JPM Vol. 28, No. 2 September 1992:67-72
Pharmacologic Action of Escherichiu coli Heat-Stable (SW Enterotoxin Floyd
C. Knoop*
and Michaela
Owens
Department of Medical Microbiology, Creighton University School of Medicine, Omaha, Nebraska, U.S.A.
Escherichia coli produces a heat-stable (STa) enterotoxin that belongs to a family of peptides that mediate several diarrhea1 diseases, including traveler’s diarrhea and epidemic diarrhea in infants and newborns. The STa enterotoxin consists of 18 or 19 amino acids and is encoded by genes specified on a transposon. Intestinal secretion is induced by specific binding to high affinity receptors that reside on the brush border cell membrane of the small intestine. Receptor activation by STa enterotoxin induces a sequence of events that culminate in the release of fluid and electrolytes into the intestinal lumen. These events include the stimulation of particulate guanylate cyclae and subsequent increase of intracellular cyclic GMP, involvement of particulate protein kinase, elevation of intracellular calcium, and activation of the phosphatidylinositol pathway. The release of arachidonic acid and production of prostaglandins and/or leukotrienes have also been implicated in the action of STa. Evidence indicates that the STa enterotoxin receptor may be a single multifunctional membrane protein. Key words: Heat-stable enterotoxin; kinase; Phosphatidylinositol
Cyclic GMP; Pharmacologic
Historical Perspectives All too often the human bowel is seeded with strains of bacteria that produce a profound diarrhea. This affliction and concomitant sequelae of electrolyte imbalance, fluid depletion, malnutrition, and secondary infection are main causes of debility and death worldwide. Frequently, the etiologic agents isolated from patients with moderate to severe diarrhea are isolates of microorganisms, including Escherichia coli, Vibrio cholerae, Klebsiella pneumoniae, Yersinia enterocolitica, Aeromonas hydrophila, and Pseudomonas spp.,
which induce a fluid response by the production of one or more enterotoxins (Gk. enteron, intestine + Gk. toxikon, poison). These substances are fluid-inducing proteins or polypeptides that have been grouped into two classes based upon heat stability (Sack, 1975). Heat-labile enterotoxins (LT) are inactivated at 56°C
Address reprint requests to Dr. F. C. Knoop, Department of Medical Microbiology, Creighton University School of Medicine, 2500 California Street, Omaha, NE 68178-0001, U.S.A. Received June 25, 1992; revised and accepted June 30, 1992. Journal of Pharmacological and Toxicological Methods 28, 67-72 (1992) 0 1992Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010
action; Calcium; Protein
for 60 min and consist of proteins with a molecular weight of -8O,OOO-100,000 daltons. Invariably, LT enterotoxins are composed of subunits, A and B, that are responsible for activity and binding, respectively. The first purified LT enterotoxin was choleragen, a protein produced by Vibrio cholerae and isolated by the methods of Finkelstein and LoSpalluto (1970). It has been demonstrated that cholera LT enterotoxin has a specific cell receptor, GM1ganglioside, and activates the adenylate cyclase system by catalyzing the NAD-dependent ADP ribosylation of G,,, the stimulatory guanine nucleotide binding protein (Gill and Meren, 1978). Heat-stable enterotoxins (ST) are not inactivated at 95°C for 60 min and consist of small polypeptides with a molecular weight of ~2000 daltons. The ST enterotoxins appear to contain a high molar ratio of cysteine and have a specific cell receptor. Two molecular species, STa and STb, of heat-stable enterotoxin have been identified from E. coli, with STa being soluble in methanol and assayed in suckling mice, and STb being insoluble in methanol and assayed in rabbit ligated ileal loops (Burgess et al., 1980).
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STa Peptide Isolation, Purification, and Molecular Structure The STa enterotoxin from both human and porcine isolates has been purified to homogeneity (Alderete and Robertson, 1978; Madsen and Knoop, 1980). Although the peptide consists of 18 or 19 amino acids, depending on the source and origin of the isolate, 14 of the 15 carboxy-terminal amino acid residues are identical (Thompson and Giannella, 1985). The toxic domain of STa produced by enterotoxigenic E. coli consists of amino acids numbered 5 through 17. The native molecule consists of NH2-ASN-THR-PHE-TYR-CYSCYS-GLU-LEU-CYS-CYS-ASN-PRO-ALA-CYSALA-GLY-CYS-TYR-COOH, and has been synthesized by the solid-phase method (Ozaki et al., 1991). The peptide has a tertiary structure with a right-hand spiral throughout the molecule, starting with the fifth amino acid at the N terminus and ending at the next to last amino acid at the C terminus, and is assembled with three B-turns. The STa molecule appears to be hydrophobic, as all amino acid side chains are oriented to the outside. Amino groups of the amino acids, except CYS6, GLU’, ALA”, and GLY16, are turned inward to the inside of the molecule (Ozaki et al., 1991). The cysteine residues form three intramolecular disulfide linkages at CYS5~‘o, CYS6,14, and CYS9,17 and are geometrically similar to those in proteins. Kubota et al. (1989) have produced a long-acting analog of STa by inserting a single o-amino acid at position 5 in the peptide chain.
