Escherichia coli cytotoxins and enterotoxins.

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Escherichia coli cytotoxins and enterotoxins CARLTON L. GYLES Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ont., Canada NIG 2 WI Received July 15, 1991

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Revision received January 15, 1992 Accepted January 20, 1992 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734 Escherichia coli verotoxins or Shiga-like toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734 History and nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734 Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736 Physicochemical features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736 Bio1,ogical activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736 Mechanism of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737 Association with diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737 Other E. coli Vero cell cytotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738 Vir cytotoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738 Cytotoxic necrotizing factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738 Cytolethal distending toxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738 Escherichia coli enterotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738 History and nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738 Association with disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738 LT-I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 739 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 739 LT-I1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 739 Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 739 Mechanism of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 740 E. coli heat-stable enterotoxin ST1 (STa) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 740 Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 740 Physicochemical features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 740 Mechanism of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 740 ST11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742 Can. J . Microbiol. 38: 734-746 (1992)

Introduction Certain strains of Escherichia coli produce protein toxins, many of which appear to play a role in disease. These include alpha hemolysin, enterohemolysin, verotoxins (Shiga-like toxins), vir cytotoxin, cytotoxic necrotising factor (CNF), cytolethal distending toxin (CLDT), and enterotoxins. Biologic effects of these toxins and their association with E. coli implicated in various diseases are shown in Table 1. This review will be limited to the verotoxins and enterotoxins but will include a brief mention of vir cytotoxin, CNF, and CLDT. Escherichia coli verotoxins or Shiga-like toxins History and nomenclature In 1977 Konowalchuk and colleagues applied the name verotoxin (VT) to an E. coli cytotoxin that was lethal for Vero cells. In a study of 136 E. coli isolates, 7 of 10 VTpositive strains were associated with diarrheal disease in humans and 1 was associated with diarrhea in pigs. In a preliminary report in 1977 (O'Brien et al. 1977) and a full report in 1982, O'Brien et al. reported their discovery that certain Printed in Canada / lmprime au Canada

enteropathogenic (EPEC) strains of E. coli produced a cytotoxin that was lethal for HeLa cells. Like Shiga toxin, this cytotoxin inhibited protein synthesis in HeLa cells, was enterotoxic in rabbit intestine, and was lethal for mice. This toxin was, therefore, called Shiga-like toxin (SLT). Among the SLT-positive strains examined by O'Brien et al. (1982) was strain H30, which had previously been shown to be VT positive (Konowalchuk et al. 1977). In 1983, O'Brien and LaVeck purified SLT and demonstrated that Shiga toxin produced by Shigella dysenteriae and SLT produced by E. coli strain H30 were identical in physicochemical characteristics and biological activities. The first study by Konowalchuk et al. (1977) demonstrated that verotoxin produced by one porcine and one human E. coli isolate was not neutralized by antiserum that neutralized the VT of strain H30 and several other strains of human origin. Subsequently, several research groups characterized two antigenic types of VT (Scotland et al. 1985; Karmali et al. 1986; Strockbine et al. 1986). These two types of VT were called VT1 and VT2 (Scotland et al. 1985) or SLT-I and SLT-I1 (Strockbine et al. 1986). Subsequently,

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TABLE1 . Association of protein toxins of E. coli with disease Toxins

Biologic effects

Alpha hemolysin

Cytotoxic for a variety of cell types Lytic for eythrocytes Damage to vascular endothelium Cytotoxic for cell cultures Cytotoxic for cell cultures Cytotoxic for cell cultures Impair electrolyte metabolism across intestinal epithelium


Enterohemolysin Verotoxins CLDT CNF Vir cytotoxin Enterotoxins

Types of disease Bacteremia, UTI HUS, HC HUS, HC, diarrhea Diarrhea Diarrhea, bacteremia Bacteremia Diarrhea

NOTE:UTI, urinary tract infection; HUS, hemolytic uremic syndrome; HC, hemorrhagic colitis; CLDT, cytolethal distending toxin; CNF, cytotoxic necrotizing factor.

