MICROBIOLOGICAL REVIEWs, Sept. 1978, p. 592-613 Vol. 42, No.3 0146-0749/78/0042-0592$02.00/0 Copyright i 1978 American Society for Printed in U.S.A. Pathophysiological Effects of cholerae and Enterotoxigenic coli and Their on Eucaryotic Cells KAREN L. RICHARDS AND STEVEN D. DOUGLAS* Departments ofMicrobiology and Medicine, University ofMinnesota 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 Production .59...... 3.53 THE ...... 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 and ...... 5...... 599 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 Lines and the Immune Response ...... 603 Fibroblasts ...... 604 Other Cell Systems ...... 604 DISCUSSION ...... 6..0.5...... 605 CONCLUSIONS .60...... 606 LITERATURE CITED .606 INTRODUCTION differences constitute the basis for further stud- and enterotoxigenic strains of ies on the structural and functional relatedness diarrheal disease in man ofthe toxins and aspects ofmechanisms ofaction and several animal species. E. coli strains can (63, 79, 80, 128). This review will analyze factors produce two enterotoxins; a low-molecular- that are requisite for localization of these orga- weight heat-stable toxin (ST) and a heat-labile nisms within the intestinal tract and toxin pro- toxin (LT) that has many properties similar to ductibn. The physicochemical properties of the those of toxin (CT). Although the mech- toxins and the effects of intestinal cells will be anism of action of E. coli ST is not known, discussed as as the response of other organ clinical symptoms due to cholera and E. coli LT and cellular systems. In addition, analogies will are the result of action of the toxins on mucosal be drawn between these toxins and other biolog- cells of the . The major action of ically active molecules. The role they may play V. cholerae and E. coli heat-labile enterotoxins in the growth of the and their use as is to activate adenylate cyclase followed by in- probes into the molecular biology of eucaryotic creases in intracellular levels of cyclic 3',5'-aden- cells will be discussed. osine monophosphate (cAMP) and hypersecre- (Part of this review was presented at the 77th tion of and water into the intestinal lumen. Annual Meeting of the American Society for There are a number offeatures distinctive to the Microbiology, 8-13 May 1977, New Orleans, exotoxins of V. cholerae and E. coli. These La.). 592 VOL. 42, 1978 EFFECTS OF E. COLI AND V. CHOLERAE TOXINS 593 PATHOPHYSIOLOGY after centrifugation. Motile show little or no movement after adherence (116). Vibrio spe- Etiology cies also possess pili (14, 197) which may be Cholera is characterized clinically by an acute involved in adhesion. Adhesion to brush borders after bacterial colonization of the small occurs at 37 and 220C but not at 40C and is intestine. Massive fluid loss frequently leads to optimum at calcium concentrations of 1 to 10 severe and . V. cholerae is a mM (114), although strontium is also effective. gram-negative, motile, curved rod that grows on Cholera vibrios also agglutinate human group simple nutrient media at alkaline pH (5, 181). O erythrocytes (115). This hemagglutination, as The has been classified into the class- well as intestinal adherence, is inhibited by L- ical and biotypes on the basis of geo- fucose and D-mannose. Nelson et al. (155) ob- graphical distribution and the ability to lyse served more rapid and uniform adhesion of V. sheep erythrocytes (61, 62). Two , In- cholerae to infant ileal loops than to adult aba and Ogawa, are distinguishable by heat-sta- rabbit ileal loops. Colonization and clearance of ble somatic . the organisms were similar in the infant and Certain strains of E. coli, commonly found in adult rabbit ileum. great numbers in the , are capable Enterotoxigenic strains of E. coli adhere to of colonizing the small intestine and have been and agglutinate guinea pig identified as causative agents of a severe cholera- and human type 0 erythrocytes (15, 50, 94). like disease in man (6, 82, 175) and domestic Certain K antigens on the surface of animals (116). Although several studies have animal isolates are important for adhesion (77, attempted to relate toxigenic strains to particu- 116, 117, 146, 162). A fimbrial demon- lar somatic antigens (84, 88), clinical disease strated in certain diarrheagenic human strains correlates more closely with the presence of acts as a colonization factor (50, 56, 84). In distinct protein capsular (K) antigens (109). In contrast to V. cholerae, adhesion is not temper- addition, certain antigenic types are often asso- ature dependent (77) and is mannose resistant ciated with a particular species; those iso- (50). K88, the adhesin of E. coli strains patho- lated from pigs possess antigens different from genic for pigs, and K99, found on isolates from those isolated from calves, lambs, or humans calves and lambs, have been isolated and puri- (117, 161). fied (88, 117, 146, 161). Agglutination of guinea pig erythrocytes by K8 is inhibited by mucous glycoproteins with terminal beta-D-galactosyl Factors in Pathogenesis residues (77). Adherence to intestinal surfaces and toxin Certain protein K antigens of pathogenic ani- production are two factors necessary for patho- mal strains and colonization factor antigens of genesis by these two . is human isolates are thin, flexible pili that are also a factor for V. cholerae (72, 115). coded for by transmissible (50, 56, 88, Motile vibrios are more virulent than nonmotile 116, 146). These are distinct from type I pili of organisms of the same (72, 85, 115) and E. coli, which are also involved in adhesion and are able to penetrate the intestinal and are receptors for specific phages (14, 157). The enter villous crypts (85, 86). Adherence is composition of type I pili is different thought to be important to enable the organisms from that of purified K88, and adhesion by type both to resist removal by peristalsis and to mul- I pili to epithelial cells and erythrocytes is in- tiply near the site of action of the toxin (86). hibited in the presence ofmannose (15, 177, 197). Studies of V. cholerae adherence to intestinal Tissue receptors for K88 in pigs are postulated epithelium by Freter and Jones (72), Jones et al. by Sellwood et al. (183) to be genetically deter- (114), and Nelson et al. (155) have demonstrated mined. Two pig phenotypes were observed, and that the organisms penetrate the intestinal mu- the presence of receptors was a dominant char- cus and attach to microvilli at the brush border acteristic; 65% ofthe offspring ofseveral matings of the epithelial cells. Several factors are impor- had K88 receptors, and littermates of particular tant determinants of adhesion of V. cholerae matings were phenotypically identical. The K and include (72), temperature, antigens of E. coli have recently been reviewed and cations (71, 114, 115). The exact nature of in detail (162). the adhesive antigen (adhesin) is not known; Genetic and Physiological Basis for Toxin however, it may be associated with the bacterial Production . Nonmotile vibrios are unable to ad- here to intestinal brush border membranes and V. cholerae synthesizes a protein . intestinal tissue slices (72); nonmotile mutants Genetic studies by Vasel et al. have demon- are unable to adhere to brush border membranes strated that toxin production is coded for by a 594 RICHARDS AND DOUGLAS MICROBIOL. REV. bacterial (201). Segregation analysis after plex is noncovalently bound to four to six B (or conjugation indicates that the tox locus is linked L) subunits (136, 143). The B subunit, which is to the locus for histidine synthesis. responsible for toxin binding to cell membranes, Finkelstein et al. (67) have reported artificially contains two residues which may form induced mutants of V. cholerae that differ from intrasubunit bridges (136). Upon storage or the wild type both in amount and in antigenic chemical treatment, the holotoxin may partially properties oftoxin produced. Nutritional studies dissociate to form other AB complexes (136, 171) of toxin production and release from V. cholerae or B subunits alone. The B subunit complex by Callahan and Richardson (18) indicate that consititutes the cholera or choleragenoid, minimal medium containing asparagine and glu- which lacks toxin activity but is capable of bind- cose supports . However, if the ing to membrane sites. Choleragenoid retains medium is not buffered properly and acid con- the antigenic properties of the native molecule ditions arise, toxin is produced but is not re- and competitively inhibits the action of the na- leased. Maximal toxin release occurs at pH 7.5 tive toxin by blocking sites. The A1 or above. subunit is a poor immunogen and is not neutral- E. coli ST and LT (203) are coded for by ized by anti-CT antisera (198). This subunit is plasmids and are often associated with the pres- responsible for all of the toxin activity of A, and ence of adhesion antigens, drug resistance fac- the A2 subunit may serve only to stabilize the A tors (91, 127), and, in some porcine strains, the subunit complex before its action on the cell (63, production of an alpha- (186). Al- 92, 100, 158). Cholera holotoxin can be disso- though surface antigens and toxin production ciated into A and B subunits in acid urea. Re- are independent characteristics coded for on dif- combination of A subunits with B subunits de- ferent plasmids, LT and ST are coded for by the rived from holotoxin or toxoid results in a mol- same or linked plasmids. Gyles (89) ecule with equivalent to that of un- showed that conjugation between Ent' donor treated toxin (65). and Ent- recipient porcine strains resulted in Enterotoxic strains of E. coli synthesize two Ent' recipients. Although some porcine strains types of enterotoxins which differ both in com- produced only ST, no strains were found to position and in cellular effects (51, 89, 111, 112, produce LT in the absence of ST. Both LT:St- 185,207). ST is a protein oflow molecular weight and ST-only-producing strains have been iso- (111). Although ST is enterotoxic, its elicits a lated from man (203). much less severe diarrheagenic response of Variation in antigenicity and in the ability to shorter duration than the higher-molecular- synthesize LT has also been observed in E. coli weight LT (207). Since early purification proce- (40, 45, 53, 66, 183). In vitro studies have defined dures were unable to separate the two toxins various parameters for optimal toxin production. and both elicit similar responses in animal These include the presence of yeast extract in models, there has been some doubt as to whether the medium (54, 154), alkaline pH (154), and ST is a precursor form of LT (112, 185) or a growth of the organisms in the presence of mi- different toxin. tomycin C (110, 154) or protein synthesis inhib- In contrast to CT, LT is a single polypeptide itors such as lincomycin (141) and polymyxin B chain of variable molecule weight which may (55). Thus, virulence by E. coli and V. cholerae exceed 100,000 when isolated from culture su- is dependent on both genetic and physiological pernatants (39, 169, 194). When E. coli is grown factors. These must be considered in the diag- at low pH in the presence of (56) or nosis and study of the pathogenesis of these when the toxin is treated with trypsin (61, 169), diseases as well as the characteristics of their an active molecule of 20,000 molecular weight is toxins. released which has properties similar to the A1 subunit of CT (40, 55, 56, 80, 168). The physical THE TOXINS and chemical properties of LT, ST, and CT are Structure presented in Tables 1 and 2. Although the physicochemical characteristics Antigen Relatedness of the Toxins of CT have been known for some time (7, 27, 100, 133, 135, 158, 167, 198), those of E. coli LT In addition to the chemical and physical sim- have only recently been elucidated (28, 39, 40, ilarities between CT and E. coli LT, the two 56, 66). CT, or choleragen, is composed of three toxins are antigenically related (89, 90, 104, 126). subunits: Al, A2, and B. The biologically active Antisera produced against choleragen and chol- A (or H) (143) subunit is composed of a single eragenoid inhibit the stimulatory effect of CT molecule of A, and A2, which are linked by a and LT on intestinal (89, 90). In addi- single bond (198). The A-subunit com- tion, the released by trypsin treatment VOL. 42, 1978 EFFECTS OF E. COLI AND V. CHOLERAE TOXINS 595 TABLE 1. Physicochemical characteristics ofE. coli and V. cholerae toxins Detennination

Toxin' Approx mol wt of Sedimentation Extinction coeffi- I coefficient cient Approx mol wt of subunits whole toxin (swm..) (E,,) pointpoect E. coli LT 100,000 5.4 11.6 6.9 20,000-35,000h E. coli ST 1,000-10,000 CT 80,000-98,000 5.54 11.4 6.6 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 in the presence of antibiotics (55) and after trypsin treatment of high-molecular-weight LT (169,180). of LT is neutralized by anti-LT, -CT, and -chol- been covalently attached to agarose beads, has eragenoid (55). The results of similar studies on been used to extract CT from solutions when the neutralization of CT activity by anti-LT present at concentrations as low as 10-" M (27). antisera are variable (48, 89, 90, 126), but recip- In addition, Gm, is capable of neutralizing the rocal neutralization has been reported (48, 126). reactivity of CT in both in vivo and in vitro E. coli LT and CT have similar amino acid assays (96,124) when incubated before the assay. composition (61, 180), and the complete amino Further evidence for Gm, as the receptor for acid composition and sequence of the B subunit the B subunit of choleragen and its toxoid has of CT have recently been reported (132, 133, been provided by King et al. (125) and Moss et 137). Although the amino acid sequence of LT al. (149). Preincubation of pigeon erythrocyte has not been determined, it is interesting to membranes with Gm, resulted in greater binding speculate on possible areas of sequence analogy of CT (63, 125). Transformed mouse fibroblasts between CT and LT to explain both differences with no detectable Gm1 in their and similarities in the functional characteristics membranes, as assayed by thin-layer chroma- of these two . tography, do not respond to CT. Incubation of cells with [3H]Gm, induced sensitivity to CT Binding Site with corresponding increases in cAMP levels within the cells (69, 149). The fact that Gm, The binding site for both LT and CT has been spontaneously incorporates into biological mem- studied by using direct binding assays (27, 96), branes and has a high affinity for these toxins, absorption studies (74), and a variety of inhibi- causing an increase in binding of the toxins to tion models (103, 165, 200). Gm, ganglioside, an modified membranes, cannot be interpreted as acidic glycolipid containing sialic acid and a definitive evidence that Gm1 is the natural mem- terminal (68) (Fig. 1) has been sug- brane receptor on these cells. The possibility gested to be the natural receptor for CT. The that CT binds to other or glycopro- most effective inhibitor ofCT is Gm, ganglioside, teins with similar moieties or that which is present in varying amounts in the Gm, is only one constituent of the natural recep- plasma membranes ofall cells examined (62,199, tor cannot be excluded. 200) and is particularly abundant in brain tissue. Although LT binding to intestinal tissue can The number of binding sites for CT on various be inhibited with Gm,, the receptor for E. coli cell types from different species ranges from less LT may not be the same as for CT (19, 96, 165). than 100 on human erythrocytes to greater than Preincubation of segments of rabbit intestine 107 on guinea pig brain cells (79). However, the with choleragenoid does not significantly de- dissociation constants for all cells reported were crease the secretory response to LT, and LT has approximately 10-' M. a lower affinity for Gm1 than CT (96,165). These CT can be effectively absorbed by various observations may be explained by differences in tissues, including brain and intestinal epithelium affinity for the same receptor or the existence of (69, 200). Indeed, the amount of bound toxin two closely related receptors. roughly correlates with the amount of ganglio- Craig and Cuatrecasas (21) have reported that side in the membrane (74, 102). Binding of CT rat lymphocytes can cap fluorescein-labeled CT. to Gm,-coated test tubes detects CT in amounts This indicates a multivalent binding of the toxin less than 1 ng/ml (96, 104), and Gm,, which has molecule to membrane receptor. 596 RICHARDS AND DOUGLAS MICROBIOL. REV. GM,: CER-GLc--Ga1-Ga1NAc-Ga1 NAN GM2: CER-G1c-Gal-GalNAc .0 IzrEE NAN GM3: CER-Glc-Gal