STa Gene Structure The LT and ST enterotoxins are specified by plasmids and, under laboratory conditions, can be transferred to other strains of bacteria (Smith and Halls, 1968; Skerman et al., 1972). These plasmids often contain antibiotic-resistance genes and specify the production of ST alone, LT alone, or ST and LT enterotoxin. Further, the ST plasmid has been shown to be a transposon. These elements are distinct DNA segments terminated by repeated sequences, mostly in inverse orientation that transfer at high frequency from replicon to replicon (plasmid t, chromosome or plasmid ++ plasmid). Transposons allow the potential for widespread dissemination of enterotoxicity with the selective pressure of antibiotic usage (So et al., 1979).
Pharmacologic Effects of STa Enterotoxin Effect of STa on Guanylate Cyclase The STa enterotoxin has been shown to stimulate particulate guanylate cyclase in intestinal epithelial
cells and thus elevate the intracellular level of cyclic GMP (Field et al., 1978; Hughes et al., 1978a). Further, it has been demonstrated that the analog 8-bromo cyclic GMP will mimic the effect of STa in animal systems (Hughes et al., 1978a). Studies by Guerrant et al. (1980) and Hughes et al. (1978b) have shown that STa specifically activates the particulate guanylate cyclase of intestinal tissue, without effect on lung, liver, pancreas, or gastric antrum. More recently, Knoop and Thomas (1983) and Forte et al. (1989) have demonstrated the production of cyclic GMP in rat basophils and opossum renal cortex, testis, and small intestine, respectively. Thus, the activity of STa enterotoxin is mediated by the intracellular second messenger cyclic GMP. Such a mechanism of action appears analogous to transmembrane signaling induced by hormones and neuromuscular transmitters.
The Receptor for STa Enterotoxin The nature of the receptor for STa enterotoxin has been studied in a number of cell systems (Giannella and Luttrell, 1982; Thomas and Knoop, 1983; Forte et al., 1989). Specific binding to cultured rat basophils was found to be inhibited by pronase and trypsin, but not by lipid- or carbohydrate-specific enzymes (Thomas and Knoop, 1983). The dissociation constant was 1.33 x 10-‘” M, and specific binding was related to the dose-dependent release of histamine. The rat intestinal receptor for STa enterotoxin was initially solubilized with 3-[(3-cholamidopropyl) dimethylammoniol-1-propanesulfonate and partially characterized (Dreyfus and Robertson, 1984). It was demonstrated that STa bound to a single brush-border membrane component with a molecular weight of -100,000 daltons. Binding was saturable, specific, and susceptible to pretreatment with trypsin and pronase, but not chymotrypsin. In other studies, Kuno et al. (1986) solubilized the STa enterotoxin receptor from rat intestinal brush-border membranes with LubrolPX. The STa enterotoxin bound specifically to a binding region consisting of polypeptides with estimated molecular weights of 60,000, 68,000, and 80,000 daltons. The solubilized receptor had a Stokes radius of 5.5 nm, a sedimentation coefficient of 7.0 S, and a pl of 5.5. Solubilized receptor was separable from particulate guanylate cyclase. Studies by Guarino et al. (1987), using the Ts4-cultured human colonic cell line, demonstrated that specific binding was time, temperature, and pH dependent. Similar dose-response relationships for specific binding and activation of guanylate cyclase indicated that the production of cyclic GMP was coupled to the occupancy of specific receptors by STa enterotoxin. de Sauvage et al. (1991) has reported the cloning and
KNOOP AND OWENS PHARMACOLOGIC ACTION OF
69 ESCHERICHZA COLI HEAT-STABLE
expression of cDNA encoding the human receptor for STa enterotoxin. The receptor was shown to contain an extracellular ligand binding site and an inward membrane-associated catalytic domain of guanylate cyclase. These observations indicate that the STa receptor is a member of the natriuretic peptide receptor family and support the concept of receptor-guanylate cyclases where the hormone binding site and the catalytic activity of guanylate cyclase reside on the same protein. The receptor family contains a single transmembrane domain that separates the molecule into a binding region on the cell surface and a signaling domain on the cytosolic surface. Results by Schulz et al. (1990), using the polymerase chain reaction to identify a unique intestinal-specific guanylate cyclase, showed that the STa enterotoxin receptor is a plasma membrane form of guanylate cyclase. Whether these observations are related to the specific binding of STa enterotoxin to other cell types, including opossum seminiferous tubules and renal cortex, is not known (Forte et al., 1989).
Effect of Pharmacologic of STa Enterotoxin
Agents on the Action
Pharmacologic agents that interrupt or inhibit the STa enterotoxin-mediated fluid response have been investigated (Madsen and Knoop, 1978; Thomas and Knoop, 1982). The cyclooxygenase inhibitor, indomethacin, when administered simultaneously, prechallenge or postchallenge, caused an inhibition of the STamediated fluid response in suckling mice, suggesting a role for prostaglandins or leukotrienes in the secretory response (Madsen and Knoop, 1978). Similar results have been obtained with zomepirac sodium and quinacrine hydrochloride, inhibitors of prostaglandin synthesis (Greenberg et al., 1982; Thomas and Knoop, 1982). These observations have led to the suggestion that the prostaglandin or leukotriene pathway is involved in the STa-mediated fluid response (Knoop and Abbey, 1981; Greenberg et al., 1982). Dreyfus et al. (1984), using radioimmunoassays, was unable to demonstrate an increase in prostaglandins EZ, Fzor or thromboxane B2 when rat intestinal epithelial cells were treated with supraoptimal doses of STa enterotoxin. The effect of calcium and/or calmodulin on the STamediated fluid response has been investigated. Wolff and Brostrom (1979) and Abbey and Knoop (1979) used chlorpromazine, which binds to calmodulin, to show a reduction in the response to STa enterotoxin in suckling mice. Other phenothiazine derivatives similar to chlorpromazine have also shown a reduction in STamediated fluid accumulation (Greenberg et al., 1980; Knoop and Abbey, 1981).