TABLE2. Nomenclature for verotoxins and Shiga-like toxins and the genes that encode them Gene product SLT terminology

VT terminology

Gene encoding cytotoxin

Reference strain(s)

SLT-I SLT-I1 SLT-IIv SLT-IIvha SLT-IIvhb SLT-IIva

VT1 VT2 VTe VT2v-a VT2v-b VTev

slt-I slt-II slt-v vtx2ha vtx2hb slt-IIva

H19, H30 933w, C600(pEB 1) 412, S1191 B2F 1 B2F 1 H.I.8

other antigenic types of SLT, all related to SLT-11, have been identified (Marques et al. 1987; Gannon et al. 1990; Ito et al. 1990). The SLT and VT systems of nomenclature for clearly characterized verotoxins and for the genes that encode them are shown in Table 2. Neutralization studies have shown that SLTs belong to two antigenic groups. Group I consists of a single antigenic type known as VT1 or SLT-I, whose activity is neutralized by antiserum to Shiga toxin (O'Brien et al. 1982). Group I1 consists of all the other VTs that have been identified so far. They constitute a group of antigenically closely related SLTs, whose cytotoxicity is not neutralized by anti-SLT-I serum or by anti-Shiga toxin antibodies. Members of group I1 include SLT-I1 variant (SLT-IIv or VTe), which is implicated in edema disease of pigs (Smith et al. 1983; Marques et al. 1987; Gannon et al. 1988; MacLeod et al. 1991b). This toxin was called a variant because, unlike SLT-11, it is inactive or weakly active on HeLa cells. SLTIIva is another variant, which is produced by an 0128 E. coli recovered from diarrhea in an infant (Gannon et al. 1990). Genetics The genes for certain VTs were first associated with temperate bacteriophages by British workers (Smith et al. 1983; Scotland et al. 1983). The genes for SLT-I were subsequently cloned from bacteriophages and their nucleotide sequence was determined (De Grandis et al. 1987; Huang et al. 1986; Jackson et al. 1987b). The SLT-I operon consists of two open reading frames which code for an A subunit and a smaller B subunit (Table 2). The deduced amino acid sequence of SLT-I differs from that of Shiga toxin by one amino acid in the B subunit (Calderwood et al. 1987; Strockbine et al. 1988). In one strain of E. coli the amino acid sequence of SLT-I is identical with that of Shiga toxin (Takao et al. 1988).

The nucleotide sequence for SLT-I1 from E. coli strain 933 was determined and compared with that of SLT-I by Jackson et al. (1987a). The structural genes for the two toxins shared 57% nucleotide sequence homology in their A subunit genes and 60% in their B subunit genes. The genes for SLT-IIv appear not to be associated with bacteriophages (Smith et al. 1983; Marques et al. 1987). The genes for SLT-IIv were cloned and sequenced from total genomic DNA of two edema disease strains of E. coli of serogroup 0139 (Gyles et al. 1988; Weinstein et al. 1988b). There was 91% overall nucleotide sequence similarity between the genes for SLT-I1 and SLT-IIv, with the A subunit genes having greater sequence similarity than the B subunit genes. Nucleotide sequence analysis of the cloned genes for SLT-IIva showed that the DNA sequence of the B subunit was 98% homologous with that of the B subunit of SLT-IIv but that the homology between the sequences of the A subunit genes was only 70.6% (Gannon et al. 1990). The slt operon for SLT-I is under the control of the fur regulatory system, and highest yields are obtained when the bacteria are grown in media of low iron concentration (Calderwood and Mekalanos 1987). In contrast, the genes for members of the SLT-I1 group do not appear to be iron regulated (Weinstein et al. 1988a; MacLeod and Gyles 1989). Initially, it appeared that strains of E. coli produced SLT-I and (or) SLT-I1 (O'Brien and Holmes 1987). Recently, it has become evident that some strains possess two operons of closely related SLTs of the SLT-I1 family. Ito et al. (1990) sequenced two separate DNA sequences from E. coli strain B2F1 (091 :H21) and compared these sequences with that of SLT-11. Results indicated that the nucleotide sequences of the two operons of B2F1 had 99% homology. The genes of the A and B subunits encoded by one operon, designated vtx2ha, had 98.6 and 95.5% nucleotide sequence homology, respectively, with the cor-

