, 157). The amino acid composition of type I pili is different from that of purified K88, and adhesion by type I pili to epithelial cells and erythrocytes is inhibited in the presence of mannose (15, 177, 197). Tissue receptors for K88 in pigs are postulated by Sellwood et al. (183) to be genetically determined. Two pig phenotypes were observed, and the presence of receptors was a dominant characteristic; 65% of the offspring of several matings had K88 receptors, and littermates of particular matings were phenotypically identical. The K antigens of E. coli have recently been reviewed in detail (162). Genetic and Physiological Basis for Toxin Production V. cholerae synthesizes a protein exotoxin. Genetic studies by Vasel et al. have demonstrated that toxin production is coded for by a

594

RICHARDS AND DOUGLAS

MICROBIOL. REV.

THE TOXINS Structure

plex is noncovalently bound to four to six B (or L) subunits (136, 143). The B subunit, which is responsible for toxin binding to cell membranes, contains two cysteine residues which may form intrasubunit bridges (136). Upon storage or chemical treatment, the holotoxin may partially dissociate to form other AB complexes (136, 171) or B subunits alone. The B subunit complex consititutes the cholera toxoid or choleragenoid, which lacks toxin activity but is capable of binding to membrane sites. Choleragenoid retains the antigenic properties of the native molecule and competitively inhibits the action of the native toxin by blocking receptor sites. The A1 subunit is a poor immunogen and is not neutralized by anti-CT antisera (198). This subunit is responsible for all of the toxin activity of A, and the A2 subunit may serve only to stabilize the A subunit complex before its action on the cell (63, 92, 100, 158). Cholera holotoxin can be dissociated into A and B subunits in acid urea. Recombination of A subunits with B subunits derived from holotoxin or toxoid results in a molecule with toxicity equivalent to that of untreated toxin (65). Enterotoxic strains of E. coli synthesize two types of enterotoxins which differ both in composition and in cellular effects (51, 89, 111, 112, 185,207). ST is a protein of low molecular weight (111). Although ST is enterotoxic, its elicits a much less severe diarrheagenic response of shorter duration than the higher-molecularweight LT (207). Since early purification procedures were unable to separate the two toxins and both elicit similar responses in animal models, there has been some doubt as to whether ST is a precursor form of LT (112, 185) or a different toxin. In contrast to CT, LT is a single polypeptide chain of variable molecule weight which may exceed 100,000 when isolated from culture supernatants (39, 169, 194). When E. coli is grown at low pH in the presence of antibiotics (56) or when the toxin is treated with trypsin (61, 169), an active molecule of 20,000 molecular weight is released which has properties similar to the A1 subunit of CT (40, 55, 56, 80, 168). The physical and chemical properties of LT, ST, and CT are presented in Tables 1 and 2.

Although the physicochemical characteristics of CT have been known for some time (7, 27, 100, 133, 135, 158, 167, 198), those of E. coli LT have only recently been elucidated (28, 39, 40, 56, 66). CT, or choleragen, is composed of three subunits: Al, A2, and B. The biologically active A (or H) (143) subunit is composed of a single molecule of A, and A2, which are linked by a single disulfide bond (198). The A-subunit com-

Antigen Relatedness of the Toxins In addition to the chemical and physical similarities between CT and E. coli LT, the two toxins are antigenically related (89, 90, 104, 126). Antisera produced against choleragen and choleragenoid inhibit the stimulatory effect of CT and LT on intestinal secretion (89, 90). In addition, the peptide released by trypsin treatment

bacterial gene (201). Segregation analysis after conjugation indicates that the tox locus is linked to the locus for histidine synthesis. Finkelstein et al. (67) have reported artificially induced mutants of V. cholerae that differ from the wild type both in amount and in antigenic properties of toxin produced. Nutritional studies of toxin production and release from V. cholerae by Callahan and Richardson (18) indicate that minimal medium containing asparagine and glucose supports bacterial growth. However, if the medium is not buffered properly and acid conditions arise, toxin is produced but is not released. Maximal toxin release occurs at pH 7.5 or above. E. coli ST and LT (203) are coded for by plasmids and are often associated with the presence of adhesion antigens, drug resistance factors (91, 127), and, in some porcine strains, the production of an alpha-hemolysin (186). Although surface antigens and toxin production are independent characteristics coded for on different plasmids, LT and ST are coded for by the same plasmid or linked plasmids. Gyles (89) showed that conjugation between Ent' donor and Ent- recipient porcine strains resulted in Ent' recipients. Although some porcine strains produced only ST, no strains were found to produce LT in the absence of ST. Both LT:Stand ST-only-producing strains have been isolated from man (203). Variation in antigenicity and in the ability to synthesize LT has also been observed in E. coli (40, 45, 53, 66, 183). In vitro studies have defined various parameters for optimal toxin production. These include the presence of yeast extract in the medium (54, 154), alkaline pH (154), and growth of the organisms in the presence of mitomycin C (110, 154) or protein synthesis inhibitors such as lincomycin (141) and polymyxin B (55). Thus, virulence by E. coli and V. cholerae is dependent on both genetic and physiological factors. These must be considered in the diagnosis and study of the pathogenesis of these diseases as well as the characteristics of their toxins.


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595

TABLE 1. Physicochemical characteristics of E. coli and V. cholerae toxins Detennination

Toxin'

E. coli LT E. coli ST CT

Approx mol wt of

Sedimentation

whole toxin

(swm..)

(E,,)

poect

5.4

11.6

6.9

5.54

11.4

6.6

100,000 1,000-10,000 80,000-98,000

Extinction coeffi-

coefficient

cient

I

point

Approx mol wt of subunits 20,000-35,000h 27,000-28,000 (A)

22,000-23,000 (A1) 2,500-5,000 (A2) 11,000-14,000 (B) 54,000-68,000 (B4 [toxoid]) aE. coli LT (40, 66); E. coli ST (111, 207); CT (61, 158, 198). b A low-molecular-weight LT is isolated from culture supernatants after trypsin treatment of high-molecular-weight LT (169,180).

of LT is neutralized by anti-LT, -CT, and -choleragenoid (55). The results of similar studies on the neutralization of CT activity by anti-LT antisera are variable (48, 89, 90, 126), but reciprocal neutralization has been reported (48, 126). E. coli LT and CT have similar amino acid composition (61, 180), and the complete amino acid composition and sequence of the B subunit of CT have recently been reported (132, 133, 137). Although the amino acid sequence of LT has not been determined, it is interesting to speculate on possible areas of sequence analogy between CT and LT to explain both differences and similarities in the functional characteristics of these two proteins.