(STa) ENTEROTOXIN
Thomas and Knoop (1982) reported that the calcium channel blockers diltiazem, nifedipine, and cromolyn sodium caused a significant decrease in the action of STa enterotoxin in infant mice. In addition, Knoop and Thomas (1983) have demonstrated that lodoxamide tromethamine and lanthanum, agents that directly block or antagonize calcium, would inhibit the ability of STa enterotoxin to elevate cyclic GMP or stimulate 45Ca2+ uptake in rat basophilic leukemia cells. Dreyfus et al. (1984), using supraoptimal doses of STa enterotoxin and inordinately high levels of calcium channel blockers, demonstrated that both basal- and toxinstimulated levels of particulate guanylate cyclase were inhibited in rat brush-border. membrane preparations, and that calcium was not involved in the STa-mediated intestinal response. More recently, Knoop et al. (1991) investigated the effect of STa enterotoxin on intracellular calcium in primary cultures of rat enterocytes. The enterocytes were loaded with the calcium indicator Indo- 1, and alterations in the level of intracellular calcium were assessed by flow cytometry. Treatment of the cells with EGTA, a calcium chelator, or ionomycin, a calcium ionophore, resulted in a decrease or increase in the level of free intracellular calcium, respectively. When STa enterotoxin was added to the cell system, an initial decrease, followed by a rapid increase, in the level of free intracellular calcium was observed. These observations occurred prior to or during an increase in the intracellular level of cyclic GMP, suggesting an effect on transmembrane regulation of the receptordependent calcium signal and/or the release of calcium from intracellular stores.
Effect of STa Enterotoxin Activity
on Protein Kinase
Several studies have suggested the involvement of an intestinal brush-border protein kinase in the action of STa enterotoxin. DeJonge (1984) observed the presence of a protein kinase in intestinal tissue that appeared to be regulated by cyclic GMP. Chinkers and Garbers (1989) have shown that on the cell surface, or just within the cell membrane, a protein kinase domain exists that is required for receptor-mediated transmembrane signaling. This domain is directly linked to the guanylate cyclase domain. Using phorbol dibutyrate, Weikel et al. (1990) examined the effect of protein kinase C activation on the cyclic GMP response to STa enterotoxin in cultured Tg4 cells. Their results demonstrated that the phorbol analog acts synergistically with STa enterotoxin to elevate intracellular cyclic GMP levels. Knoop et al. (1990) have demonstrated that particulate protein kinase activity in rat enterocytes was increased by STa enterotoxin during a time period when intracellular cyclic GMP increased tenfold.
70
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These results suggest that the action of STa enterotoxin may be related to particulate protein kinase activity, or that protein kinase activity is a requisite for the activation of guanylate cyclase. In other studies, Banik and Ganguly (1988, 1989) have demonstrated that STa enterotoxin stimulates the formation of physiologically active phosphoinositides and diacylglycerol in rat enterocytes. The production of these messengers results in the mobilization of internal calcium from storage sites and the stimulation of protein kinase C activity (Berridge, 1984).
Conclusion The heat-stable
enterotoxin
produced
by Escheresulting in diarrhea1 illness and disease in both humans and animals. Elucidation of sequences in the cascade of events involved in the pharmacologic action of STa enterotoxin has enhanced studies related to cellular transduction and signal amplification. The STa enterorichia coli induces intestinal fluid accumulation,
toxin exists on a transposon that codes for a low-molecular weight polypeptide that binds to a specific cell membrane receptor (Giannella et al., 1983; Thomas and Knoop, 1983; Forte et al., 1989), stimulates membrane bound guanylate cyclase (Field et al., 1978; Hughes et al., 1978b), and induces a cellular response (Sack, 1975; Burgess et al., 1980). A number of studies have revealed important steps in the pharmacologic action of STa enterotoxin (Figure 1). These studies include the STa-activated breakdown of phosphoinositides, resulting in production of the second messengers inositol 1,4,5_trisphosphate and diacylglycerol (Banik and Ganguly, 1988), the release of arachidonic acid related to the metabolism of diacylglycerol (Banik and Ganguly, 1989), and the elevation of protein kinase activity and intracellular calcium levels (Knoop et al., 1990, 1991). These observations and others (Chinkers and Garbers, 1989; Weikel et al., 1990) suggest that the receptor for STa enterotoxin is a particulate guanylate cyclase composed of a protein kinase-like portion and a catalytic portion (Figure 1).