CAN. J. MICROBIOL. VOL. 38, 1992

TABLE3. Structural components of Shiga toxin, SLTs, CT, and LTs


Approximate molecular weights Toxin

Holotoxin

A subunit

A1 fragment

A2 fragment

B subunit

Shiga SLTs CT 1,T-I LT-I1

70 000 71 000 85 000 88 000 83 000

32 000 33 000 27 000 28 000 28 000

27 000 27 000 22 000 22 000 21 000

5000 6000 5 500 5 500 7000

7 700 7000 1 1 600 12 000 1 1 800

responding genes of the slt-II operon. Similarly, Schmitt and et al. (1991) demonstrated that 5 of 19 E. coli strains that produced SLT of the SLT-I1 group had two copies of genes that hybridized with a DNA probe derived from slt-II. Nucleotide sequencing of the genes for two toxins from one strain (E325 11) showed that one operon was virtually identical with the SLT-I1 operon and the other encoded an A subunit that was identical with that of SLT-I1 and a B subunit that was identical with that of vtx2ha from strain B2F1 reported by Ito et al. in 1990. This second operon was designated slt-IIc. Escherichia coli strain E325 11, a reference strain, had previously been thought to encode a single type of SLT, called VT2 (Scotland et al. 1985). Structure The A-B subunit structure of Shiga-like toxins has been demonstrated by determination of the identity of SLT-I with Shiga toxin (Takao et al. 1988), for which this structural organization has been clearly demonstrated (Donohue-Rolfe et al. 1984); by nucleotide sequence data (De Grandis et al. 1987; Gyles et al. 1988; Weinstein et al. 1988b; Gannon et al. 1990) and by studies on purified toxins (O'Brien and LaVeck 1983; Yutsudo et al. 1987; Takao et al. 1988; Downes et al. 1988; Ito et al. 1988; Oku et al. 1989; Macleod and Gyles 1990). SLTs all consist of an A subunit of approximately 33 kDa and several B subunits of approximately 7.5 kDa (Table 3). The number of B subunits associated with each A subunit has been shown to be five for Shiga toxin (Donohue-Rolfe et al. 1984), and it is likely that a similar arrangement holds for SLTs. When the A subunit of Shiga toxin or SLT is subjected to proteolysis and to reduction of a disulfide bond, a large N-terminal A1 fragment, which possesses enzymatic activity, and a small C-terminal fragment are produced (Olsnes et al. 1981;MacLeod et al. 1991a). The A subunits of Shiga toxin and SLT-I recovered from the bacteria may be nicked (O'Brien and LaVeck 1983) or unnicked (Ito et al. 1988; Yutsudo et al. 1987; MacLeod and Gyles 1990). The B subunit is involved in binding SLT to a glycolipid receptor. The receptor for SLT-I and SLT-I1 is globotriosyl ceramide (Gb3) (Lingwood et al. 1987), whereas the preferred receptor for SLT-IIv is globotetraosyl ceramide (Gb4) (De Grandis et al. 1989; Samuel et al. 1990). Differences in receptor distribution in endothelial cells in various tissues and in various animal species may be responsible for differences in target organs, and remarkable differences in clinical syndromes. Physicochemical features The pIs for SLT-I, SLT-11, and SLT-IIv have been reported to be 7.0,4.1 or 5.1, and 9.0, respectively (O'Brien and LaVeck 1983; Downes et al. 1988; Macleod et al.

1991a). Oku et al. (1989) reported their purification of a SLT immunologically related to SLT-11. The toxin was derived from E. coli strain B2F1, implicated in hemolytic uremic syndrome, and was considered to be a variant of SLT-I1 because it was much more active on Vero than on HeLa cells. This toxin had a pI of 6.1, a feature that helped to distinguish it from the SLT-IIv of porcine edema disease isolates. pI may be an additional useful property for differentiation of SLTs. There is strong evidence that Shiga toxin occupies a periplasmic location in S. dysenteriae (Donohue-Rolfe and Keusch 1983). There is similar evidence to support the same location for SLTs (MacLeod and Gyles 1989; MacLeod et al. 1991a), but there are differences among the toxins with respect to their secretion. SLT-I is highly cell associated, whereas SLT-I1 is less highly cell associated (Strockbine et al. 1986). SLT-IIv is reported to be secreted to the exterior of the cell and the B subunit appeared to be responsible for secretion (Weinstein et al. 1989). Vero cell cytotoxicity of SLT-IIv was lost after heating at 65°C for 30 min or after exposure to 2-mercaptoethanol or dithiothreitol but was stable between pH 6 and 11 and increased after exposure to trypsin (MacLeod et al. 1991a). Biological activities All SLTs are cytotoxic for Vero cells, lethal for mice, and enterotoxic in rabbit intestine. SLTs cause Vero cells to take on a round but shrivelled appearance, which becomes evident at around 24 h and is maximal at 3-4 days (Konowalchuk et al. 1977). However, inhibition of protein synthesis is detectable as early as 1 h after exposure of cells to toxin (MacLeod et al. 1991a). SLTs are also cytotoxic for vascular endothelial cells, and the signs and symptoms of disease can be related to this effect in target tissues (Karmali 1989; Kavi et al. 1987; MacLeod et al. 1991a). Interestingly, there may be some species specificity in the action of SLTs on vascular endothelium. A. Valdivieso-Garcia, C .L. Gyles, D.L. MacLeod, and R.C. Clarke (personal communication) have shown that SLT-IIv is markedly more active on vascular endothelial cells of porcine origin than on similar cells of bovine origin. Mice inoculated with SLT exhibit hind-limb paralysis before death, and it is presumed that damage to the central nervous system is responsible for death. There is no direct evidence to support this supposition, but in rabbits and pigs (Barrett et al. 1989; MacLeod et al. 1991b) appearance of extensive focal hemorrhagic capillary lesions and edema in the brain in response to SLTs is consistent with death due to central nervous system damage. Several researchers have administered SLT intragastrically to infant rabbits in controlled experiments and have shown