> X NAN + + + GD1b: CER-G1c-Ga1-Ga1NAc-Ga1 NAN-NAN +I GT1: CER-G1c-Ga1-Ga1NAc-Ga1 NAN-NAN NAN FIG. 1. General structure of selected gangliosides (68). Abbreviations: CER, ceramide; Gal, galactose; t,*+ + N-acetylgalactosgamine; Glc, ; NAN, Z.D N-acetylneuraminic acid. Action of Toxins on Adenylate Cyclase O bD ~++ I+I + 0 Both LT and CT elicit a -dependent ac- n tivation of the membrane-bound ade- ~~~~~~~~GalNAc,nylate cyclase (EC 4.6.1.1), which converts aden- 0 + osine 5-triphosphate (ATP) to cAMP (9, 10, 52, $4; > + 176). CT increases basal activity in both the ileum and jejunum and enhances prostaglandin, but not sodium fluoride, activation in these tis- sues (184). Studies of CT activation of adenylate w8.>+ + cyclase in erythrocyte membranes (78) indicate 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 approxi- cli mately the same level and neither is enhanced X z beyond a maximal level by the subsequent ad- < D dition of the other toxin of sodium fluoride. E t s Adenylate cyclase activation by CT or LT is distinct from that caused by epinephrine, pros- taglandins, glucagon (10), catecholamines, or ad- renocorticotropin (ACTH) (206). The addition -;_ _ of CT or LT potentiates hormonal stimulation in most cell systems. However, there are differ- ences 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 wash- ing. Further, intestinal secretion after LT treat- ment is substantially less than that observed after CT exposure (66). The sequence of events

_ after toxin to a membrane re- E_; cm HensuingE_v ceptor, and during thebindingactivation of adenylate VOL. 42, 1978 EFFECTS OF E. COLI AND V. CHOLERAE TOXINS 597 cyclase, is largely unknown. In studies utilizing ribosylation of arginine (152, 196). Recent stud- '251-labeled CT, Holmgren and Lonnroth (101) ies have shown the ADP- to be bound to have shown that the toxin does not accumulate the A subunit after the hydrolysis. It is not in large quantities within the of the known whether the activation of adenylate cy- cell, but remains associated with the cell mem- clase is due to its direct interaction with the brane. Therefore it is probable that events lead- ribosylated A subunit or whether an acceptor ing to adenylate cyclase activation occur at or protein is ribosylated and causes enzyme acti- within the plasma membrane. vation. The A subunit would then act as a trans- fer protein (150). Studies by Gill et al. (80) Solubilized Adenylate Cyclase and Toxins indicate that NAD and ATP are also necessary Recent studies on the specific events leading for LT activation of adenylate cyclase in eryth- to the activation of adenylate cyclase have been rocyte ghosts. done with whole-cell particulate fractions, puri- Adenylate cyclase activation is viewed as oc- fied membranes, and solubilized enzyme from curring in three stages (206). The first, or prep- cells in the absence of cytoplasmic compo- aratory phase, involves the unbound toxin in the nents (8, 10, 70, 151, 176). Bennet et al. (8) presence of a reducing agent and NAD and may reported the stability of solubilized rat liver involve the formation of toxin-NAD complexes. membrane adenylate cyclase activity after CT The second, or dissociation phase, includes the exposure. Activation of adenylate cyclase in binding of the toxin to a membrane site and the membrane fractions of isolated fat cells by hol- dissociation of an active subunit from the com- otoxin occurred after a 25-min lag phase in an plex. During the third, or activation phase, NAD ATP-generating system containing ATP, gua- is utilized during the activation of adenylate nosine 5'-triphosphate (GTP), and magnesium cyclase. (176). Toxin-adenylate cyclase complexes formed by CT during incubation of whole cells Involvement of Guanosine 5'- or membrane fractions remained complexed Monophosphate (GMP) after solubilization by nonionic detergents, and The requirements for the complete activation cyclase activity was precipitated from solution of solubilized adenylate cyclase from brain by by antisera to the CT and the A subunit (8, 176). CT requires cAMP and a protein of Activated cyclase present in solubilized mem- phosphodiesterase (151). This brane preparations could be neutralized by an- preparation was seen to partially replace the titoxin and anti-A antisera. The possible exist- need for cell supernatants. Direct involvement ence of two classes of toxin receptors, some of of GTP and guanosine 5'-diphosphate as pri- which act on adenylate cyclase, was excluded by mary regulators of the activation of adenylate blocking with the toxoid. A large number of cyclase has been suggested by studies utilizing independent and equivalent receptors for toxin neuroblastoma cells and their isolated mem- were proposed which must come into favorable branes (140). The activation of the enzyme by orientation after binding of toxin. CT or hormones in the presence of nucleotide triphosphates, NAD, and magnesium closely re- Cofactors Required for Toxin Activity sembles stimulation by guanyl-5'-yl imidodi- When broken-cell preparations (78,80,81) are phosphate. Activation by CT in the presence of used to study toxin activity, there is no lag 10 mM magnesium requires the presence of gua- period between binding of LT or CT and ade- nine nucleotide triphosphates which may be es- nylate cyclase activation. Significant cyclase ac- sential for regulation of activity by fixing the tivation occurs within 1 min in erythrocyte enzyme in an active state. Quantitation of cyclic ghosts (80) in the presence of CT. Gill (78) GMP (cGMP)/cAMP ratios suggests that a pri- showed that activation requires the presence of mary cGMP elevation occurs before a cAMP cofactors-nicotinamide adenine dinucleotide elevation (139). The formation of GTP-enzyme (NAD), ATP, and a protein from the cell cyto- complexes may represent an event that occurs plasm. In addition, reducing agents such as glu- during the lag phase characteristic of both LT tathione or dithiothreitol are necessary for CT and CT. It is pertinent to note here that Hughes activation, possibly to release the A1 subunit et al. (107) have shown increases in cGMP by from the toxin molecule. Similar enhanced activ- ST in rabbit intestine without a rise in cAMP ity occurs in the presence of sodium dodecyl levels. Furthermore, 8-bromo-5'-GMP causes sulfate. fluid secretion comparable to that due to ST. NAD has been shown to be hydrolyzed by CT These data support the hypothesis that LT and or its A subunit to adenosine 5'-diphosphate ST are distinctly different toxins with different (ADP)-ribose and nicotinamide followed by the mechanisms ofaction and that cyclic nucleotides 598 RICHARDS AND DOUGLAS MICROBIOL. REV. are important in the regulation of intestinal se- Variability in Activation of Adenylate cretion. Cyclase Differences in the kinetics ofadenylate cyclase Effect of Increased Intracellular Cyclic activation and cAMP production after exposure Adenosine 3',5'-Monophosphate to CT and LT have been observed. These differ- There are a number of secondary cellular ef- ences may be due to a number of factors, both fects that are the result ofincreased intracellular in the microorganisms and in the host, and in- cAMP. Of these, increased intestinal secretion is clude genetic variability in the nature or amount a primary factor in the pathogenesis due to these of toxin produced by a particular organism, dif- organisms. Water and electrolyte secretion and ferences in the binding of the toxins to their cyclic nucleotides have been reviewed by Strew- receptors, in the receptors with age of the host ler and Orloff (190) and Field (58, 59). Under (46, 120), or in modulation of the intracellular normal conditions, there is low spontaneous