Figure 1. Schematic model for the induction of a cellular response by Escherichia coli STa enterotoxin. The mechanisms for an increase in calcium level, stimulation of guanylate cyclase, and elevation of inositol trisphosphate, diacylglycerol, and protein kinase activity are depicted. Ca+ +, calcium; R, receptor domain; PK-L, protein kinase-like domain; C, catalytic domain; GTP, guanosine 5’-triphosphate; cGMP, cyclic 3’,5’-guanosine monophosphate; [II-PDE, inactive phosphodiesterase; [A]-PDE, active phosphodiesterase; PtdIns 4,5P2, phosphatidylinositol 4,5-diphosphate; Ins 1,4,5P3, inositol 1,4,5 trisphosphate; DAG, diacylglycerol; PKC, protein kinase C; [A]-Ca+ + PK, calcium-activated protein kinase; AA, arachidonic acid; and 8BR-cGMP, 8-bromo cyclic 3’,5’-guanosine monophosphate. S’l‘a hterotoxin
CELL EXTERIOR
PtdIns [I]-POE
q [A]-PDE
4,5P2
/
Ins 1,4,5P3 JI Internal
+
Ca++ (+ )
DAG JI PKC r
\k [ A]-Ca++ PK -
CELL CYTOSOL
KNOOP AND OWENS PHARMACOLOGIC ACTION OF ESCHERICHZA COLI HEAT-STABLE
It would appear that receptor mediation stimulates an initial uptake in calcium. The activation of guanylate cyclase leads, directly or indirectly, to the activation of phosphodiesterase and the subsequent breakdown of polyphosphatidylinositol to form diacylglycerol and inositol 1,4,Wrisphosphate. These mediators result in the stimulation of protein kinase C, increase of arachidonic acid, and release of internal calcium from cytosolic storage sites. The activation of protein kinase C and release of arachidonic acid, resulting in the production of prostagladins, leukotrienes, and thromboxane, may promote the cellular response and thus release of fluid from the cytosolic compartment. It is now recognized that each step of the pathway plays an integral part in the cascade of events that leads to the induction of a cellular response.
References
71 (STa) ENTEROTOXIN
Forte LR, Krause WJ, Freeman RH (1989) Escherichiu coli enterotoxin receptors: Localization in opossum kidney, intestine and testis. Am J Physiol257:F874-F881. Giannella RA, Luttrell M (1982) Binding of heat-stable enterotoxin to small intestinal brush border receptors. Gustroenterology 82: 1065. Giannella RA, Luttrell M, Thompson M (1983) Binding of Escherichia coli heat-stable enterotoxin to receptors on rat intestinal cells. Am J Physiol 245:G492-G498. Gill DM, Meren R (1978) ADP-ribosylation of membrane proteins catalyzed by cholera toxin: Basis of the activation of adenylate cyclase. Proc Nut1 Acud Sci USA 75:3050-3054. Greenberg RN, Chang B, Murad F, Guerrant RL (1980) Inhibition of E. co/i heat-stable enterotoxin by phenothiazine derivatives and indomethacin. Clin Res 28:369A. Greenberg RN, Guerrant RL, Chang B, Robertson DC, Murad F (1982) Inhibition of Escherichia coli heat-stable enterotoxin effects on intestinal guanylate cyclase and fluid secretion by quinacrine. Biochem Pharmucol31:2005-2009. Guarino A, Cohen M, Thompson M, Charmsathaphom K, Giannella R (1987) Tad cell receptor binding and guanylate cyclase activation by Escherichia coli heat-stable toxin. Am J Physiol 253: G775-780.
Abbey DM, Knoop FC (1979) Effect of chlorpromazine on the secretory activity of Escherichia coli heat-stable enterotoxin. Infect Zmmun 26:1000-1003.
Guerrant RL, Hughes JM, Chang B, Robertson DC, Murad R (1980) Activation of rat and rabbit intestinal guanylate cyclase by the heat-stable enterotoxin of Escherichiu coli: Studies of tissue specificity, potential receptors and intermediates. J Infect Dis 142:220-228.
Alderete JF, Robertson DC (1978) Purification and chemical characterization of the heat-stable enterotoxin produced by porcine strains of enterotoxigenic Escherichia coli. Infect Immun 19: 1021-1030.
Hughes JM, Murad F, Guerrant RL (1978a) Studies to elucidate the mechanism of action of heat-stable enterotoxin of Escherichiu coli. Clin Res 26:524A.
Banik N, Ganguly U (1988) Stimulation of phosphoinositides breakdown by the heat-stable E. colt’ enterotoxin in rat intestinal epithelial cells. FEBS Lett 236:489-492. Banik ND, Ganguly U (1989) Diacylglycerol breakdown in plasma membrane of rat intestinal epithelial cells. Effect of E. coli heatstable enterotoxin. FEBS Lett 236:201-204. Berridge MJ (1984) Inositol trisphosphate and diacylglycerol as second messengers. Biochem J 220:345-360. Burgress MN, Mullan NA, Newsome PM (1980) Heat-stable enterotoxins from Escherichiu co/i. Infect Zmmun 28:1038-1040. Chinkers M, Garbers DL (1989) The protein kinase domain of the ANP receptor is requried for signaling. Science 245: 1392-1394. DeJonge HR (1984) The mechanism of action of Escherichia co/i heat-stable toxin. Biochem Sot Truns 12:180-184. de Sauvage FJ, Camerato TR, Goeddel DV (1991) Primary structure and functional expression of the human receptor for Escherichia coii heat-stable enterotoxin. J Biol Chem 266:17912-17918.
Hughes JM, Murad F, Chang B, Guerrant RL (1978b) Role of cyclic GMP in the action of heat-stable enterotoxin of Escherichiu co/i. Nature 2711755-756. Knoop FC, Abbey DM (1981) Effect of chemical and pharmacological agents on the secretory activity induced by Escherichia coli heat-stable enterotoxin. Can J Microbial 27:754-758. Knoop FC, Thomas DD (1983) Stimulation of calcium uptake and cyclic GMP synthesis in rat basophilic leukemia cells by Escherichiu coli heat-stable enterotoxin. Infect Immun 41:971-977. Knoop FC, Martig RJ, Boehm WJ (1990) The effect of Escherichiu coli heat-stable enterotoxin on protein kinase activity. Toxicon 28:493-500. Knoop FC, Owens M, Marcus JN, Murphy B (1991) Elevation of calcium in rat enterocytes by Escherichia coli heat-stable (STa) enterotoxin. Curr Microbial 23:291-296. Kubota H, Hidaka Y, Ozaki H, Ito H, Hirayama T, Takeda Y, Shimonishi Y (1989) A long-acting heat-stable enterotoxin analog of enterotoxigenic Escherichia coli with a single o-amino acid. Biochem Biophys Res Comm 161:229-235.