REVIEW / SYNTHESE

that diarrhea and morphologic changes, particularly in the large intestine, are attributable to the toxin (Pai et al. 1986). Others have also demonstrated development of lesions in the large intestine attributable to SLT deposited in ligated segments of rabbit intestine (Keenan et al. 1986) or administered intravenously (Barrett et al. 1989). There is no alteration in levels of adenyl cyclase or guanyl cyclase in enterocytes in rabbits injected intraintestinally with SLT. Enterotoxicity may be due to impaired absorption caused by damage to and premature expulsion of mature columnar absorptive enterocytes at the tips of villi (Keenan et al. 1986). These workers suggested that there was a direct toxic effect on the epithelial cells. Kandel et al. (1989) have convincingly shown that Shiga toxin affects intestinal electrolyte transport in rabbit jejunal epithelium, causing an inhibition in absorption of NaCl, but no alteration in active anion secretion. They also demonstrated that villus cells, with more toxin binding sites, were more susceptible to inhibition of protein synthesis by Shiga toxin than were crypt cells. Other mechanisms may also be involved since damage to villus epithelial cells is observed when SLT-IIv is administered intravenously to pigs and appears to be the result of damage to the underlying vasculature (MacLeod et al. 1991b). SLT-IIv is poorly enterotoxic compared with the other SLTs, and pig intestine does not seem to respond to SLTs with a fluid response as seen in rabbit intestine (Gannon and Gyles 1990; MacLeod et al. 1991a). Diarrhea, which is seen in some outbreaks of edema disease in pigs, is probably due to enterotoxins produced by the E. coli. A platelet aggregating activity which can be inhibited by prostacyclin has been ascribed to SLT (Rose et al. 1985), and Karch et al. (1988) have reported that SLTs decrease synthesis of prostacyclin by endothelial cells. It seems, therefore, that some effects observed after exposure to SLTs are secondary to the damage to endothelial cells. Mechanism of action Both SLT-I and the SLT-I1 group of toxins inhibit protein synthesis in eukaryotic cells as a result of RNA N-glycosidase activity (Igarashi et al. 1987; Endo et al. 1988; Ogasawara et al. 1988; Saxena et al. 1989; Furutani et al. 1990). SLTs cleave a specific N-glycosidic bond in 28s ribosomal RNA, resulting in release of a single adenine residue and failure of elongation factor 1 dependent binding of aminoacyl tRNA to ribosomes. The mechanism of action of SLTs is identical with that of the plant toxin ricin (Igarashi et al. 1987; Saxena et al. 1989). Comparison of the amino acid sequences of the A subunits of ricin and SLTs has identified two regions of high homology (Hovde et al. 1988; Jackson 1990). Examination of the ricin molecule by X-ray crystallography has demonstrated that these two regions lie within a cleft in the A chain which may contain the active site of the molecule (Montfort et al. 1987; Frankel et al. 1989). Studies evaluating the effect on enzymatic activity of changes in specific amino acids within these regions have so far been inconclusive (Hovde et al. 1988; Frankel et al. 1989; Schlossman et al. 1989; Jackson et al. 1990a). Jackson et al. (1990b) compared amino acid sequences of SLT-I, SLT-11, and SLT-IIv and subjected certain conserved regions in the B subunit to site-specific mutagenesis. They demonstrated that a hydrophilic region near the N-terminus of the B subunit was involved in binding and that a glutamine