Binding Site The binding site for both LT and CT has been studied by using direct binding assays (27, 96), absorption studies (74), and a variety of inhibition models (103, 165, 200). Gm, ganglioside, an acidic glycolipid containing sialic acid and a terminal galactose (68) (Fig. 1) has been suggested to be the natural receptor for CT. The most effective inhibitor of CT is Gm, ganglioside, which is present in varying amounts in the plasma membranes of all cells examined (62,199, 200) and is particularly abundant in brain tissue. The number of binding sites for CT on various cell types from different species ranges from less than 100 on human erythrocytes to greater than 107 on guinea pig brain cells (79). However, the dissociation constants for all cells reported were approximately 10-' M. CT can be effectively absorbed by various tissues, including brain and intestinal epithelium (69, 200). Indeed, the amount of bound toxin roughly correlates with the amount of ganglioside in the membrane (74, 102). Binding of CT to Gm,-coated test tubes detects CT in amounts less than 1 ng/ml (96, 104), and Gm,, which has

in the presence of antibiotics (55) and

been covalently attached to agarose beads, has been used to extract CT from solutions when present at concentrations as low as 10-" M (27). In addition, Gm, is capable of neutralizing the reactivity of CT in both in vivo and in vitro assays (96,124) when incubated before the assay. Further evidence for Gm, as the receptor for the B subunit of choleragen and its toxoid has been provided by King et al. (125) and Moss et al. (149). Preincubation of pigeon erythrocyte membranes with Gm, resulted in greater binding of CT (63, 125). Transformed mouse fibroblasts with no detectable Gm1 ganglioside in their membranes, as assayed by thin-layer chromatography, do not respond to CT. Incubation of cells with [3H]Gm, induced sensitivity to CT with corresponding increases in cAMP levels within the cells (69, 149). The fact that Gm, spontaneously incorporates into biological membranes and has a high affinity for these toxins, causing an increase in binding of the toxins to modified membranes, cannot be interpreted as definitive evidence that Gm1 is the natural membrane receptor on these cells. The possibility that CT binds to other gangliosides or glycoproteins with similar carbohydrate moieties or that Gm, is only one constituent of the natural receptor cannot be excluded. Although LT binding to intestinal tissue can be inhibited with Gm,, the receptor for E. coli LT may not be the same as for CT (19, 96, 165). Preincubation of segments of rabbit intestine with choleragenoid does not significantly decrease the secretory response to LT, and LT has a lower affinity for Gm1 than CT (96,165). These observations may be explained by differences in affinity for the same receptor or the existence of two closely related receptors. Craig and Cuatrecasas (21) have reported that rat lymphocytes can cap fluorescein-labeled CT. This indicates a multivalent binding of the toxin molecule to membrane receptor.

596

n~ ~GalNAc,


GM,:

.0 rEE Iz

GM2: GM3:

NAN

CER-G1c-Gal-GalNAc NAN CER-Glc-Gal

NAN

+ + +

GD1b:

+I

+ + t , * Z.D

GT1:

CER-G1c-Ga1-Ga1NAc-Ga1 NAN-NAN

CER-G1c-Ga1-Ga1NAc-Ga1

NAN-NAN NAN FIG. 1. General structure of selected gangliosides (68). Abbreviations: CER, ceramide; Gal, galactose; N-acetylgalactosgamine; Glc, glucose; NAN, N-acetylneuraminic acid.

Action of Toxins on Adenylate Cyclase Both LT and CT elicit a dose-dependent activation of the membrane-bound enzyme adenylate cyclase (EC 4.6.1.1), which converts aden+ osine 5-triphosphate (ATP) to cAMP (9, 10, 52, 0 176). CT increases basal activity in both the $4; > + ileum and jejunum and enhances prostaglandin, but not sodium fluoride, activation in these tissues (184). Studies of CT activation of adenylate + + cyclase in erythrocyte membranes (78) indicate w 8.> that a minimum of one molecule per cell is sufficient to achieve significant activation over base-line levels. Both toxins are presumed to :+ 1+ activate adenylate cyclase at the same binding site, since cyclase activity is raised to approximately the same level and neither is enhanced z beyond a maximal level by the subsequent addition of the other toxin of sodium fluoride. D ts Adenylate cyclase activation by CT or LT is distinct from that caused by epinephrine, prostaglandins, glucagon (10), catecholamines, or adrenocorticotropin (ACTH) (206). The addition of CT or LT potentiates hormonal stimulation -;_ _ in most cell systems. However, there are differences in the kinetics of the elevation of cAMP elicited by the two toxins. In whole-cell studies, CT has its maximal effect after 1 to 3 h, and cAMP levels remain elevated for up to 12 h after X Xremoval of the toxin by washing. In contrast, LT v4 001> exhibits a maximal effect in 15 to 90 min, and e _ cAMP elevation is reversed 30 min after washing. Further, intestinal secretion after LT treatment is substantially less than that observed after CT exposure (66). The sequence of events after toxin binding to a membrane reE_ _ E_ Hensuing ; cm v ceptor, and during the activation of adenylate O

bD

0

X < E

CER-GLc--Ga1-Ga1NAc-Ga1

X

>

cli

MICROBIOL. REV.

~+ I+ +

I

+

VOL. 42, 1978


cyclase, is largely unknown. In studies utilizing '251-labeled CT, Holmgren and Lonnroth (101) have shown that the toxin does not accumulate in large quantities within the cytoplasm of the cell, but remains associated with the cell membrane. Therefore it is probable that events leading to adenylate cyclase activation occur at or within the plasma membrane.

Solubilized Adenylate Cyclase and Toxins Recent studies on the specific events leading to the activation of adenylate cyclase have been done with whole-cell particulate fractions, purified membranes, and solubilized enzyme from liver cells in the absence of cytoplasmic components (8, 10, 70, 151, 176). Bennet et al. (8) reported the stability of solubilized rat liver membrane adenylate cyclase activity after CT exposure. Activation of adenylate cyclase in membrane fractions of isolated fat cells by holotoxin occurred after a 25-min lag phase in an ATP-generating system containing ATP, guanosine 5'-triphosphate (GTP), and magnesium (176). Toxin-adenylate cyclase complexes formed by CT during incubation of whole cells or membrane fractions remained complexed after solubilization by nonionic detergents, and cyclase activity was precipitated from solution by antisera to the CT and the A subunit (8, 176). Activated cyclase present in solubilized membrane preparations could be neutralized by antitoxin and anti-A antisera. The possible existence of two classes of toxin receptors, some of which act on adenylate cyclase, was excluded by blocking with the toxoid. A large number of independent and equivalent receptors for toxin were proposed which must come into favorable orientation after binding of toxin.

Cofactors Required for Toxin Activity When broken-cell preparations (78,80,81) are used to study toxin activity, there is no lag period between binding of LT or CT and adenylate cyclase activation. Significant cyclase activation occurs within 1 min in erythrocyte ghosts (80) in the presence of CT. Gill (78) showed that activation requires the presence of cofactors-nicotinamide adenine dinucleotide (NAD), ATP, and a protein from the cell cytoplasm. In addition, reducing agents such as glutathione or dithiothreitol are necessary for CT activation, possibly to release the A1 subunit from the toxin molecule. Similar enhanced activity occurs in the presence of sodium dodecyl sulfate. NAD has been shown to be hydrolyzed by CT or its A subunit to adenosine 5'-diphosphate (ADP)-ribose and nicotinamide followed by the

597

ribosylation of arginine (152, 196). Recent studies have shown the ADP-ribose to be bound to the A subunit after the hydrolysis. It is not known whether the activation of adenylate cyclase is due to its direct interaction with the ribosylated A subunit or whether an acceptor protein is ribosylated and causes enzyme activation. The A subunit would then act as a transfer protein (150). Studies by Gill et al. (80) indicate that NAD and ATP are also necessary for LT activation of adenylate cyclase in erythrocyte ghosts. Adenylate cyclase activation is viewed as occurring in three stages (206). The first, or preparatory phase, involves the unbound toxin in the presence of a reducing agent and NAD and may involve the formation of toxin-NAD complexes. The second, or dissociation phase, includes the binding of the toxin to a membrane site and the dissociation of an active subunit from the complex. During the third, or activation phase, NAD is utilized during the activation of adenylate cyclase.