Dreyfus LA, Robertson DC (1984) Solubilization and partial characterization of the intestinal receptor for Escherichiu coli heat-stable enterotoxin. Infect Zmmun 46~537-543.
Kuno T, Kamisaki Y, Waldman SA, Gariepy J, Schoolnik G, Murad F (1986) Characterization of the receptor for heat-stable enterotoxin from Escherichiu coli in rat intestine. J Biol Chem 261: 1470-1476.
Dreyfus LA, Jaso-Friedman L, Robertson DC (1984) Characterization of the mechanism of action of Escherichiu co/i heat-stable enterotoxin. Infect lmmun 44:493-501.
Madsen GL, Knoop FC (1978) Inhibition of the secretory activity of Escherichia coli heat-stable enterotoxin by indomethacin. Infect Immun 22:143-147.
Field M, Graf LH Jr., Laird WJ, Smith PL (1978) Heat-stable enterotoxin of Escherichiu cob: in vitro effects on guanylate cyclase, cyclic GMP concentration, and ion transport in small intestine. Proc Nut1 Acad Sci USA 75:28OO-2804.
Madsen GL, Knoop FC (1980) Physiochemical properties of a heatstable enterotoxin produced by Escherichiu coli of human origin. Infect Immun 28:1051-1053.
Finkelstein RA, LoSpalluto JJ (1970) Production of highly purified choleragen and choleragenoid. J Infect Dis 121(Suppl): S63-S72.
Ozaki H, Sato T, Kubota H, Hata Y, Katsube Y, Shimonishi Y (1991) Molecular structure of the toxic domain of heat-stable enterotoxin produced by a pathogenic strain of Escherichia coli. J Biol Chem 266:5934-5941.
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Sack RB (1975) Human diarrhea1 diseases caused by enterotoxigenic Escherichia coli. Ann Rev Microbial 29~333-353. Schulz S, Green CK, Yuen PST, Garbers DL (1990) Guanylyl cyclase is a heat-stable enterotoxin receptor. Ce// 63:941-948. Skerman FJ, Formal SB, Falkow S (1972) Plasmid-associated enterotoxin production in a strain of Escherichia coli isolated from humans. Infect Immun 51622-624. Smith HW, Halls S (1968) The transmissible nature of the genetic factor in Escherichia coli that controls enterotoxin production. J Gen Microbial 52:319-334. So M, Heffron F, McCarthy B (1979) The E. coli gene encoding heatstable toxin is a bacterial transposon flanked by inverted repeats of ISI. Nature 2771453-456. Thomas DD, Knoop FC (1982) The effect of calcium and prostaglan-
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din inhibitors on the intestinal fluid response to heat-stable enterotoxin of Escherichia co/i. J Infect Dis 145:141-147. Thomas DD, Knoop FC (1983) Effect of heat-stable enterotoxin of Escherichia coli on cultured mammalian cells. J Infect Dis 147: 450-459. Thompson MR, Giannella RA (1985) Revised amino acid sequence for a heat-stable enterotoxin produced by an Escherichia co/i (18D) that is pathogenic for humans. Infect Zmmun 47:834-836. Weikel CS, Spann CL, Chambers CP, Crane JK, Linden J, Hewlett EL (1990) Phorbol esters enhance the cyclic GMP response of Ts4 cells to the heat-stable enterotoxin of Escherichia coli (STa). Infect Immun 58:1402-1407. Wolff DJ, Brostrom CO (1979) Properties and functions of the calcium-dependent regulator protein. Adv Cyclic Nucleotide Protein Phosphotylation Res 11:27-88.
Pharmacologic Action of Escherichiu coli Heat-Stable (SW Enterotoxin Floyd
C. Knoop*
and Michaela
Owens
Department of Medical Microbiology, Creighton University School of Medicine, Omaha, Nebraska, U.S.A.