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residue near the C-terminus was involved in extracellular localization. Perera et al. (1991) identified three amino acid residues in the B subunit of SLT-I1 that were essential for toxin activity. Association with diseases Karmali (1989) has reviewed the evidence that implicates SLT-producing E. coli as a cause of hemolytic uremic syndrome (HUS) and hemorrhagic colitis in humans. HUS is characterized by a combination of acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia and is often preceeded by diarrheal disease. Hemorrhagic colitis results in a dysenteric syndrome. Mild diarrhea and subclinical infection are other forms of disease associated with SLT-producing E. coli. There is a strong body of epidemiologic data that associates the presence of SLT-producing E. coli in the feces with disease in patients (Karmali et al. 1985; Grandsen et al. 1986; Karmali 1989). Karmali (1989) has also pointed to the remarkable similarity in clinicopathological and radiologic features of HUS and hemorrhagic colitis and made the argument that these syndromes are different expressions of the same toxemia. SLT is produced in vivo and a specific immune response to SLT has been demonstrated in patients. Interestingly, neutralizing antibodies to SLT-I but not to SLT-I1 appear to be widespread in the human population (Ashkenazi et al. 1988). Similar observations have been made for sera from cattle in Ontario: approximately 80% of the samples had neutralizing antibodies to SLT-I and about 5% had neutralizing antibodies to SLT-I1 (R.C. Clarke, personal communication). Escherichia coli serotype 0 157:H7 has been particularly associated with outbreaks of hemorrhagic colitis but is also involved in sporadic cases of the disease. This serotype and other serotypes of verotoxigenic E. coli which cause hemorrhagic colitis are referred to as enterohemorrhagic E. coli (EHEC). In outbreaks, HUS may develop in a percentage of individuals with hemorrhagic colitis. Hamburger and milk have been implicated as vehicles of infection, and cattle appear to be a reservoir of organisms that cause disease in humans (Martin et al. 1986; Borczyk et al. 1987; Clarke et al. 1989; Read et al. 1990). Emphasis on serotype 0157:H7 sometimes obscures the fact that over 50 other serotypes of SLT-producing E. coli have been recovered from humans (reviewed by Karmali, 1989). Most of these serotypes also exist in the intestine of normal cattle (Clarke et al. 1989; Read et al. 1990). Bettelheim et al. (1990) showed that E. coli strains that produced E. coli heat-labile enterotoxin (LT) or SLT were present in the intestine of 21 of 46 infants who had died of sudden infant death syndrome (SIDS), whereas toxigenic E. coli were absent from the feces of 24 normal infants who lived in the same area at the same time. It is conceivable that SLT may be a cause of SIDS. Hemorrhagic colitis and diarrhea in calves have been associated with SLT-producing E. coli (Chanter et al. 1986; Mainil et al. 1987a; Schoonderwoerd et al. 1988). Disease has been reproduced by inoculation of calves with isolates of the organisms recovered from diseased calves (Moxley and Francis 1986). Edema disease in pigs has long been known to be a toxemia involving a heat-labile toxin produced by certain strains of E. coli. In recent years, edema disease strains of E. coli have been shown to produce SLT (Smith et al. 1983;


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Marques et al. 1987; Gannon et al. 1988), and this toxin has been shown to be SLT-IIv (Gannon and Gyles 1990; MacLeod et al. 1991b). Intravenous injection of pigs with purified SLT-IIv has reproduced the signs and symptoms of edema disease (MacLeod et al. 1991b) . Both experimental and natural edema disease have the same essential features of other diseases in which SLTproducing E. coli have been implicated (MacLeod et al. 1991b). These features resolve around damage to small blood vessels in target organs. In edema disease, the most common targets are the stomach, the eyelids, the cerebellum, and the colon. Grossly, edema and hemorrhage are observed and microscopic lesions consist of edema, hemorrhage, and vasculitis. There is little information on SLT-producing E. coli in other species. However, these organisms have been implicated in diarrhea in cats (Abaas et al. 1989). Other E. coli Vero cell cytotoxins Vir cytotoxin Smith (1974) and Smith and Huggins (1976) reported that certain bovine and ovine septicemic strains of E. coli of serogroup 078:K80 produced a toxin that was lethal for mice, rabbits, and chickens. Synthesis of this toxin was plasmid mediated and was associated with the presence of a surface antigen. The plasmid responsible for production of toxin and the surface antigen was called Vir. Both Smith and Huggins (1976) and Lopez-Alvarez and Gyles (1980) demonstrated that the Vir phenotype was uncommon among septicemic strains of E. coli. The latter researchers (Lopez-Alvarez et al. 1980) showed that the Vir plasmid behaved as an episome, that the surface antigen that it encoded was a pilus, and that some strains of E. coli produced toxin but no Vir pili. The lethal effects of vir cytotoxin suggest that this toxin may play a role in bacteremic disease in calves and lambs. However, the low frequency of its occurrence suggests that the role is not a critical one. Oswald et al. (1989) termed the Vir toxin vir cytotoxin because it causes a cytopathic effect in HeLa cells, characterized by multinucleation.