Involvement of Guanosine 5'Monophosphate (GMP) The requirements for the complete activation of solubilized adenylate cyclase from brain by CT requires cAMP and a protein activator of cyclic nucleotide phosphodiesterase (151). This preparation was seen to partially replace the need for cell supernatants. Direct involvement of GTP and guanosine 5'-diphosphate as primary regulators of the activation of adenylate cyclase has been suggested by studies utilizing neuroblastoma cells and their isolated membranes (140). The activation of the enzyme by CT or hormones in the presence of nucleotide triphosphates, NAD, and magnesium closely resembles stimulation by guanyl-5'-yl imidodiphosphate. Activation by CT in the presence of 10 mM magnesium requires the presence of guanine nucleotide triphosphates which may be essential for regulation of activity by fixing the enzyme in an active state. Quantitation of cyclic GMP (cGMP)/cAMP ratios suggests that a primary cGMP elevation occurs before a cAMP elevation (139). The formation of GTP-enzyme complexes may represent an event that occurs during the lag phase characteristic of both LT and CT. It is pertinent to note here that Hughes et al. (107) have shown increases in cGMP by ST in rabbit intestine without a rise in cAMP levels. Furthermore, 8-bromo-5'-GMP causes fluid secretion comparable to that due to ST. These data support the hypothesis that LT and ST are distinctly different toxins with different mechanisms of action and that cyclic nucleotides

598


are important in the regulation of intestinal se-

cretion. Effect of Increased Intracellular Cyclic Adenosine 3',5'-Monophosphate There are a number of secondary cellular effects that are the result of increased intracellular cAMP. Of these, increased intestinal secretion is a primary factor in the pathogenesis due to these organisms. Water and electrolyte secretion and cyclic nucleotides have been reviewed by Strewler and Orloff (190) and Field (58, 59). Under normal conditions, there is low spontaneous transmembrane electrical potential in the intestinal epithelium which is believed to account for its high passive permeability. Unidirectional lumen-to-blood fluxes of sodium and chloride occur and are in part balanced by absorption in the opposite direction, resulting in a slight net flux toward the serosa. Movement of ions, primarily bicarbonate, occurs toward the lumen. Elevation of intracellular cAMP by the addition of theophylline causes a transient increase in the electrical potential of the membrane and decreased absorption of sodium leading to a net secretion. In addition, the direction of chloride transport reverses. These events result in an efflux of water by osmosis. The action of CT and LT on intestinal epithelium is similar (58, 59) to the effect of theophylline, and net sodium secretion has been reported (31). Both the brush border and crypt cells are involved in secretion after exposure to CT (29). There is no evidence for gross mechanical damage to the epithelium during the secretory phase in vivo, although the presence of small lesions in the plasma membrane of the brush border and mitochondrial swelling has been reported (164). Trivalent cations have been shown to inhibit toxin-induced intestinal secretion in rabbits (139). Pretreatment with lanthanum chloride resulted in a twofold reduction in secretion due to CT and a 10fold reduction after LT exposure. This effect is postulated to be due to the inhibition of a protein kinase system, resulting in a rise in cGMP (139) and subsequent cAMP elevation after adenylate cyclase activation (139, 144). DeLorenzo et al. (30) reported the decrease in a phosphorylated protein toad bladder membrane in response to antidiuretic hormone or cAMP treatment. The decrease in this protein was correlated with increased sodium transport and water permeability, and they postulated the inhibition of a protein kinase or the stimulation of protein phosphatases in the membrane. Whether these observations are related to the ribosylation of membrane components by CT remains to be elucidated.

MICROBIOL. REV.

Variability in Activation of Adenylate Cyclase Differences in the kinetics of adenylate cyclase activation and cAMP production after exposure to CT and LT have been observed. These differences may be due to a number of factors, both in the microorganisms and in the host, and include genetic variability in the nature or amount of toxin produced by a particular organism, differences in the binding of the toxins to their receptors, in the receptors with age of the host (46, 120), or in modulation of the intracellular effects after toxin binding (45).

Similarities to Glycoprotein Hormones and Other Bacterial Toxins The functional and structural properties of CT are similar to those of many glycoprotein hormones in their subunit structure, membrane binding site, and adenylate cyclase activation (33, 137, 138). Computer comparison of partial and complete sequences of CT and four glycoprotein hormones (thyrotropin [TSH], luteinizing hormone [LH], human chorionic gonadotropin [HCG], and follicle-stimulating hormone [FSH]) has revealed that a short segment of the B subunit of CT was analogous to a similar segment of TSH, LH, HCG, and FSH (Table 3). Quantitative measurement of similarities indicates significant analogy between CT, TSH, LH, and HCG, but not FSH. Structural analogy also exists between the A, subunit of CT and alpha subunits of these same hormones (138). The evolutionary significance of these findings is of great interest. The similarities in binding subunit structure, membrane receptors, and the activation of adenylate cyclase by proteins from such biologically unrelated groups suggest possible evolutionary convergence in both structure and function and are not without precedent. For example, the active sites of chymotrypsin and subtilisin are comparable to each other in composition and function. The similarity between the subunit structure of CT and other bacterial and plant toxins has also been noted (12, 160, 179, 191). Abrin, ricin, ricinus agglutinin, and botulinal and diphtheria toxins are all comprised of separate binding and enzymatic subunits. Moss et al. (150) have suggested similar functional similarities between cholera and diphtheria toxins. Both exhibit nicotinamide adenine dinucleotidase activity and cause ADP-ribosylation of an acceptor protein. In addition, gangliosides constitute part of the membrane receptors for the glycoprotein hormones, tetanus toxin, botulinal toxins, serotonin, and interferon. The similarity between LT, CT (68), and other


VOL. 42, 1978

599

biologically active molecules offers the molecu-

lar biologist a potent probe into the mechanisms of hormone action and other cAMP-mediated events in mammalian cells (7, 119).