effects after toxin binding (45). transmembrane electrical potential in the intes- tinal epithelium which is believed to account for Similarities to Glycoprotein Hormones its high passive permeability. Unidirectional lu- and Other Bacterial Toxins men-to- fluxes of sodium and chloride oc- The functional and structural properties of cur and are in part balanced by absorption in CT are similar to those of many glycoprotein the opposite direction, resulting in a slight net hormones in their subunit structure, membrane flux toward the serosa. Movement of ions, pri- binding site, and adenylate cyclase activation marily bicarbonate, occurs toward the lumen. (33, 137, 138). Computer comparison of partial Elevation of intracellular cAMP by the addition and complete sequences of CT and four glyco- oftheophylline causes a transient increase in the protein hormones (thyrotropin [TSH], luteiniz- electrical potential of the membrane and de- ing hormone [LH], human chorionic gonadotro- creased absorption of sodium leading to a net pin [HCG], and follicle-stimulating hormone secretion. In addition, the direction of chloride [FSH]) has revealed that a short segment of the transport reverses. These events result in an B subunit of CT was analogous to a similar of water by osmosis. The action of CT and segment of TSH, LH, HCG, and FSH (Table 3). LT on intestinal epithelium is similar (58, 59) to Quantitative measurement of similarities indi- the effect oftheophylline, and net sodium secre- cates significant analogy between CT, TSH, LH, tion has been reported (31). Both the brush and HCG, but not FSH. Structural analogy also border and crypt cells are involved in secretion exists between the A, subunit of CT and alpha after exposure to CT (29). There is no evidence subunits of these same hormones (138). The for gross mechanical damage to the epithelium evolutionary significance of these findings is of during the secretory phase in vivo, although the great interest. The similarities in binding sub- presence of small lesions in the plasma mem- unit structure, membrane receptors, and the ac- brane of the brush border and mitochondrial tivation of adenylate cyclase by proteins from swelling has been reported (164). Trivalent cat- such biologically unrelated groups suggest pos- ions have been shown to inhibit toxin-induced sible evolutionary convergence in both structure intestinal secretion in (139). Pretreat- and function and are not without precedent. For ment with lanthanum chloride resulted in a two- example, the active sites of chymotrypsin and fold reduction in secretion due to CT and a 10- subtilisin are comparable to each other in com- fold reduction after LT exposure. This effect is position and function. postulated to be due to the inhibition ofa protein The similarity between the subunit structure kinase system, resulting in a rise in cGMP (139) of CT and other bacterial and toxins has and subsequent cAMP elevation after adenylate also been noted (12, 160, 179, 191). , , cyclase activation (139, 144). DeLorenzo et al. ricinus agglutinin, and botulinal and (30) reported the decrease in a phosphorylated toxins are all comprised of separate binding and protein toad bladder membrane in response to enzymatic subunits. Moss et al. (150) have sug- antidiuretic hormone or cAMP treatment. The gested similar functional similarities between decrease in this protein was correlated with in- cholera and diphtheria toxins. Both exhibit nic- creased sodium transport and water permeabil- otinamide adenine dinucleotidase activity and ity, and they postulated the inhibition of a pro- cause ADP-ribosylation of an acceptor protein. tein kinase or the stimulation of protein phos- In addition, gangliosides constitute part of the phatases in the membrane. Whether these ob- membrane receptors for the glycoprotein hor- servations are related to the ribosylation of mones, toxin, botulinal toxins, serotonin, membrane components by CT remains to be and interferon. elucidated. The similarity between LT, CT (68), and other VOL. 42, 1978 EFFECTS OF E. COLI AND V. CHOLERAE TOXINS 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 infec- co 0 !0 w w $tions by enterotoxigenic strains ofE. coli in both man and domestic animals has led to a search for effective vaccines. Knowledge of the struc- ture and functional properties of the toxins and % virulence factors of the organisms together with information gained from a variety of animal models has led to a varied approach to clinical vaccination programs. The use of killed vibrios as immunizing agents against cholera in man has met with limited success, primarily due to the short duration of o protection (61). However, V. cholerae L-fonns .I and b-fonn lysates do elicit protective immune responses in rabbits (2-4). Oral administration ofL-forms of V. cholerae results in the successful X 0000Ucz U cz immunization of rabbits (2). L-form lysates also induce an immune response when given paren- .g_Et E tE$EW terally, and agglutinating as well as vibriocidal activites are present in these anximals for many '4... 3 o O O c: weeks. Oral administration of L-form lysates in .tc ~rabbits and humans (3) results in the production of copro and serum to homologous and heterologous strains and, in conjunction with alcohol- or phenol-precipitated ribonucleic acid (RNA) from lysates, induces enhanced cel- lular immunity in rabbits. Delayed hypersensi- I I,,,, tivity to cholera lysate antigens, the inhibition of migration of leukocytes and in the presence oflysate antigens, and X , agglutination have been reported (4). Crude flag- ellar 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 i' , production of coproantibodies of the immuno- 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). Cy-

q tophilic IgG and on the surface of macro- E phages (1) and IgMopsonins are also produced. These antibodies are directed toward m 0 0 0 g constituents of the organisms (47, 71, 86), which 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 =-:c 8 = a 166) and humans (64, 75) with variable results. U FE- 5x Formalin-treated toxoid has been demonstrated 600 RICHARDS AND DOUGLAS MICROBIOL. REV. to partially revert to a toxic product upon stor- humans, which may be protective in nursing age (106). Glutaraldehyde-treated toxin does not infants (97, 189). revert to active toxin, but leads to a poor im- These studies have shown that not only is the mune response (75, 106). Aggregated, purified B immunization agent important in protection, but subunits have been used as an immunogen and the route of immunization may also play a role found to be very effective in protecting against in the level of protection obtained. Enteral and whole-cell challenge as well as toxin challenge in parenteral administration of vaccines have both rabbits (106). given protection in animals; however, the mech- Parenteral immunization using B-subunit tox- anisms of protection may be different. Paren- oid results in increased titers of serum teral administration leads to higher serum anti- antibodies and protection against intraintestinal body levels and in vitro vibriocidal activity, but challenge in intestinal loops in the absence of may not enhance local defense in the intestine coproantibodies (60). Toxoid- or prevent bacterial adherence or action of tox- (LPS) preparations gave the best protection ins on epithelial cells (204). Multiple enteral after challenge in humans (61) and rabbits (193). administration of bacterial vaccines in combi- Killed whole-cell plus B-subunit toxoid nation with toxoid preparations may yield the was also effective in clinical studies using human most potent immunization schedule. volunteers (75). These combinations do not lead to serum levels of antivibrio antibodies higher ENTEROTOXINS IN IN VIVO SYSTEMS than with either antigen alone; thus other mech- Whole-Animal Models anisms which interfere with bacterial adhesion, toxin binding, or both are important. Vaccina- Whole-animal and intestinal loop models were tion with combined preparations gives longer- used extensively in the early work to assay for lasting immunity than with any