Cytotoxic necrotizing factor A cytotoxic necrotizing factor (CNF) which induces formation of giant, polynucleated cells in HeLa and Vero cultures has been detected in extracts of E. coli from diarrheal diseases in humans, pigs, and calves (Caprioli et al. 1984; Gonzalez and Blanco 1985; De Rycke et al. 1987, 1989), as well as in E. coli from septicemia and urinary tract infections in humans (Alonso et al. 1987; Caprioli et al. 1987). Production of CNF is often associated with possession of P fimbriae and with production of alpha hemolysin (Blanco et al. 1990; Jacobsen et al. 1990). CNF is immunologically related to vir cytotoxin (De Rycke et al. 1987; Oswald et al. 1989), and recent evidence indicates that these cytotoxins are sufficiently related as to be called CNFl and CNF2, respectively (De Rycke et al. 1990). Both toxins are lethal for mice and both toxins induce multinucleation in HeLa cells and necrosis in rabbit skin, but CNF2 is less potent in causing multinucleation and much more potent in causing skin necrosis than is CNF1. The toxicity of each type of CNF is partially neutralized by antibodies against the other type, but it is possible to distinguish between the two by an enzyme-linked immunosorbent assay (Tabouret and De Rycke 1990). CNFl is

associated with a 115-kDa entity recognized by SDS-PAGE (De Rycke et al. 1990), whereas CNF2 is associated with a 110-kDa protein (Oswald and de Rycke 1990).

Cytolethal distending toxin A heat-labile cytolethal distending toxin (CLDT) has been reported in culture filtrates of certain E. coli strains associated with diarrheal disease in humans (Johnson and Lior 1988) as well as in Shigella dysenteriae type 2 and Shigella boydii (Johnson and Lior 1987). CLDT causes cytotoxic damage and cell distension in Vero, HeLa, HEp-2, and CHO cells and is produced in some isolates that also produce verotoxin. The roles of CNF and CLDT in disease are not known. Escherichia coli en terotoxins History and nomenclature In the search for an enterotoxin that might account for diarrhea caused by porcine strains of E. coli, Smith and Halls (1967) in England were the first to identify a cell-free heat-stable preparation that caused fluid accumulation in ligated segments of pig intestine. Shortly thereafter, Gyles and Barnum (1969) in Canada identified an enterotoxin that was also associated with E. coli strains implicated in piglet diarrhea, but was heat-labile and antigenically related to cholera toxin (CT). Later Smith and Gyles (1970) compared the two toxins and called them ST (stable toxin) and LT (labile toxin). LTs derived from E. coli of human and porcine origin are sometimes differentiated by the terms LTh and LTp (Holmes and Twiddy 1983), hLT and pLT (Yamamoto and Yokota (1983). A second type of LT (LT-11), which differed in its antigenicity from the established LT (LT-I), was identified by Green et al. in 1983. Two antigenic types of this LT have been identified as LT-IIa and LT-IIb (Guth et al. 1986). In 1978, Burgess and colleagues demonstrated that there were two types of ST: one (STa) was methanol soluble and active in the intestine of infant mice; the other (STb) was methanol insoluble and inactive in the infant mouse intestine, but active in the intestine of weaned pigs. The terms ST1 and ST11 were later used as synonyms for STa and STb, respectively (So and McCarthy 1980). When two types of ST1 were distinguished, they were called STIa and STIb (Moseley et al. 1983a, 1983b). STIa consists of 18 amino acids, whereas STIb is made up of 19 amino acids. Enterotoxigenic E. coli (ETEC) may produce LT alone, ST alone, or combinations of LT and ST. Association with disease In adult humans, ETEC is one of the most common etiologic agents of traveller's diarrhea and may account for 30-50070 of cases (Levine 1983, 1987). Infants in third world countries are also prone to developing diarrhea due to ETEC infection. Diarrheal episodes due to ETEC may occur in sporadic cases as well as in outbreaks (Sack 1975; Riordan et al. 1985). Only a limited number of serotypes of E. coli are consistently implicated in ETEC diarrhea, but other serotypes are occasionally causes of diseases. This likely represents established clones of pathogens on the one hand and serotypes that have more recently acquired virulence attributes by horizontal transfer. ETEC of human origin produce LT and ST1 but do not appear to produce ST11 (Weikel et al. 1986). Interestingly, human ileal mucosa, in contrast to pig jejunum, does not respond electrogenically to ST11 (Weikel et al. 1986).


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Young domestic animals are susceptible to diarrhea due to ETEC, but adults of these species do not seem to be affected. Neonatal calves and lambs and newborn and recently weaned pigs are all vulnerable to ETEC infections (reviewed by Holland, 1990). ETEC recovered from calves and lambs typically produce ST1 only, whereas those recovered from pigs produce various combinations of LT, STI, and STII. ETEC have also been isolated from dogs with diarrhea and from chickens. The canine ETEC identified in one study (Olson et al. 1985) produced both LT and ST, and the LT was antigenically different from LTh-I. In another study (Wasteson et al. 1988) all five ETEC isolated from the feces of dogs with diarrhea produced only STI. Japanese workers (Sekizaki et al. 1985) have shown that some ETEC recovered from chickens produce LTh-I, whereas others produce LTp-I. Genes coding for LT-I1 have been detected in E. coli from humans, cattle, and buffalo (Seriwatana et al. 1988). L T-I LT-I was first recognized by its ability to induce fluid accumulation in ligated segments of pig intestine (Gyles and Barnum 1969). LT-I produced by a wide variety of E. coli strains from pigs appeared to be antigenically identical or closely related. Subsequently, LT-I was associated with E. coli strains from diarrheal disease in humans (reviewed by Sack, 1975). LTp-I and LTh-I are very similar, having more than 95% nucleotide sequence similarity.