_

>

Vaccines and Immunity The severity of clinical cholera and the infecco 0 !0 w w $tions by enterotoxigenic strains of E. coli in both man and domestic animals has led to a search for effective vaccines. Knowledge of the structure and functional properties of the toxins and % virulence factors of the organisms together with information gained from a variety of animal models has led to a varied approach to clinical vaccination programs. The use of killed vibrios as immunizing agents against cholera in man has met with limited success, primarily due to the short duration of o protection (61). However, V. cholerae L-fonns .I and b-fonn lysates do elicit protective immune responses in rabbits (2-4). Oral administration ofL-forms of V. cholerae results in the successful immunization of rabbits (2). L-form lysates also X 0000U cz U cz induce an immune response when given parenterally, and agglutinating as well as vibriocidal .g _ Et E tE$EW activites are present in these anximals for many '4... 3 o O O c: weeks. Oral administration of L-form lysates in .t c ~rabbits and humans (3) results in the production of copro and serum antibodies to homologous and heterologous strains and, in conjunction with alcohol- or phenol-precipitated ribonucleic acid (RNA) from lysates, induces enhanced cellular immunity in rabbits. Delayed hypersensitivity to cholera lysate antigens, the inhibition I I,,,, of migration of leukocytes and macrophages in the presence of lysate antigens, and macrophage X , agglutination have been reported (4). Crude flagellar preparations of vibrios as well as purified I , flagella also induce immunity in mice which can be passively transferred to suckling mice (47, 86). Vaccination with V. cholerae leads to the production of coproantibodies of the immunoi' , globulin G (IgG) and IgA classes which inhibit X6 < Q w @ @ tbacterial adhesion (71) and motility (86) and are vibriocidal within the intestine (1, 71, 175). Cytophilic IgG and on the surface of macroq E phages (1) and IgM opsonins are also produced. These antibodies are directed toward cell wall constituents of the organisms (47, 71, 86), which m000g are associated with the flagella or pili (47). Various preparations of cholera toxoid have been used in both aniInal and human studies (16). Fornalin- and glutaraldehyde-treated tox°o g s g | :> oids, heat-aggregated toxoid, and toxoid subunit preparations have been used in animals (60, 106, E-4 166) and humans (64, 75) with variable results. =-:c 8= U FE- 5x Formalin-treated toxoid has been demonstrated a

600


to partially revert to a toxic product upon storage (106). Glutaraldehyde-treated toxin does not revert to active toxin, but leads to a poor immune response (75, 106). Aggregated, purified B subunits have been used as an immunogen and found to be very effective in protecting against whole-cell challenge as well as toxin challenge in rabbits (106). Parenteral immunization using B-subunit toxoid results in increased titers of serum antitoxin antibodies and protection against intraintestinal challenge in intestinal loops in the absence of coproantibodies (60). Toxoid-lipopolysaccharide (LPS) preparations gave the best protection after challenge in humans (61) and rabbits (193). Killed whole-cell vaccine plus B-subunit toxoid was also effective in clinical studies using human volunteers (75). These combinations do not lead to serum levels of antivibrio antibodies higher than with either antigen alone; thus other mechanisms which interfere with bacterial adhesion, toxin binding, or both are important. Vaccination with combined preparations gives longerlasting immunity than with any of the conventional vaccines (106). Both enteral and parenteral immunization of rabbits with CT and toxoid protects against subsequent challenge with toxin and live vibrios (105). Intestinal IgG and IgA levels are increased by enteral and, to a lesser extent, parenteral immunization. Serum IgG levels are elevated only after parenteral

immunization. Immunization of rats with CT by combined intraperitoneal and oral immunization results in increased serum IgG but not IgA (214). Eighty percent of intestinal antibody activity is IgGl. Furthermore, binding of radiolabeled CT to intestinal microvilli and intestinal secretion was depressed in immunized animals. Although the role of specific antibody classes in immunity to cholera was not addressed by these investigations, antitoxin antibodies of the IgG class appeared to be involved in protection at this site. Infection by enterotoxigenic strains of E. coli results in the production of serum anti-LT antibodies in animals (174) and man (57, 175, 204). However, the correlation between rises in antitoxin levels and protection is not clear. Anti-LT titers vary and are dependent on prior infections, severity of disease, and enhancement due to exposure to V. cholerae and its antigenically related toxin (204). Prior immunization of rats with CT or cholera toxoid protects against both LT and CT challenge (166). Quantitative measurement of neutralizing antibody titers in human milk indicates that enteral exposure to both enterotoxigenic E. coli and V. cholerae can induce the production of secretory IgA and IgG in

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humans, which may be protective in nursing infants (97, 189). These studies have shown that not only is the immunization agent important in protection, but the route of immunization may also play a role in the level of protection obtained. Enteral and parenteral administration of vaccines have both given protection in animals; however, the mechanisms of protection may be different. Parenteral administration leads to higher serum antibody levels and in vitro vibriocidal activity, but may not enhance local defense in the intestine or prevent bacterial adherence or action of toxins on epithelial cells (204). Multiple enteral administration of bacterial vaccines in combination with toxoid preparations may yield the most potent immunization schedule.

ENTEROTOXINS IN IN VIVO SYSTEMS Whole-Animal Models Whole-animal and intestinal loop models were used extensively in the early work to assay for both CT and E. coli toxins (17, 76). These assays provided the first information on the physiological effects and mechanisms of action of these toxins. The infant rabbit was used in many studies where bacterial suspensions or crude culture filtrates were given orally. Other animal systems used in similar studies included dogs, cats, rodents, pigs, and chickens. Transmural injection of E. coli ST into the stomach of suckling mice leads to intestinal fluid accumulation; this has been used as an assay for ST (76). The obvious economic limitations of whole-animal studies, as well as difficulty in the quantitation of results, led to the development of organ system assays. Of these, the ileal loop and skin permeability systems are discussed. Ileal Loop Assay The rabbit ileal loop assays, originally used in the early 1950s (64), involves the introduction of the material to be tested (bacterial suspension, culture filtrates, toxins, or toxin subunits) into ligated segments of the ileum of adult rabbits or swine (147). Examination of the intestinal tissue at the light or electron microscopic level revealed intestinal morphology resembling the clinical disease. The focal lesions observed were judged to be caused by increased hydrostatic pressure from the lumen, and the goblet cells actively discharged mucus. There was no evidence for direct damage to epithelial cells due to toxin exposure. After an appropriate time interval, usually 18 h, separate ileal segments were excised, and both the length of the segment and the fluid accumulated within it were measured

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(17, 49, 53, 105, 166). Early studies using viable microorganisms (17) revealed that the secretory response was the result of toxemia rather than bacteremia. As few as 10 viable organisms led to infection and secretion within hours after inoculation. Fluid secretion in response to ST is immediate and maximal within 6 h irrespective of dose, although the net secretion is less than with LT (49). Onset of secretion after exposure to LT is dose dependent, reaching maximal levels later than for ST. Higher doses of LT led to sustained fluid secretion, whereas low doses did not. These findings paralleled those observed after CT exposure.