of the conven- both CT and E. coli toxins (17, 76). These assays tional vaccines (106). Both enteral and paren- provided the first information on the physiolog- teral immunization of rabbits with CT and tox- ical effects and mechanisms of action of these oid protects against subsequent challenge with toxins. The infant rabbit was used in many stud- toxin and live vibrios (105). Intestinal IgG and ies where bacterial suspensions or crude culture IgA levels are increased by enteral and, to a filtrates were given orally. Other animal systems lesser extent, parenteral immunization. Serum used in similar studies included dogs, cats, ro- IgG levels are elevated only after parenteral dents, pigs, and chickens. Transmural injection immunization. of E. coli ST into the stomach of suckling mice Immunization of rats with CT by combined leads to intestinal fluid accumulation; this has intraperitoneal and oral immunization results in been used as an assay for ST (76). The obvious increased serum IgG but not IgA (214). Eighty economic limitations of whole-animal studies, as percent of intestinal activity is IgGl. well as difficulty in the quantitation of results, Furthermore, binding of radiolabeled CT to in- led to the development of organ system assays. testinal microvilli and intestinal secretion was Of these, the ileal loop and skin permeability depressed in immunized animals. Although the systems are discussed. role of specific antibody classes in immunity to cholera was not addressed by these investiga- Ileal Loop Assay tions, antitoxin antibodies of the IgG class ap- The rabbit ileal loop assays, originally used in peared to be involved in protection at this site. the early 1950s (64), involves the introduction of by enterotoxigenic strains of E. coli the material to be tested (bacterial suspension, results in the production of serum anti-LT an- culture filtrates, toxins, or toxin subunits) into tibodies in animals (174) and man (57, 175, 204). ligated segments of the ileum of adult rabbits or However, the correlation between rises in anti- swine (147). Examination of the intestinal tissue toxin levels and protection is not clear. Anti-LT at the light or electron microscopic level re- titers vary and are dependent on prior , vealed intestinal morphology resembling the severity of disease, and enhancement due to clinical disease. The focal lesions observed were exposure to V. cholerae and its antigenically judged to be caused by increased hydrostatic related toxin (204). Prior immunization of rats pressure from the lumen, and the goblet cells with CT or cholera toxoid protects against both actively discharged mucus. There was no evi- LT and CT challenge (166). Quantitative mea- dence for direct damage to epithelial cells due to surement of titers in hu- toxin exposure. After an appropriate time inter- man indicates that enteral exposure to both val, usually 18 h, separate ileal segments were enterotoxigenic E. coli and V. cholerae can in- excised, and both the length of the segment and duce the production of secretory IgA and IgG in the fluid accumulated within it were measured VOL. 42, 1978 EFFECTS OF E. COLI AND V. CHOLERAE TOXINS 601

(17, 49, 53, 105, 166). Early studies using viable yield PF activity. When PF is synthesized, it is microorganisms (17) revealed that the secretory always found to be associated with . response was the result of toxemia rather than The assay system itself was also found to be a bacteremia. As few as 10 viable organisms led to factor in the detection of PF activity. Older, infection and secretion within hours after inoc- larger rabbits used in this assay were found to ulation. be less reactive to PF than younger animals. In Fluid secretion in response to ST is immediate addition, the characteristics of bluing after E. and maximal within 6 h irrespective of dose, coli PF exposure are different from those for V. although the net secretion is less than with LT cholerae PF exposure (66). Bluing is less intense (49). Onset of secretion after exposure to LT is and is surrounded by an area of without dose dependent, reaching maximal levels later bluing. Purification of E. coli PF on agarose than for ST. Higher doses of LT led to sustained A5M and polyacrylamide P-150 columns led to fluid secretion, whereas low doses did not. These the of two factors: a blanching factor, findings paralleled those observed after CT ex- which is heat stable, and a bluing factor, which posure. is heat labile and elutes slightly behind blanch- Preincubation of CT with antitoxin results in ing factor on P-150. Histological examination of a reduction of the secretory response and cor- areas of blanching revealed early eosinophilic, relates with the amount of antiserum used (96). histiocyte infiltration and dermal thickening, Gm, inhibits the response to CT, and 100 times leading to liquefication and involving less ganglioside is required to inhibit the CT hair follicles and blood vessels. This reaction response than the LT response. Pretreatment continued to increase after 72 h without any with choleragenoid blocks the CT but not LT indication of resolution. The area of bluing was response in the loop assay. Prior immunization shown to consist of a mixed infiltrate at 18 h, of rabbits with culture filtrate from toxigenic leading to mononuclear infiltrate at 24 h con- strains of E. coli (174) and with CT (105) results taining eosinophilic proteinaceous edema which in an increased resistance to oral challenge with began to resolve at 72 h. The possibility that homologous bacterial suspensions and crude en- blanching factor contains LPS and that blanch- terotoxin. Immunization ofrats with cholera tox- ing was incidental to the bluing effect of PF oid gives equal protection against CT and LT cannot be excluded. (166). Neutralization studies (48) showed that PF activity of both V. cholerae and E. coli is in- Skin Permeability Assay hibited by homologous and heterologous anti- Studies on the action of culture filtrates of V. sera. This finding is in contrast to the results of cholerae after intradermal demon- neutralization studies on the toxigenic effects of strates that a heat-labile component causes er- the two toxins in intestinal models. PF activity ythema, edema, and induration at the site of of both toxins gave equivalent results in rabbit injection (11, 20, 48, 54). This procedure has skin. been modified to include a later intravenous ENTEROTOXINS IN IN VITRO SYSTEMS dose of a protein-binding dye, such as Evan's blue, which enters the indurated area. The ac- Various in vitro cell systems have been used tive component was called "skin toxin," vascular to quantitate the biological activity of both E. permeability factor, or PF (61-63). It was coli LT and CT. These systems have also yielded thought to be identical to or associated with CT. information regarding the mechanisms of action Several investigators showed that the two activ- of these toxins and have provided valuable tools ities were co-isolated (associated) at various for the study of many aspects of eucaryotic cell stages ofpurification and were neutralized in the growth and development. presence of antitoxin antisera. However, treat- ment of CT at low pH and subsequent neutrali- Erythrocyte Ghosts zation was shown to selectively destroy skin The use of erythrocyte ghosts to determine permeability activity without loss of its diar- the cofactor requirements for cyclase activation rheagenic or toxic activity in animal assays (11). has already been mentioned. Additional infor- Vascular permeability factor has been isolated mation has been obtained with this system to from enterotoxigenic strains of E. coli (48, 54), study membrane receptors and the role of Gm, and the culture conditions during bacterial (125). Pretreatment of pigeon erythrocyte mem- growth appear to be important in its synthesis branes with purified Gm, results in increased (54). Yeast extract and aeration were found to binding of CT and increased activation of ade- be necessary for synthesis, and media commonly nylate cyclase. Correlation of the amount of used to promote LT production did not always toxin