Escherichia coli produces a heat-stable (STa) enterotoxin that belongs to a family of peptides that mediate several diarrhea1 diseases, including traveler’s diarrhea and epidemic diarrhea in infants and newborns. The STa enterotoxin consists of 18 or 19 amino acids and is encoded by genes specified on a transposon. Intestinal secretion is induced by specific binding to high affinity receptors that reside on the brush border cell membrane of the small intestine. Receptor activation by STa enterotoxin induces a sequence of events that culminate in the release of fluid and electrolytes into the intestinal lumen. These events include the stimulation of particulate guanylate cyclae and subsequent increase of intracellular cyclic GMP, involvement of particulate protein kinase, elevation of intracellular calcium, and activation of the phosphatidylinositol pathway. The release of arachidonic acid and production of prostaglandins and/or leukotrienes have also been implicated in the action of STa. Evidence indicates that the STa enterotoxin receptor may be a single multifunctional membrane protein. Key words: Heat-stable enterotoxin; kinase; Phosphatidylinositol
Cyclic GMP; Pharmacologic
Historical Perspectives All too often the human bowel is seeded with strains of bacteria that produce a profound diarrhea. This affliction and concomitant sequelae of electrolyte imbalance, fluid depletion, malnutrition, and secondary infection are main causes of debility and death worldwide. Frequently, the etiologic agents isolated from patients with moderate to severe diarrhea are isolates of microorganisms, including Escherichia coli, Vibrio cholerae, Klebsiella pneumoniae, Yersinia enterocolitica, Aeromonas hydrophila, and Pseudomonas spp.,
which induce a fluid response by the production of one or more enterotoxins (Gk. enteron, intestine + Gk. toxikon, poison). These substances are fluid-inducing proteins or polypeptides that have been grouped into two classes based upon heat stability (Sack, 1975). Heat-labile enterotoxins (LT) are inactivated at 56°C
Address reprint requests to Dr. F. C. Knoop, Department of Medical Microbiology, Creighton University School of Medicine, 2500 California Street, Omaha, NE 68178-0001, U.S.A. Received June 25, 1992; revised and accepted June 30, 1992. Journal of Pharmacological and Toxicological Methods 28, 67-72 (1992) 0 1992Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010
action; Calcium; Protein
for 60 min and consist of proteins with a molecular weight of -8O,OOO-100,000 daltons. Invariably, LT enterotoxins are composed of subunits, A and B, that are responsible for activity and binding, respectively. The first purified LT enterotoxin was choleragen, a protein produced by Vibrio cholerae and isolated by the methods of Finkelstein and LoSpalluto (1970). It has been demonstrated that cholera LT enterotoxin has a specific cell receptor, GM1ganglioside, and activates the adenylate cyclase system by catalyzing the NAD-dependent ADP ribosylation of G,,, the stimulatory guanine nucleotide binding protein (Gill and Meren, 1978). Heat-stable enterotoxins (ST) are not inactivated at 95°C for 60 min and consist of small polypeptides with a molecular weight of ~2000 daltons. The ST enterotoxins appear to contain a high molar ratio of cysteine and have a specific cell receptor. Two molecular species, STa and STb, of heat-stable enterotoxin have been identified from E. coli, with STa being soluble in methanol and assayed in suckling mice, and STb being insoluble in methanol and assayed in rabbit ligated ileal loops (Burgess et al., 1980).
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STa Peptide Isolation, Purification, and Molecular Structure The STa enterotoxin from both human and porcine isolates has been purified to homogeneity (Alderete and Robertson, 1978; Madsen and Knoop, 1980). Although the peptide consists of 18 or 19 amino acids, depending on the source and origin of the isolate, 14 of the 15 carboxy-terminal amino acid residues are identical (Thompson and Giannella, 1985). The toxic domain of STa produced by enterotoxigenic E. coli consists of amino acids numbered 5 through 17. The native molecule consists of NH2-ASN-THR-PHE-TYR-CYSCYS-GLU-LEU-CYS-CYS-ASN-PRO-ALA-CYSALA-GLY-CYS-TYR-COOH, and has been synthesized by the solid-phase method (Ozaki et al., 1991). The peptide has a tertiary structure with a right-hand spiral throughout the molecule, starting with the fifth amino acid at the N terminus and ending at the next to last amino acid at the C terminus, and is assembled with three B-turns. The STa molecule appears to be hydrophobic, as all amino acid side chains are oriented to the outside. Amino groups of the amino acids, except CYS6, GLU’, ALA”, and GLY16, are turned inward to the inside of the molecule (Ozaki et al., 1991). The cysteine residues form three intramolecular disulfide linkages at CYS5~‘o, CYS6,14, and CYS9,17 and are geometrically similar to those in proteins. Kubota et al. (1989) have produced a long-acting analog of STa by inserting a single o-amino acid at position 5 in the peptide chain.
STa Gene Structure The LT and ST enterotoxins are specified by plasmids and, under laboratory conditions, can be transferred to other strains of bacteria (Smith and Halls, 1968; Skerman et al., 1972). These plasmids often contain antibiotic-resistance genes and specify the production of ST alone, LT alone, or ST and LT enterotoxin. Further, the ST plasmid has been shown to be a transposon. These elements are distinct DNA segments terminated by repeated sequences, mostly in inverse orientation that transfer at high frequency from replicon to replicon (plasmid t, chromosome or plasmid ++ plasmid). Transposons allow the potential for widespread dissemination of enterotoxicity with the selective pressure of antibiotic usage (So et al., 1979).
Pharmacologic Effects of STa Enterotoxin Effect of STa on Guanylate Cyclase The STa enterotoxin has been shown to stimulate particulate guanylate cyclase in intestinal epithelial
cells and thus elevate the intracellular level of cyclic GMP (Field et al., 1978; Hughes et al., 1978a). Further, it has been demonstrated that the analog 8-bromo cyclic GMP will mimic the effect of STa in animal systems (Hughes et al., 1978a). Studies by Guerrant et al. (1980) and Hughes et al. (1978b) have shown that STa specifically activates the particulate guanylate cyclase of intestinal tissue, without effect on lung, liver, pancreas, or gastric antrum. More recently, Knoop and Thomas (1983) and Forte et al. (1989) have demonstrated the production of cyclic GMP in rat basophils and opossum renal cortex, testis, and small intestine, respectively. Thus, the activity of STa enterotoxin is mediated by the intracellular second messenger cyclic GMP. Such a mechanism of action appears analogous to transmembrane signaling induced by hormones and neuromuscular transmitters.