Preincubation of CT with antitoxin results in reduction of the secretory response and correlates with the amount of antiserum used (96). Gm, inhibits the response to CT, and 100 times less ganglioside is required to inhibit the CT response than the LT response. Pretreatment with choleragenoid blocks the CT but not LT response in the loop assay. Prior immunization of rabbits with culture filtrate from toxigenic strains of E. coli (174) and with CT (105) results in an increased resistance to oral challenge with homologous bacterial suspensions and crude enterotoxin. Immunization of rats with cholera toxoid gives equal protection against CT and LT (166). a

Skin Permeability Assay Studies on the action of culture filtrates of V. cholerae after intradermal inoculation demonstrates that a heat-labile component causes erythema, edema, and induration at the site of injection (11, 20, 48, 54). This procedure has been modified to include a later intravenous dose of a protein-binding dye, such as Evan's blue, which enters the indurated area. The active component was called "skin toxin," vascular permeability factor, or PF (61-63). It was thought to be identical to or associated with CT. Several investigators showed that the two activities were co-isolated (associated) at various stages of purification and were neutralized in the presence of antitoxin antisera. However, treatment of CT at low pH and subsequent neutralization was shown to selectively destroy skin permeability activity without loss of its diarrheagenic or toxic activity in animal assays (11). Vascular permeability factor has been isolated from enterotoxigenic strains of E. coli (48, 54), and the culture conditions during bacterial growth appear to be important in its synthesis (54). Yeast extract and aeration were found to be necessary for synthesis, and media commonly used to promote LT production did not always

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yield PF activity. When PF is synthesized, it is always found to be associated with enterotoxin. The assay system itself was also found to be a factor in the detection of PF activity. Older, larger rabbits used in this assay were found to be less reactive to PF than younger animals. In addition, the characteristics of bluing after E. coli PF exposure are different from those for V. cholerae PF exposure (66). Bluing is less intense and is surrounded by an area of edema without bluing. Purification of E. coli PF on agarose A5M and polyacrylamide P-150 columns led to the isolation of two factors: a blanching factor, which is heat stable, and a bluing factor, which is heat labile and elutes slightly behind blanching factor on P-150. Histological examination of areas of blanching revealed early eosinophilic, histiocyte infiltration and dermal thickening, leading to liquefication and necrosis involving hair follicles and blood vessels. This reaction continued to increase after 72 h without any indication of resolution. The area of bluing was shown to consist of a mixed infiltrate at 18 h, leading to mononuclear infiltrate at 24 h containing eosinophilic proteinaceous edema which began to resolve at 72 h. The possibility that blanching factor contains LPS and that blanching was incidental to the bluing effect of PF cannot be excluded. Neutralization studies (48) showed that PF activity of both V. cholerae and E. coli is inhibited by homologous and heterologous antisera. This finding is in contrast to the results of neutralization studies on the toxigenic effects of the two toxins in intestinal models. PF activity of both toxins gave equivalent results in rabbit skin.

ENTEROTOXINS IN IN VITRO SYSTEMS Various in vitro cell systems have been used to quantitate the biological activity of both E. coli LT and CT. These systems have also yielded information regarding the mechanisms of action of these toxins and have provided valuable tools for the study of many aspects of eucaryotic cell growth and development.

Erythrocyte Ghosts The use of erythrocyte ghosts to determine the cofactor requirements for cyclase activation has already been mentioned. Additional information has been obtained with this system to study membrane receptors and the role of Gm, (125). Pretreatment of pigeon erythrocyte membranes with purified Gm, results in increased binding of CT and increased activation of adenylate cyclase. Correlation of the amount of toxin bound with intracellular cAMP levels in-

602 RICHARDS AND DOUGLAS dicates that more receptors are present than are necessary for maximal stimulation ofthe enzyme in both treated and untreated cells. In addition, the membrane receptor may be more complex than Gm, alone. Binding after Gm, incorporation was greater than expected, possibly due to the formation of complexes containing Gm, and other membrane components, which have a greater affinity for CT than does Gm, alone. The direct interaction of CT with adenylate cyclase in the presence of NAD and dithiothreitol or the A1 subunit in the presence of NAD alone indicates a direct membrane interaction by the active subunit (212). The need for cytoplasmic components reported in other studies may be supplied by a membrane-associated factor, such as GTP, in these studies. The greatly reduced lag period using erythrocyte membranes and dissociated subunits emphasizes the conclusion that the lag period involves events related to proper binding, orientation, and dissociation of the subunits at the cell surface. Further evidence comes from the use of the A1 subunit in a 30-fold molar excess over holotoxin with intact cells, which can activate adenylate cyclase after a longer lag period. These results indicate the role of the B and A2 subunits for "focusing" toxin on the membrane surface, stabilization during binding, and facilitating possible membrane insertion (212). Adrenal Cells The Y1 cell line, which is responsive to LT, CT, and ACTH derived from mouse adrenal cortex, and the OS-3 mutant, which is unresponsive to ACTH, have been used to study these toxins (32, 33, 35, 38, 129, 134, 173, 209-211, 213, 215). Y1 cells, when grown as confluent monolayers, undergo morphological and metabolic changes after exposure to ACTH, cAMP, CT, or LT. The cells round up and detach from the substrate and secrete increased amounts of delta-4,3-ketosteroids, which can be quantitated

spectrophotometrically or fluorometrically.

These changes correlate with increased levels of intracellular cAMP (35). Rounding up is more sensitive to changes in cAMP and requires only 10% of the increase necessary for elevated steroidogenesis (211). The effects of ACTH are different from those of toxin treatment with respect to the nature of the binding sites, kinetics of the response, and ion requirement. There are also differences in the effect of inhibitors in this system when compared with in vivo systems. The response of Y1 to ACTH is rapid: only a few minutes are required for cell rounding. A lag period of 1 to 3 h is required to obtain the same response with LT or CT (35-37, 135). The re-

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sponse to CT is specific, can be saturated, and reaches plateau levels at approximately 1 ng/ml. Two or more hours are required before increases of steroid production above base-line levels are observed. Increases in steroidogenesis are related to the concentration of the toxin used (35). The duration of the response to CT is longer than for ACTH and can last as long as 72 h. CT subunit A induces changes similar to those induced by whole toxin (34). The morphological changes after addition of cAMP commence after a lag similar to that found with CT but revert to normal after 24 h. ACTH is thought to bind to membrane receptors different from the toxins. OS-3 cells, responsive to ACTH by both morphological and metabolic criteria, respond to LT, CT, and cAMP (32, 211). The response in these cells is also characterized by a lag period. Further, half-maximal stimulating doses of ACTH and CT are additive in Y1 cells. Pretreatment with maximally stimulating doses of CT potentiates the subsequent response to ACTH. In addition, calcium is required for the action of ACTH, but not for the action of either toxin (211). The lag period after toxin exposure is not due to differences in the rate of binding of the toxins to the cell surface. Maximal stimulation is observed after a 10-min incubation followed by extensive washing. Preincubation of the cells with toxoid neutralizes binding of the toxin, and subsequent stimulation and preincubation of the cells with Gm, ganglioside increases binding of the toxin (38). Pretreatment of the toxin with either antitoxin or antitoxoid neutralizes the response, and pretreatment of the toxin with levels of Gm, as low as 1:1 molar ratios has the same effect (210). Exposure of the cells to antitoxin during the lag period partially inhibits the response by these cells (211); addition of antitoxin 15 min after toxin exposure significantly decreases both morphological and steroidogenic responses. Addition of antitoxin as late as 2 h after toxin results in as much as 20% decrease in response (210). The results from studies using adrenal cells indicate many characteristics similar to those of in vivo studies usingrwhole animals or intestinal loops. The characteristic lag period is seen for both LT and CT. These toxins probably bind to the same receptor site, Gm,, which is distinct from that for hormones by several criteria, even though both have similar effects on cells. The final response can be inhibited to varying degrees by the addition of antitoxin to the system during the lag period, even though binding is complete during the first 10 min. This inhibition may be related to a reversible step in the interaction of the toxin with membrane components

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and may in part account for the lag period. If so, the lag period may include dissociation of the toxin complex, toxin processing, and/or interaction with a membrane protein intermediate or the adenylate cyclase itself. In addition, the toxins appear to interact with or in some way stimulate adenylate cyclase molecules distinct from those which are acted upon by ACTH, since toxin and ACTH potentiate each other's effects. If cyclase molecules sensitive to the action of these toxins represent a subset of total cyclase within the membranes, the proper orientation of the toxin with an appropriate cyclase molecule may also occur during the lag period.