bound with intracellular cAMP levels in- 602 RICHARDS AND DOUGLAS MICROBIOL. REV. dicates that more receptors are present than are sponse to CT is specific, can be saturated, and necessary for maximal stimulation ofthe enzyme reaches plateau levels at approximately 1 ng/ml. in both treated and untreated cells. In addition, Two or more hours are required before increases the membrane receptor may be more complex of steroid production above base-line levels are than Gm, alone. Binding after Gm, incorpora- observed. Increases in steroidogenesis are re- tion was greater than expected, possibly due to lated to the concentration of the toxin used (35). the formation of complexes containing Gm, and The duration of the response to CT is longer other membrane components, which have a than for ACTH and can last as long as 72 h. CT greater affinity for CT than does Gm, alone. subunit A induces changes similar to those in- The direct interaction of CT with adenylate duced by whole toxin (34). The morphological cyclase in the presence of NAD and dithiothre- changes after addition of cAMP commence after itol or the A1 subunit in the presence of NAD a lag similar to that found with CT but revert to alone indicates a direct membrane interaction normal after 24 h. by the active subunit (212). The need for cyto- ACTH is thought to bind to membrane recep- plasmic components reported in other studies tors different from the toxins. OS-3 cells, respon- may be supplied by a membrane-associated fac- sive to ACTH by both morphological and met- tor, such as GTP, in these studies. The greatly abolic criteria, respond to LT, CT, and cAMP reduced lag period using erythrocyte membranes (32, 211). The response in these cells is also and dissociated subunits emphasizes the conclu- characterized by a lag period. Further, half-max- sion that the lag period involves events related imal stimulating doses of ACTH and CT are to proper binding, orientation, and dissociation additive in Y1 cells. Pretreatment with maxi- of the subunits at the cell surface. Further evi- mally stimulating doses of CT potentiates the dence comes from the use of the A1 subunit in a subsequent response to ACTH. In addition, cal- 30-fold molar excess over holotoxin with intact cium is required for the action of ACTH, but not cells, which can activate adenylate cyclase after for the action of either toxin (211). a longer lag period. These results indicate the The lag period after toxin exposure is not due role of the B and A2 subunits for "focusing" to differences in the rate of binding of the toxins toxin on the membrane surface, stabilization to the cell surface. Maximal stimulation is ob- during binding, and facilitating possible mem- served after a 10-min incubation followed by brane insertion (212). extensive washing. Preincubation of the cells with toxoid neutralizes binding of the toxin, and Adrenal Cells subsequent stimulation and preincubation ofthe cells with Gm, ganglioside increases binding of The Y1 cell line, which is responsive to LT, the toxin (38). Pretreatment of the toxin with CT, and ACTH derived from mouse adrenal either antitoxin or antitoxoid neutralizes the re- cortex, and the OS-3 mutant, which is unrespon- sponse, and pretreatment ofthe toxin with levels sive to ACTH, have been used to study these of Gm, as low as 1:1 molar ratios has the same toxins (32, 33, 35, 38, 129, 134, 173, 209-211, 213, effect (210). Exposure of the cells to antitoxin 215). Y1 cells, when grown as confluent mono- during the lag period partially inhibits the re- layers, undergo morphological and metabolic sponse by these cells (211); addition of antitoxin changes after exposure to ACTH, cAMP, CT, or 15 min after toxin exposure significantly de- LT. The cells round up and detach from the creases both morphological and steroidogenic substrate and secrete increased amounts of responses. Addition of antitoxin as late as 2 h delta-4,3-ketosteroids, which can be quantitated after toxin results in as much as 20% decrease in spectrophotometrically or fluorometrically. response (210). These changes correlate with increased levels of The results from studies using adrenal cells intracellular cAMP (35). Rounding up is more indicate many characteristics similar to those of sensitive to changes in cAMP and requires only in vivo studies usingrwhole animals or intestinal 10% of the increase necessary for elevated ste- loops. The characteristic lag period is seen for roidogenesis (211). The effects of ACTH are both LT and CT. These toxins probably bind to different from those of toxin treatment with the same receptor site, Gm,, which is distinct respect to the nature ofthe binding sites, kinetics from that for hormones by several criteria, even of the response, and ion requirement. There are though both have similar effects on cells. The also differences in the effect of inhibitors in this final response can be inhibited to varying de- system when compared with in vivo systems. grees by the addition of antitoxin to the system The response of Y1 to ACTH is rapid: only a during the lag period, even though binding is few minutes are required for cell rounding. A lag complete during the first 10 min. This inhibition period of 1 to 3 h is required to obtain the same may be related to a reversible step in the inter- response with LT or CT (35-37, 135). The re- action of the toxin with membrane components VOL. 42, 1978 EFFECTS OF E. COLI AND V. CHOLERAE TOXINS 603 and may in part account for the lag period. If so, and the lipolytic response (25, 26). Fetuin, a the lag period may include dissociation of the glycoprotein, also inhibits binding. The inhibi- toxin complex, toxin processing, and/or interac- tion is less effective with increasing time due to tion with a intermediate or the dissociation of the toxin-inhibitor complex. the adenylate cyclase itself. In addition, the tox- Prior incubation with Gm, was also inhibitory, ins appear to interact with or in some way stim- and reversal of inhibition was not seen due to ulate adenylate cyclase molecules distinct from the high affinity of the ganglioside for the toxin. those which are acted upon by ACTH, since Lipolysis is also neutralized in the presence of toxin and ACTH potentiate each other's effects. antitoxin. If cyclase molecules sensitive to the action of The natural receptor for LT and CT on all these toxins represent a subset of total cyclase cells studied has been concluded to be Gm1, within the membranes, the proper orientation of based on neutralization and inhibition studies. the toxin with an appropriate cyclase molecule However, quantitation of the components of fat may also occur during the lag period. cell membranes has shown that they contain no detectable Gm,; the major gangliosides were Isolated Fat Cells found to be Gmn. and Gm2. The general struc- Isolated fat cells are obtained from rat epidid- tures of these gangliosides are compared with ymal fat pads by digestion of the tissue with that of Gm, in Fig. 1. Although exogenously and cell flotation. These cells are added Gm, was seen to be incorporated into similar to adrenal cells in their response (23-26, adipocyte membranes, the binding of CT by 52, 119, 144, 148, 172, 202, 208). They can be these modified membranes may be nonspecific, stimulated by a variety of hormones and drugs since these membranes did not show saturation to release glycerol and free fatty acids into the kinetics. In addition, although Gm, incorpora- medium with the simultaneous increase in intra- tion increased the rate of lipolysis, it did not cellular cAMP. The magnitude of the response increase the maximum level of stimulation or is measured by glycerol release into the medium lead to responses to substimulating doses of and is quantitated spectrophotometrically (172, toxin. Exogenously added Gm, may, therefore, 208). ACTH, TSH, glucagon, epinephrine, and serve only to bind toxin to the membrane on a isoproterenol elicit this response, as do theo- nonspecific manner and not represent the true phylline, cAMP, dibutyryl cAMP, CT, and LT membrane receptor for toxin on these particular (93). Lipolysis with or