Structure LT-I is an A-B subunit toxin whose molecular organisation is identical with that of CT and very similar to that of Shiga toxin and SLTs (Table 3). The A subunit, synthesized as a single polypeptide, is subsequently proteolyzed (nicked) to form two peptides, A, and A2, which remain connected by a single disulfide bond involving one cysteine residue in the A1 fragment and one in the A2. Reduction of this bond releases the enzymatically active Al peptide. Molecular weights of LTs and their components are given in Table 3. A lag in the activity of LT is due, at least in part, to the delay required for processing of the A fragment. Purified A1 fragment activates adenyl cyclase in broken but not intact pigeon erythrocytes, indicating the requirement of the B subunit for internalization of the A subunit. The number of B subunits per A subunit is estimated to be 5 (Hofstra and Witholy 1985). This is based on crosslinking studies and determination of molecular weights of A and B subunits and holotoxin. The B subunits serve to bind the toxin to the cell surface receptor galactosyl-Nacetylgalactosaminyl-[Nacetylneuraminyl]-galactosylglucosylceramide (GM,). Gangliosides in human milk have been shown to inhibit the enterotoxic activity of CT and LT (Otnaess et al. 1983). Two cysteine residues in the B subunit are located at the N- and C-terminals and form a S-S bond joining the two ends of the molecule. The B subunits are likely arranged in the form of a ring. Probably, the A subunit is partially inserted into the B ring by the A2 portion. The data are supported by electron microscopic studies. Deletion or substitutions of the last few amino acids at the C-terminus of the B subunit markedly affect release of the B subunit from the cytoplasmic membrane (Sandkvist et al. 1990). Immunoprecipitation tests show that the A and B subunits are antigenically distinct (Gilligan et al. 1983). Both subunits

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are also markedly different in immunogenicity. Immunization with intact toxin induces antibodies that react strongly with the B subunit and weakly or not at all with the A. There have been extensive studies on antigenic relationships among members of the serogroup I CT-LT toxin family (Marchlewicz and Finkelstein 1983; Takeda et al. 1983; Finkelstein et al. 1987; Kazemi and Finkelstein 1990). These toxins have common and unique antigenic determinants, and enterotoxinspecific antibodies may sometimes account for a major portion of the neutralizing activity. Small differences in primary structure are sometimes associated with marked differences in neutralizing antibody specificities. LT-I is a periplasmic protein. Pre-LT A and pre-LT B are formed in the cytoplasm, transported through the cytoplasmic membrane, processed to form A and B subunits, which are then assembled into holotoxin molecules (Hofstra and Witholy 1985). L T-II LT-I1 differs from LT-I in that it is not neutralized by anti-CT or anti-LT-I. A variant of LT-I1 (LT-IIb) has been detected that is distinguishable in antigenicity and other characteristics from the prototype LT-IIa. LT-I1 has a structure that is similar to that of LT-I (Table 3; Guth et al. 1986; Holmes et al. 1986). The prototype LT-IIa consists of A and B polypeptides similar in size and in susceptibility to proteolytic cleavage compared with LT-I, LT-IIa and LT-IIb are similar in structure and are antigenically related but differ in some of their biologic activities and in their PIS. The specific activity of LT-IIa in Y1 mouse adrenal tumour cells is 25-50 times that of LT-I (Holmes et al. 1986). However, E. coli cells that produce LT-IIa do so at a level less than 1% of the level of LT-I associated with LT-Ipositive E. coli. LT-IIa is similar to LT-I in enzymatic activity and mode of activation of adenylate cyclase (Holmes et al. 1986) but binds to a receptor that is different from GM1 ganglioside. Whereas LT-I binds best to GM1, LT-IIa binds best to GDl b, and LT-IIb binds best to GD 1a (Fukuta et al. 1988). Based on these observations as well as binding to other gangliosides, Fukuta and colleagues have suggested that LT-I binds to the terminal sugar sequence Galpl -3GalNAcpl4(NeuAcar2-3)Gal, where GalNAc is N-acetylgalactosamine and NeuAc is N-acetylneuraminic acid; that LT-IIa binds to a sequence that is similar to that bound by LT-I except that it has a second NeuAc; and that LT-IIb binds a terminal NeuAcar2-3Galp 1-4GalNAc sequence. LT-IIa is not enterotoxic in rabbit, even in relatively large doses, and its role in disease has not been established.