Isolated Fat Cells Isolated fat cells are obtained from rat epididymal fat pads by digestion of the tissue with collagenase and cell flotation. These cells are similar to adrenal cells in their response (23-26, 52, 119, 144, 148, 172, 202, 208). They can be stimulated by a variety of hormones and drugs to release glycerol and free fatty acids into the medium with the simultaneous increase in intracellular cAMP. The magnitude of the response is measured by glycerol release into the medium and is quantitated spectrophotometrically (172, 208). ACTH, TSH, glucagon, epinephrine, and isoproterenol elicit this response, as do theophylline, cAMP, dibutyryl cAMP, CT, and LT (93). Lipolysis with or without stimulation can be inhibited in the presence of insulin or prostaglandin El. Lipolysis after, hormone stimulation, but not toxin exposure, requires the presence of calcium, magnesium, and potassium (202). Fat cells incubated with honnones and catecholamines show immediate increases in intracellular cAMP. Increases after CT exposure were delayed for 1 to 3 h and for shorter periods after LT treatment (93, 144). Addition of theophylline shortened the lag period and also resulted in higher cAMP levels. Significant activation of adenylate cyclase without a lag occurs when intact cells are exposed to the A subunit of cholera toxin (176). Exposure to cholera toxin did not cause a potentiation of cAMP accumulation in response to isoproterenol in intact cells. The response to CT is dose dependent and can be saturated (202), and its binding occurs rapidly and tightly. Dissociation constants of 10-`0 M have been reported (23). Glycerol release after LT exposure occurred after 5 min, whereas glycerol release after CT took 1 h. The response lasted several hours after stimulation with either toxin, but could be reversed in LT-treated cells after washing (93). Prior incubation of CT with mixed gangliosides but not the hormones inhibits both binding

603

and the lipolytic response (25, 26). Fetuin, a glycoprotein, also inhibits binding. The inhibition is less effective with increasing time due to the dissociation of the toxin-inhibitor complex. Prior incubation with Gm, was also inhibitory, and reversal of inhibition was not seen due to the high affinity of the ganglioside for the toxin. Lipolysis is also neutralized in the presence of antitoxin. The natural receptor for LT and CT on all cells studied has been concluded to be Gm1, based on neutralization and inhibition studies. However, quantitation of the components of fat cell membranes has shown that they contain no detectable Gm,; the major gangliosides were found to be Gmn. and Gm2. The general structures of these gangliosides are compared with that of Gm, in Fig. 1. Although exogenously added Gm, was seen to be incorporated into adipocyte membranes, the binding of CT by these modified membranes may be nonspecific, since these membranes did not show saturation kinetics. In addition, although Gm, incorporation increased the rate of lipolysis, it did not increase the maximum level of stimulation or lead to responses to substimulating doses of toxin. Exogenously added Gm, may, therefore, serve only to bind toxin to the membrane on a nonspecific manner and not represent the true membrane receptor for toxin on these particular cell types (118). Fat cells can be metabolically stimulated in the presence of enterotoxins, and this stimulation is related to an increase in cAMP (93, 144). The toxins exhibit a lag period of 1 h or more, and the response can be inhibited in the presence of antitoxins, or antitoxoid or by pretreatment of the toxin with Gm, ganglioside.

Lymphocyte and Lymphoid Cell Lines and the Immune Response Lymphocyte and lymphoid cell lines are responsive to CT, and these cells have been utilized in studies of toxin action. CT localization has been investigated by using rat lymphocytes incubated with fluoresceinated toxin (21, 22, 170, 182). The observation that diffuse localization at 0°C progresses to a cap at one pole of the cell after warming to 37°C (21) is evidence for ligandtriggered plasma membrane receptor mobility. Moreover, several types of evidence suggest that ligands must be bivalent to induce capping. The demonstration that choleragen-treated lymphocytes bind to Gm, agarose beads also suggests that CT can bind to at least two receptors (22). Similar studies by Sedlacek et al. (182) using a fluorescent-labeled synthetic sialoglycolipid (dansyl ganglioside) demonstrated that this dansyl label binds to protein I of CT (B subunit) and to the lymphocyte membrane. Ligand-in-

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duced mobility was shown to be temperature dependent and to co-cap with lymphocyte antiimmunoglobulin receptors (182). Douglas et al. (42) and Zuckerman and Douglas (217, 218) have studied membrane receptors and the freeze-fracture topography of a murine lymphoblastoid cell line treated with CT. These studies have demonstrated that CT inhibits the receptor for the Fc portion of IgG and also alters the distribution of intramembrane particles. Treatment of two lymphoid cell lines with CT for up to 24 h resulted in significant increases in intracellular cAMP and mitochondrial swelling. Similar treatment of mononuclear phagocytes resulted in an increase in cAMP levels without morphological changes in mitochondria (43). Further increases in cAMP levels by incubation of toxintreated mononuclear phagocytes with aminophylline had no effect on cellular morphology. CT and E. coli LT bind to and produce a delayed activation of adenylate cyclase in isolated mouse thymocytes (216). Both toxins bind rapidly and tightly to these cells at 37°C, but CT was seen to have a higher affiniy than LT at 8°C. CT also inhibits the synthesis of DNA, RNA, and protein by isolated mouse spleen cells and thymocytes cultured with concanavalin A, LPS, or phytohemagglutinin (98, 99, 192, 205) without affecting viability. Inhibition was correlated with cAMP levels and paralleled the concentration of toxin (98), and doses that were submaximal for cyclase activation completely inhibited DNA synthesis. The effect of CT on antibody production in both primary and secondary responses was variable and dependent on the time of administration relative to antigen exposure. Increased numbers of plaque-forming cells were detected when the toxin was given simultaneously with antigen, and plaque-forming cells were reduced in number if given after antigen administration. Both spleen and thymus weights were reduced in mice given intravenous injections of CT. This effect was related to the effect of CT on the adrenal glands. In vitro studies have shown a reduction of both immediate and delayed hypersensitivity as judged by histamine release from leukocytes and cell-mediated lympholysis (142). Cytotoxic activity of toxin-treated cells both in vivo and in vitro prolong allograft survival in CT-treated animals (98,142). Lymphocytes from CT-treated donors are more able to produce a graft-versushost reaction and the "allogenic effect" in recipient mice, possibly by inhibiting a suppressor population (98). Theta-antigen co-caps with choleragen on mouse thymocytes (195), but bound toxin does not interfere with the ability of these cells to be labeled with anti-Thy 1.2 or with cytolysis by toxin-coated cells. Thus, al-