without stimulation can cell types (118). Fat cells can be metabolically be inhibited in the presence of or pros- stimulated in the presence of enterotoxins, and taglandin El. Lipolysis after, hormone stimula- this stimulation is related to an increase in tion, but not toxin exposure, requires the pres- cAMP (93, 144). The toxins exhibit a lag period ence of calcium, magnesium, and potassium of 1 h or more, and the response can be inhibited (202). in the presence of , or antitoxoid or by Fat cells incubated with honnones and cate- pretreatment of the toxin with Gm, ganglioside. cholamines show immediate increases in intra- cellular cAMP. Increases after CT exposure Lymphocyte and Lymphoid Cell Lines and were delayed for 1 to 3 h and for shorter periods the Immune Response after LT treatment (93, 144). Addition of theo- Lymphocyte and lymphoid cell lines are re- phylline shortened the lag period and also re- sponsive to CT, and these cells have been uti- sulted in higher cAMP levels. Significant acti- lized in studies of toxin action. CT localization vation of adenylate cyclase without a lag occurs has been investigated by using rat lymphocytes when intact cells are exposed to the A subunit incubated with fluoresceinated toxin (21, 22, 170, of (176). Exposure to cholera toxin 182). The observation that diffuse localization at did not cause a potentiation of cAMP accumu- 0°C progresses to a cap at one pole of the cell lation in response to isoproterenol in intact cells. after warming to 37°C (21) is evidence for ligand- The response to CT is dose dependent and can triggered plasma membrane receptor mobility. be saturated (202), and its binding occurs rapidly Moreover, several types of evidence suggest that and tightly. Dissociation constants of 10-`0 M ligands must be bivalent to induce capping. The have been reported (23). Glycerol release after demonstration that choleragen-treated lympho- LT exposure occurred after 5 min, whereas glyc- cytes bind to Gm, agarose beads also suggests erol release after CT took 1 h. The response that CT can bind to at least two receptors (22). lasted several hours after stimulation with either Similar studies by Sedlacek et al. (182) using a toxin, but could be reversed in LT-treated cells fluorescent-labeled synthetic sialoglycolipid after washing (93). (dansyl ganglioside) demonstrated that this dan- Prior incubation of CT with mixed ganglio- syl label binds to protein I of CT (B subunit) sides but not the hormones inhibits both binding and to the lymphocyte membrane. Ligand-in- 604 RICHARDS AND DOUGLAS MICROBIOL. REV. duced mobility was shown to be temperature though the toxin receptor undergoes redistribu- dependent and to co-cap with lymphocyte anti- tion with Thy 1.2, the association with theta is immunoglobulin receptors (182). Douglas et al. indirect. (42) and Zuckerman and Douglas (217, 218) have Fibroblasts studied membrane receptors and the freeze-frac- The effect of CT on Chinese hamster ovary ture topography of a murine lymphoblastoid cell (CHO) cells and other fibroblasts in culture is to line treated with CT. These studies have dem- increase collagen synthesis (87), cell elongation, onstrated that CT inhibits the receptor for the and adhesion to a substrate (113, 156) and to Fc portion of IgG and also alters the distribution elevate cAMP (144). This effect is also seen after of intramembrane particles. Treatment of two treatment with cAMP, dibutyryl cAMP, and lymphoid cell lines with CT for up to 24 h theophylline. Increased adhesion occurred after resulted in significant increases in intracellular a 60-min lag and was related to concentration of cAMP and mitochondrial swelling. Similar CT. The mechanism by which CT enhances treatment of mononuclear resulted substrate adhesion by CHO cells may be due to in an increase in cAMP levels without morpho- an effect on microtubules and microfilaments in logical changes in mitochondria (43). Further CHO cells. The effect seen in fibroblasts is the increases in cAMP levels by incubation of toxin- reverse of that observed with adrenal cells in treated mononuclear phagocytes with amino- culture, which emphasizes the pleiotropic effect phylline had no effect on cellular morphology. of cAMP in various cell types. Normal fibro- CT and E. coli LT bind to and produce a blasts exhibit characteristic growth patterns that delayed activation of adenylate cyclase in iso- are absent or altered in transformed cells, which lated mouse thymocytes (216). Both toxins bind may be due to the disorganization of cAMP- rapidly and tightly to these cells at 37°C, but dependent cytoskeletal elements found in eucar- CT was seen to have a higher affiniy than LT at yotic cells. Increased cAMP levels have other 8°C. CT also inhibits the synthesis of DNA, effects on fibroblasts in culture (130, 131, 168). RNA, and protein by isolated mouse spleen cells Transport of nucleotides, amino acids, and pro- and thymocytes cultured with , tein synthesis is inhibited. Stimulation of RNA LPS, or phytohemagglutinin (98, 99, 192, 205) and DNA synthesis by epidermal growth factor without affecting viability. Inhibition was cor- is inhibited by CT, dibutyryl cAMP, and theo- related with cAMP levels and paralleled the phylline (95); cGMP, B, colcemid, concentration of toxin (98), and doses that were and vinblastine reverse this inhibition. submaximal for cyclase activation completely inhibited DNA synthesis. The effect of CT on Other Cell Systems antibody production in both primary and sec- CT and LT have been shown to alter mor- ondary responses was variable and dependent phology and in a variety of other on the time of administration relative to antigen cell types. CT enhances the binding of TSH to exposure. Increased numbers of plaque-forming thyroid cells at low concentrations and inhibits cells were detected when the toxin was given binding at high concentrations (153). Both simultaneously with antigen, and plaque-form- agents activate adenylate cyclase, but TSH does ing cells were reduced in number if given after not require NAD for activation. Both agents antigen administration. Both spleen and thymus bind to cell membranes via ganglioside recep- weights were reduced in mice given intravenous tors: TSH to GD1b, GT1, and, to a lesser extent, injections of CT. This effect was related to the Gm, (Fig. 1). effect of CT on the adrenal glands. CT increases glycogenolysis in liver cells and In vitro studies have shown a reduction of (83) and inhibits histamine release, mi- both immediate and delayed hypersensitivity as crobicidal activity, and enzyme release and judged by histamine release from leukocytes and from neutrophils (13, 142). Purified cell-mediated lympholysis (142). Cytotoxic ac- LT has been demonstrated to inhibit neutrophil tivity of toxin-treated cells both in vivo and in chemotaxis in vitro. This effect appears to be vitro prolong allograft survival in CT-treated related to increased intracellular cAMP (9). If animals (98,142). Lymphocytes from CT-treated so, this effect could be of selective advantage to donors are more able to produce a graft-versus- the organism in inhibiting neutrophil migration. host reaction and the "allogenic effect" in recip- Both- LT and CT cause adenylate cyclase acti- ient mice, possibly by inhibiting a suppressor vation in thyroid slices (145), in a human embry- population (98). Theta-antigen co-caps with onic intestinal cell line (121), as well as in two choleragen on mouse thymocytes (195), but epidermal carcinoma cell lines, KB and HEp-2. bound toxin does not interfere with the ability Neuroblastoma, Yoshita ascites hepatoma, and of these cells to be labeled with anti-Thy 1.2 or sarcoma 180 cells (206) are rendered unrespon- with cytolysis by toxin-coated cells. Thus, al- sive to catecholamines by CT. In addition, the VOL. 42, 1978 EFFECTS OF E. COLI AND V. CHOLERAE TOXINS 605 CT response can be altered without affecting the of the lag period and membrane events that response to epinephrine. The pathways by which occur before and during adenylate cyclase acti- activation by each occurs therefore appear to be vation. Further, the structural