The Receptor for STa Enterotoxin The nature of the receptor for STa enterotoxin has been studied in a number of cell systems (Giannella and Luttrell, 1982; Thomas and Knoop, 1983; Forte et al., 1989). Specific binding to cultured rat basophils was found to be inhibited by pronase and trypsin, but not by lipid- or carbohydrate-specific enzymes (Thomas and Knoop, 1983). The dissociation constant was 1.33 x 10-‘” M, and specific binding was related to the dose-dependent release of histamine. The rat intestinal receptor for STa enterotoxin was initially solubilized with 3-[(3-cholamidopropyl) dimethylammoniol-1-propanesulfonate and partially characterized (Dreyfus and Robertson, 1984). It was demonstrated that STa bound to a single brush-border membrane component with a molecular weight of -100,000 daltons. Binding was saturable, specific, and susceptible to pretreatment with trypsin and pronase, but not chymotrypsin. In other studies, Kuno et al. (1986) solubilized the STa enterotoxin receptor from rat intestinal brush-border membranes with LubrolPX. The STa enterotoxin bound specifically to a binding region consisting of polypeptides with estimated molecular weights of 60,000, 68,000, and 80,000 daltons. The solubilized receptor had a Stokes radius of 5.5 nm, a sedimentation coefficient of 7.0 S, and a pl of 5.5. Solubilized receptor was separable from particulate guanylate cyclase. Studies by Guarino et al. (1987), using the Ts4-cultured human colonic cell line, demonstrated that specific binding was time, temperature, and pH dependent. Similar dose-response relationships for specific binding and activation of guanylate cyclase indicated that the production of cyclic GMP was coupled to the occupancy of specific receptors by STa enterotoxin. de Sauvage et al. (1991) has reported the cloning and
KNOOP AND OWENS PHARMACOLOGIC ACTION OF
69 ESCHERICHZA COLI HEAT-STABLE
expression of cDNA encoding the human receptor for STa enterotoxin. The receptor was shown to contain an extracellular ligand binding site and an inward membrane-associated catalytic domain of guanylate cyclase. These observations indicate that the STa receptor is a member of the natriuretic peptide receptor family and support the concept of receptor-guanylate cyclases where the hormone binding site and the catalytic activity of guanylate cyclase reside on the same protein. The receptor family contains a single transmembrane domain that separates the molecule into a binding region on the cell surface and a signaling domain on the cytosolic surface. Results by Schulz et al. (1990), using the polymerase chain reaction to identify a unique intestinal-specific guanylate cyclase, showed that the STa enterotoxin receptor is a plasma membrane form of guanylate cyclase. Whether these observations are related to the specific binding of STa enterotoxin to other cell types, including opossum seminiferous tubules and renal cortex, is not known (Forte et al., 1989).
Effect of Pharmacologic of STa Enterotoxin
Agents on the Action
Pharmacologic agents that interrupt or inhibit the STa enterotoxin-mediated fluid response have been investigated (Madsen and Knoop, 1978; Thomas and Knoop, 1982). The cyclooxygenase inhibitor, indomethacin, when administered simultaneously, prechallenge or postchallenge, caused an inhibition of the STamediated fluid response in suckling mice, suggesting a role for prostaglandins or leukotrienes in the secretory response (Madsen and Knoop, 1978). Similar results have been obtained with zomepirac sodium and quinacrine hydrochloride, inhibitors of prostaglandin synthesis (Greenberg et al., 1982; Thomas and Knoop, 1982). These observations have led to the suggestion that the prostaglandin or leukotriene pathway is involved in the STa-mediated fluid response (Knoop and Abbey, 1981; Greenberg et al., 1982). Dreyfus et al. (1984), using radioimmunoassays, was unable to demonstrate an increase in prostaglandins EZ, Fzor or thromboxane B2 when rat intestinal epithelial cells were treated with supraoptimal doses of STa enterotoxin. The effect of calcium and/or calmodulin on the STamediated fluid response has been investigated. Wolff and Brostrom (1979) and Abbey and Knoop (1979) used chlorpromazine, which binds to calmodulin, to show a reduction in the response to STa enterotoxin in suckling mice. Other phenothiazine derivatives similar to chlorpromazine have also shown a reduction in STamediated fluid accumulation (Greenberg et al., 1980; Knoop and Abbey, 1981).
(STa) ENTEROTOXIN
Thomas and Knoop (1982) reported that the calcium channel blockers diltiazem, nifedipine, and cromolyn sodium caused a significant decrease in the action of STa enterotoxin in infant mice. In addition, Knoop and Thomas (1983) have demonstrated that lodoxamide tromethamine and lanthanum, agents that directly block or antagonize calcium, would inhibit the ability of STa enterotoxin to elevate cyclic GMP or stimulate 45Ca2+ uptake in rat basophilic leukemia cells. Dreyfus et al. (1984), using supraoptimal doses of STa enterotoxin and inordinately high levels of calcium channel blockers, demonstrated that both basal- and toxinstimulated levels of particulate guanylate cyclase were inhibited in rat brush-border. membrane preparations, and that calcium was not involved in the STa-mediated intestinal response. More recently, Knoop et al. (1991) investigated the effect of STa enterotoxin on intracellular calcium in primary cultures of rat enterocytes. The enterocytes were loaded with the calcium indicator Indo- 1, and alterations in the level of intracellular calcium were assessed by flow cytometry. Treatment of the cells with EGTA, a calcium chelator, or ionomycin, a calcium ionophore, resulted in a decrease or increase in the level of free intracellular calcium, respectively. When STa enterotoxin was added to the cell system, an initial decrease, followed by a rapid increase, in the level of free intracellular calcium was observed. These observations occurred prior to or during an increase in the intracellular level of cyclic GMP, suggesting an effect on transmembrane regulation of the receptordependent calcium signal and/or the release of calcium from intracellular stores.