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though the toxin receptor undergoes redistribution with Thy 1.2, the association with theta is indirect. Fibroblasts The effect of CT on Chinese hamster ovary (CHO) cells and other fibroblasts in culture is to increase collagen synthesis (87), cell elongation, and adhesion to a substrate (113, 156) and to elevate cAMP (144). This effect is also seen after treatment with cAMP, dibutyryl cAMP, and theophylline. Increased adhesion occurred after a 60-min lag and was related to concentration of CT. The mechanism by which CT enhances substrate adhesion by CHO cells may be due to an effect on microtubules and microfilaments in CHO cells. The effect seen in fibroblasts is the reverse of that observed with adrenal cells in culture, which emphasizes the pleiotropic effect of cAMP in various cell types. Normal fibroblasts exhibit characteristic growth patterns that are absent or altered in transformed cells, which may be due to the disorganization of cAMPdependent cytoskeletal elements found in eucaryotic cells. Increased cAMP levels have other effects on fibroblasts in culture (130, 131, 168). Transport of nucleotides, amino acids, and protein synthesis is inhibited. Stimulation of RNA and DNA synthesis by epidermal growth factor is inhibited by CT, dibutyryl cAMP, and theophylline (95); cGMP, cytochalasin B, colcemid, and vinblastine reverse this inhibition. Other Cell Systems CT and LT have been shown to alter morphology and metabolism in a variety of other cell types. CT enhances the binding of TSH to thyroid cells at low concentrations and inhibits binding at high concentrations (153). Both agents activate adenylate cyclase, but TSH does not require NAD for activation. Both agents bind to cell membranes via ganglioside receptors: TSH to GD1b, GT1, and, to a lesser extent, Gm, (Fig. 1). CT increases glycogenolysis in liver cells and platelets (83) and inhibits histamine release, microbicidal activity, and enzyme release and chemotaxis from neutrophils (13, 142). Purified LT has been demonstrated to inhibit neutrophil chemotaxis in vitro. This effect appears to be related to increased intracellular cAMP (9). If so, this effect could be of selective advantage to the organism in inhibiting neutrophil migration. Both- LT and CT cause adenylate cyclase activation in thyroid slices (145), in a human embryonic intestinal cell line (121), as well as in two epidermal carcinoma cell lines, KB and HEp-2. Neuroblastoma, Yoshita ascites hepatoma, and sarcoma 180 cells (206) are rendered unresponsive to catecholamines by CT. In addition, the

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CT response can be altered without affecting the response to epinephrine. The pathways by which activation by each occurs therefore appear to be distinct; one can be blocked without affecting the other. These studies indicate that in this cell system there may be a common catalytic event but distinct activation pathways. There are numerous cells that are sensitive to the action of both CT and E. coli LT (41, 121, 159, 187). The diverse changes in metabolism, morphology, and response to extracellular molecules such as hormones, catecholamines, and prostaglandins all follow changes in intracellular cAMP levels. These systems have been used to study the diverse functions of these extracellular substances and their relationship to cyclic nucleotides. Keusch and Donta (122) have pointed out that not all bacterial toxins exert their effect through cyclic nucleotides, and the distinction between cytotoxic and cytotonic effects should be recognized. Therefore, caution must be exercised in interpreting these results, especially when extrapolating to in vivo systems, where the observed effects may be indirect. DISCUSSION There are many common factors in the pathogenesis of diarrheal disease caused by E. coli and V. cholerae. Both gram-negative organisms possess specialized antigens for adhesion to intestinal epithelium. Both produce protein exotoxins that have similar physical and chemical characteristics and immunological cross-reactivity. These toxins rapidly bind to membrane receptors composed of or similar to Gm, ganglioside. The biological activity of these toxins is the result of interaction with membrane-bound adenylate cyclase, leading to persistent elevation in intracellular levels of cAMP. Stimulation of adenylate cyclase in intact cells always occurs after a characteristic lag phase. Hypersecretion of salts and water is the result of a change in membrane transport. Although the effect of these toxins is restricted to the intestine in vivo, many cell types are sensitive to their action when given systemically in vivo or to isolated organs and cells in vitro. The response observed in these systems may have no pathological significance but serves to underscore the central role of cyclic nucleotides in cellular regulation. Equally intriguing are the differences between these two toxins. The length and the degree of stimulation of adenylate cyclase point to possible differences in affinity, dissociation, and mechanism of activation of the cyclase molecule. There are three areas in which the study of these two toxins has left unanswered questions. Future studies must address the problem of the nature

605

of the lag period and membrane events that occur before and during adenylate cyclase activation. Further, the structural and evolutionary relatedness of the two toxins to themselves, other toxins, and hormones has not been clarified. In addition, the role these molecules may play in the life cycle of the organisms themselves has not been examined. It seems improbable that events such as dissociation and diffusion across the lipid bilayer can fully account for the length of lag observed in the cell systems discussed here. More subtle events at or within the membrane must be evoked and may include complex associations of toxin with membrane lipid and protein components. Although there is evidence of direct interaction of toxin with adenylate cyclase, the existence of accessory or intermediate membrane molecules has not been ruled out. The significance of the similarities between the two proteins has yet to be determined. The isolation of purified LT and complete amino acid sequence is needed to resolve the nature of their relatedness. The role of the various subunits of CT has been well documented. The possibility that the LT molecule, a single polypeptide chain, contains two domains, one for binding and one for cyclase activation, is attractive. The necessity for dissociation of CT and release of the A subunit before enzyme activation has support from various studies. The need for proteolytic cleavage of LT before enzyme activation, postulated by some investigators, needs further study. Analogies have been drawn between these toxins and diphtheria, botulinal, and tetanus toxins (199). Similarities in membrane receptors, subunit function, interaction with membrane or cytoplasmic components after subunit dissociation, and long-term alteration of normal cellular function are the bases for these analogies. The observation that such similar proteins are coded for in one organism by a chromosomal gene and in another by a plasmid poses further questions about their evolutionary relationship. Analogies to eucaryotic glycoprotein hormones leads one to speculate on the possibilities of convergent evolution. The many studies of the nature and actions of enterotoxins produced by E. coli and V. cholerae already discussed have dealt with the effects on eucaryotic cells and not with the role these proteins play in the growth of the organisms themselves. Many organisms, both gram positive and gram negative, have a membrane-associated adenylate cyclase system (108, 123), and cAMP is elevated within the cell and released into the medium during growth under various conditions (163). In E. coli, cAMP is involved in the regu-

606


lation of several genes by both positive and negative control. The gene products are involved in the synthesis of enzymes for carbohydrate metabolism, amino acid synthesis, and flagellar elements as well as the regulation of the life cycle of some phages. cAMP regulation of the expression of the lac operon has been most thoroughly studied. V. cholerae also has been shown to possess an adenylate cyclase system. Extracellular cAMP levels as well as toxin production vary depending on the strain, carbon source, and growth stage (73). The possibility that these toxins act as signals for the regulation of metabolic events within the cells has not been excluded.