and evolutionary distinct; one can be blocked without affecting relatedness of the two toxins to themselves, the other. These studies indicate that in this cell other toxins, and hormones has not been clari- system there may be a common catalytic event fied. In addition, the role these molecules may but distinct activation pathways. play in the life cycle ofthe organisms themselves There are numerous cells that are sensitive to has not been examined. the action of both CT and E. coli LT (41, 121, It seems improbable that events such as dis- 159, 187). The diverse changes in metabolism, sociation and diffusion across the lipid bilayer morphology, and response to extracellular mol- can fully account for the length of lag observed ecules such as hormones, catecholamines, and in the cell systems discussed here. More subtle prostaglandins all follow changes in intracellular events at or within the membrane must be cAMP levels. These systems have been used to evoked and may include complex associations of study the diverse functions ofthese extracellular toxin with membrane lipid and protein compo- substances and their relationship to cyclic nu- nents. Although there is evidence of direct inter- cleotides. Keusch and Donta (122) have pointed action of toxin with adenylate cyclase, the exist- out that not all bacterial toxins exert their effect ence of accessory or intermediate membrane through cyclic nucleotides, and the distinction molecules has not been ruled out. between cytotoxic and cytotonic effects should The significance of the similarities between be recognized. Therefore, caution must be exer- the two proteins has yet to be determined. The cised in interpreting these results, especially isolation ofpurified LT and complete amino acid when extrapolating to in vivo systems, where sequence is needed to resolve the nature of their the observed effects may be indirect. relatedness. The role of the various subunits of CT has been well documented. The possibility DISCUSSION that the LT molecule, a single polypeptide chain, There are many common factors in the path- contains two domains, one for binding and one ogenesis of diarrheal disease caused by E. coli for cyclase activation, is attractive. The neces- and V. cholerae. Both gram-negative organisms sity for dissociation of CT and release of the A possess specialized antigens for adhesion to in- subunit before enzyme activation has support testinal epithelium. Both produce protein exo- from various studies. The need for proteolytic toxins that have similar physical and chemical cleavage of LT before enzyme activation, pos- characteristics and immunological cross-reactiv- tulated by some investigators, needs further ity. These toxins rapidly bind to membrane re- study. ceptors composed of or similar to Gm, ganglio- Analogies have been drawn between these side. The biological activity of these toxins is the toxins and diphtheria, botulinal, and tetanus result of interaction with membrane-bound ade- toxins (199). Similarities in membrane receptors, nylate cyclase, leading to persistent elevation in subunit function, interaction with membrane or intracellular levels ofcAMP. Stimulation of ade- cytoplasmic components after subunit dissocia- nylate cyclase in intact cells always occurs after tion, and long-term alteration of normal cellular a characteristic lag phase. Hypersecretion of function are the bases for these analogies. salts and water is the result of a change in The observation that such similar proteins are membrane transport. coded for in one organism by a chromosomal Although the effect ofthese toxins is restricted gene and in another by a plasmid poses further to the intestine in vivo, many cell types are questions about their evolutionary relationship. sensitive to their action when given systemically Analogies to eucaryotic glycoprotein hormones in vivo or to isolated organs and cells in vitro. leads one to speculate on the possibilities of The response observed in these systems may convergent . have no pathological significance but serves to The many studies of the nature and actions of underscore the central role of cyclic nucleotides enterotoxins produced by E. coli and V. cholerae in cellular regulation. already discussed have dealt with the effects on Equally intriguing are the differences between eucaryotic cells and not with the role these these two toxins. The length and the degree of proteins play in the growth of the organisms stimulation of adenylate cyclase point to possi- themselves. Many organisms, both gram positive ble differences in affinity, dissociation, and and gram negative, have a membrane-associated mechanism of activation ofthe cyclase molecule. adenylate cyclase system (108, 123), and cAMP There are three areas in which the study ofthese is elevated within the cell and released into the two toxins has left unanswered questions. Future medium during growth under various conditions studies must address the problem of the nature (163). In E. coli, cAMP is involved in the regu- 606 RICHARDS AND DOUGLAS MICROBIOL. REV. lation of several by both positive and cell adherent immune factor. Infect. Immun. negative control. The gene products are involved 7:35-38. in the synthesis of for carbohydrate 2. Agarwal, S. C., and N. K. Ganguly. 1972. metabolism, amino acid synthesis, and flagellar Experimental oral immunization with L-forms of Vibrio cholerae. Infect. Immun. 5:31-34. elements as well as the regulation of the life 3. Agarwal, S. C., and N. K. Ganguly. 1972. Oral cycle of some phages. cAMP regulation of the immunization with L-forms of Vibrio cholerae expression of the lac has been most in human volunteers. Infect. Immun. 6:17-20. thoroughly studied. 4. Agarwal, S. C., and T. Sundaraj. 1976. Cell- V. cholerae also has been shown to possess an mediated immunity in Vibrio cholerae with adenylate cyclase system. Extracellular cAMP ribonucleic acid-protein fractions of V. chol- levels as well as toxin production vary depending erae L-form lysates. Infect. Immun. 14: on the strain, carbon source, and growth stage 363-367. (73). The possibility that these toxins act as 5. Bailey, W. R., and E. G. Scott. 1970. , 3rd ed. The C. V. Mosby Co., St. signals for the regulation of metabolic events Louis. within the cells has not been excluded. 6. Banwell, J. G., S. L. Gorbach, N. F. Pierce, R. Mitra, and A. Mondal. 1971. Acute undif- CONCLUSIONS ferentiated human in the tropics. II. The discovery of a variety of cellular systems Alterations in intestinal fluid and electrolyte that are exquisitely sensitive to the action of movements. J. Clin. Invest. 50:890-900. enterotoxin V. and enter- 7. Bennet, V., S. Craig, M. D. Hollenberg, E. produced by cholerae O'Keefe, N. Sahyoun, and P. Cuatrecasas. opathogenic strains of E. coli has led to the 1976. Structure and function of cholera toxin purification and characterization of these toxins and hormone receptors. J. Supramol. Struct. and the elucidation of many aspects of their 4:99-120. mode of action and pathology. They have ena- 8. Bennet, V., E. O'Keefe, and P. Cuatrecasas. bled investigators to assess the purity of toxin 1975. Mechanism of action of cholera toxin and preparations during isolation procedures and the mobile receptor theory of hormone recep- have become useful diagnostic tools for the clin- tor-adenylate cyclase interactions. Proc. Natl. ical researcher. In addition, isolated tissue and Acad. Sci. U.S.A. 72:33-37. cellular have to the detailed mech- 9. Bergman, M. J., R. L. Guerrant, F. Murad, S. systems led H. Richardson, D. Weaver, and G. L Man- anism of action of these toxins by dissecting the dell. 1978. Interaction of polymorphonuclear numerous events that occur during their action neutrophils with Escherichia coli. Effect of in vivo. Further knowledge obtained from these enterotoxin on , killing, chemo- studies is being utilized to design effective vac- taxis and cyclic AMP. J. Clin. Invest. cination programs. Aside from the better under- 61:227-234. standing and treatment of diarrheal diseases 10. Berkenbile, F., and R. Delaney. 1976. 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