Effect of STa Enterotoxin Activity
on Protein Kinase
Several studies have suggested the involvement of an intestinal brush-border protein kinase in the action of STa enterotoxin. DeJonge (1984) observed the presence of a protein kinase in intestinal tissue that appeared to be regulated by cyclic GMP. Chinkers and Garbers (1989) have shown that on the cell surface, or just within the cell membrane, a protein kinase domain exists that is required for receptor-mediated transmembrane signaling. This domain is directly linked to the guanylate cyclase domain. Using phorbol dibutyrate, Weikel et al. (1990) examined the effect of protein kinase C activation on the cyclic GMP response to STa enterotoxin in cultured Tg4 cells. Their results demonstrated that the phorbol analog acts synergistically with STa enterotoxin to elevate intracellular cyclic GMP levels. Knoop et al. (1990) have demonstrated that particulate protein kinase activity in rat enterocytes was increased by STa enterotoxin during a time period when intracellular cyclic GMP increased tenfold.
70
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These results suggest that the action of STa enterotoxin may be related to particulate protein kinase activity, or that protein kinase activity is a requisite for the activation of guanylate cyclase. In other studies, Banik and Ganguly (1988, 1989) have demonstrated that STa enterotoxin stimulates the formation of physiologically active phosphoinositides and diacylglycerol in rat enterocytes. The production of these messengers results in the mobilization of internal calcium from storage sites and the stimulation of protein kinase C activity (Berridge, 1984).
Conclusion The heat-stable
enterotoxin
produced
by Escheresulting in diarrhea1 illness and disease in both humans and animals. Elucidation of sequences in the cascade of events involved in the pharmacologic action of STa enterotoxin has enhanced studies related to cellular transduction and signal amplification. The STa enterorichia coli induces intestinal fluid accumulation,
toxin exists on a transposon that codes for a low-molecular weight polypeptide that binds to a specific cell membrane receptor (Giannella et al., 1983; Thomas and Knoop, 1983; Forte et al., 1989), stimulates membrane bound guanylate cyclase (Field et al., 1978; Hughes et al., 1978b), and induces a cellular response (Sack, 1975; Burgess et al., 1980). A number of studies have revealed important steps in the pharmacologic action of STa enterotoxin (Figure 1). These studies include the STa-activated breakdown of phosphoinositides, resulting in production of the second messengers inositol 1,4,5_trisphosphate and diacylglycerol (Banik and Ganguly, 1988), the release of arachidonic acid related to the metabolism of diacylglycerol (Banik and Ganguly, 1989), and the elevation of protein kinase activity and intracellular calcium levels (Knoop et al., 1990, 1991). These observations and others (Chinkers and Garbers, 1989; Weikel et al., 1990) suggest that the receptor for STa enterotoxin is a particulate guanylate cyclase composed of a protein kinase-like portion and a catalytic portion (Figure 1).
Figure 1. Schematic model for the induction of a cellular response by Escherichia coli STa enterotoxin. The mechanisms for an increase in calcium level, stimulation of guanylate cyclase, and elevation of inositol trisphosphate, diacylglycerol, and protein kinase activity are depicted. Ca+ +, calcium; R, receptor domain; PK-L, protein kinase-like domain; C, catalytic domain; GTP, guanosine 5’-triphosphate; cGMP, cyclic 3’,5’-guanosine monophosphate; [II-PDE, inactive phosphodiesterase; [A]-PDE, active phosphodiesterase; PtdIns 4,5P2, phosphatidylinositol 4,5-diphosphate; Ins 1,4,5P3, inositol 1,4,5 trisphosphate; DAG, diacylglycerol; PKC, protein kinase C; [A]-Ca+ + PK, calcium-activated protein kinase; AA, arachidonic acid; and 8BR-cGMP, 8-bromo cyclic 3’,5’-guanosine monophosphate. S’l‘a hterotoxin
CELL EXTERIOR
PtdIns [I]-POE
q [A]-PDE
4,5P2
/
Ins 1,4,5P3 JI Internal
+
Ca++ (+ )
DAG JI PKC r
\k [ A]-Ca++ PK -
CELL CYTOSOL
KNOOP AND OWENS PHARMACOLOGIC ACTION OF ESCHERICHZA COLI HEAT-STABLE
It would appear that receptor mediation stimulates an initial uptake in calcium. The activation of guanylate cyclase leads, directly or indirectly, to the activation of phosphodiesterase and the subsequent breakdown of polyphosphatidylinositol to form diacylglycerol and inositol 1,4,Wrisphosphate. These mediators result in the stimulation of protein kinase C, increase of arachidonic acid, and release of internal calcium from cytosolic storage sites. The activation of protein kinase C and release of arachidonic acid, resulting in the production of prostagladins, leukotrienes, and thromboxane, may promote the cellular response and thus release of fluid from the cytosolic compartment. It is now recognized that each step of the pathway plays an integral part in the cascade of events that leads to the induction of a cellular response.
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71 (STa) ENTEROTOXIN
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