CONCLUSIONS The discovery of a variety of cellular systems that are exquisitely sensitive to the action of enterotoxin produced by V. cholerae and enteropathogenic strains of E. coli has led to the purification and characterization of these toxins and the elucidation of many aspects of their mode of action and pathology. They have enabled investigators to assess the purity of toxin preparations during isolation procedures and have become useful diagnostic tools for the clinical researcher. In addition, isolated tissue and cellular systems have led to the detailed mechanism of action of these toxins by dissecting the numerous events that occur during their action in vivo. Further knowledge obtained from these studies is being utilized to design effective vaccination programs. Aside from the better understanding and treatment of diarrheal diseases caused by these organisms, these toxins offer to cellular and molecular biologists potent tools for the study of a wide variety of questions in eucaryotic systems (119). The studies of cell growth and differentiation as well as mechanisms of hormone and enzyme action represent areas where these toxins may provide valuable and exciting results.

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2. 3. 4.

5. 6.

7.

8.

9.

10.

11.

12.

ACKNOWLEDGMENTS These studies were supported by grant Al 12478 from the Public Health Service and by grants from the Kroc Foundation, Santa Ynez, Calif., and the National Leukemia Association. K. L. R. is a predoctoral candidate in microbiology supported by Public Health Service training grant CA 09138 from the National Cancer Institute. We thank Henry D. Isenberg and Albert Balows for their interest and encouragement during the preparation of this manuscript.

13.

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cell adherent immune factor. Infect. Immun. 7:35-38. Agarwal, S. C., and N. K. Ganguly. 1972. Experimental oral immunization with L-forms of Vibrio cholerae. Infect. Immun. 5:31-34. Agarwal, S. C., and N. K. Ganguly. 1972. Oral immunization with L-forms of Vibrio cholerae in human volunteers. Infect. Immun. 6:17-20. Agarwal, S. C., and T. Sundaraj. 1976. Cellmediated immunity in Vibrio cholerae with ribonucleic acid-protein fractions of V. cholerae L-form lysates. Infect. Immun. 14: 363-367. Bailey, W. R., and E. G. Scott. 1970. Diagnostic microbiology, 3rd ed. The C. V. Mosby Co., St. Louis. Banwell, J. G., S. L. Gorbach, N. F. Pierce, R. Mitra, and A. Mondal. 1971. Acute undifferentiated human diarrhea in the tropics. II. Alterations in intestinal fluid and electrolyte movements. J. Clin. Invest. 50:890-900. Bennet, V., S. Craig, M. D. Hollenberg, E. O'Keefe, N. Sahyoun, and P. Cuatrecasas. 1976. Structure and function of cholera toxin and hormone receptors. J. Supramol. Struct. 4:99-120. Bennet, V., E. O'Keefe, and P. Cuatrecasas. 1975. Mechanism of action of cholera toxin and the mobile receptor theory of hormone receptor-adenylate cyclase interactions. Proc. Natl. Acad. Sci. U.S.A. 72:33-37. Bergman, M. J., R. L. Guerrant, F. Murad, S. H. Richardson, D. Weaver, and G. L Mandell. 1978. Interaction of polymorphonuclear neutrophils with Escherichia coli. Effect of enterotoxin on phagocytosis, killing, chemotaxis and cyclic AMP. J. Clin. Invest. 61:227-234. Berkenbile, F., and R. Delaney. 1976. Stimulation of adenylate cyclase by Vibrio cholerae toxin and its active subunit. J. Infect. Dis. Suppl. 133:S82-S88. Bhattacharjee, A. K., and W. H. Mosley. 1973. Selective destruction of skin permeability activity in cholera toxin: effect of acid on cholera enterotoxin. Infect. Immun. 8:133-136. Boquet, P., and A. M. Pappenheimer, Jr. 1976. Interaction of diphtheria toxin with mammalian cell membranes. J. Biol. Chem. 251:5770-5778. Bourne, H. R., R. I. Lehrer, L M. Uichtenstein, G. Weissmann, and R. Zurier. 1973. Effects of cholera enterotoxin on adenosine 3',5'-monophosphate and neutrophil function. Comparison with other compounds which stimulate leukocyte adenyl cyclase. J. Clin. Invest. 52:698-708. Brinton, C. C., Jr. 1966. Contributions of pili to the specificity of the bacterial surface, p. 37-70. In B. D. Davis and L. Warren (ed.), The specificity of cell surfaces. Prentice-Hall, Englewood Cliffs, N.J. Burrows, M. R., R. Seliwood, and R. A. Gibbons. 1976. Haemagglutinating and adhesive properties associated with the K99 antigen of

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DNA damage and prophage induction and toxicity of nitrofurantoin in Escherichia coli and Vibrio cholerae cells.

Stimulation of cyclic AMP secretion in Vero cells by enterotoxins of Escherichia coli and Vibrio cholerae.

Uropathogenic Escherichia coli-Associated Exotoxins.

Animal Enterotoxigenic Escherichia coli.

The adjuvant effect of Vibrio cholerae and Escherichia coli heat-labile enterotoxins is linked to their ADP-ribosyltransferase activity.

Antibacterial activity of silver and zinc nanoparticles against Vibrio cholerae and enterotoxic Escherichia coli.

Demonstration of shared and unique immunological determinants in enterotoxins from Vibrio cholerae and Escherichia coli.

Interactions of choleragenoid and GM1 ganglioside with enterotoxins of Vibrio cholerae and Escherichia coli in cultured adrenal cells.

Functional analysis of Vibrio vulnificus RND efflux pumps homologous to Vibrio cholerae VexAB and VexCD, and to Escherichia coli AcrAB.

Induction of Escherichia coli and Vibrio cholerae enterotoxins by an inhibitor of protein synthesis.

Enterotoxigenic Escherichia coli and travellers' diarrhoea.

The effect of attapulgite and charcoal on enterotoxicity of Vibrio cholerae and Escherichia coli enterotoxins in rabbits.

A double-blind, controlled trial of bioflorin (Streptococcus faecium SF68) in adults with acute diarrhea due to Vibrio cholerae and enterotoxigenic Escherichia coli.

Immunity to enterotoxigenic Escherichia coli.

Effects of lng Mutations on LngA Expression, Processing, and CS21 Assembly in Enterotoxigenic Escherichia coli E9034A.

Pathogenic mechanisms of a non-agglutinable Vibrio cholerae strain: demonstration of invasive and enterotoxigenic properties.

Antimicrobial susceptibility of enterotoxigenic Escherichia coli.

Virulence regulons of enterotoxigenic Escherichia coli.

Colonization factors of enterotoxigenic Escherichia coli.

The protease from Vibrio cholerae nicks arginine at position 192 from the N-terminus of the heat-labile enterotoxin a subunit from enterotoxigenic Escherichia coli.

Novel antigens for enterotoxigenic Escherichia coli vaccines.

Effects of chitosan on intestinal inflammation in weaned pigs challenged by enterotoxigenic Escherichia coli.

Clinical cholera caused by enterotoxigenic Escherichia coli.

Rapid screening method for enterotoxigenic Escherichia coli.