Genetics The structural genes for LT-I are on plasmids (Gyles et al. 1974), but genes in the chromosome as well as DNA of the flanking regions may affect the level of expression of the plasmid-encoded genes (Neil1 et al. 1983; Katayama et al. 1990). The plasmids sometimes also encode resistance to antibacterial drugs (Gyles et al. 1977). Nucleotide sequences of the genes for LT-I (Yamamoto et al. 1983, 1984) have confirmed the close relationship of LTh-I and LTp-I. Codon usage and G + C content have suggested that the E. coli LT genes were derived from Vibrio cholerae. Among porcine ETEC, LT-I genes are typically found in strains that also have genes for the K88 antigen. Among ETEC of human

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origin, LT-I genes are often associated with CFA-I1 (Echeverria et al. 1986). The structural genes for LT-I1 are chromosomal. The LT-IIa and LTh-I A subunit genes have 57% nucleotide sequence similarity, but the B subunit genes lack significant sequence homology (Pickett et al. 1989).

Mechanism of action Bacterial toxins may alter normal bowel function by selective destruction of normal absorptive cells, as in the case of SLTs. Alternatively, they may stimulate normal secretory pathways or inhibit normal absorptive pathways to produce abnormal net secretion. Escherichia coli enterotoxins act by the second mechanism. LTs act in an manner identical with that of CT (Fishman 1990): they stimulate membrane-bound adenylate cyclase in intestinal epithelial cells. LTs activate adenylate cyclase by catalyzing the transfer of ADP-ribose from NAD to an amino acid in the stimulatory guanine nucleotide binding regulatory protein (G,), leading to activation of the cyclase catalytic unit and increased CAMP levels. This stimulation results in persistent elevation of cyclic AMP, which leads to hypersecretion of electrolytes and water, manifested as watery diarrhea. There is no detectable gut damage, since there is simply overstimulation of normal mechanisms. LTs are, therefore, said to be cytotonic toxins, in contrast to cytotoxic toxins. They cause impaired cell function without any evidence of structural damage. Unlike CT, in which the A region is totally nicked (as a result of proteolytic action on the secreted toxin), E. coli LT is unnicked, and its activity is increased by trypsin treatment (Clements and Finkelstein 1979). The activity of LT is equivalent to that of CT in bioassays. E. coli heat-stable enterotoxin ST1 (STa) The two major types of ST appear to be completely unrelated and will therefore be discussed separately. What they have in common are heat stability, low molecular weight, secretion from the cell, and ability to interfere with water and electrolyte movement across the gut epithelium.

Genetics Genes for ST1 are typically found on plasmids of a wide range of molecular sizes (Gyles et al. 1974; Harnett and Gyles 1985). Among porcine and bovine ETEC it is common to find genes for STI, colonizing fimbriae, drug resistance, and production of colicin on a single plasmid (Harnett and Gyles 1985). Plasmids with genes for drug resistance and ST1 have been reported from ETEC of human and animal origins (Gyles et al. 1977); Echeverria et al. 1978). ST1 genes may be lost from an ETEC by loss of plasmid or by deletion of a DNA segment from the plasmid. Under the selective pressure applied by K99 antibodies in the intestine, genes for K99 and for ST1 may be lost during infection of pigs with ETEC (Mainil et al. 1987b). Genetic studies demonstrated the existence of two types of STI. A plasmid gene for STI, called STIa, was cloned from a bovine isolate of E. coli and shown to be part of a transposon, Tn1681, which is flanked by inverted repeats of IS1 (So and McCarthy 1980). Subsequently, ST1 genes from bovine, porcine, and avian ETEC were also shown to be part of the same transposon (Sekizaki et al. 1985). A DNA probe from a procine STIa gene failed to detect many ST-producing E. coli isolates of human origin

(Moseley et al. 1980), which were subsequently detected by a second DNA probe from a different ST gene (Moseley et al. 1983a). The ST detected by this second probe was called STIb. Genes for both STIa and STIb may be carried by a single strain of ETEC of human origin (Moseley et al. 1983b). Nucleotide sequence data and purification of the ST product showed that the gene for STIa encoded an 18 amino acid product and the gene for STIb encoded a 19 amino acid product (Aimoto et al. 1982; Moseley et al. 1983b; Takao et al. 1983; Sekizaki et al. 1985; Stieglitz et al. 1988; Okamoto and Takahara 1990). STIa is sometimes referred to as STp (porcine STI) but is produced by animal and human isolates. STIb is sometimes called STh (human STI) and is produced by human isolates only. DNA sequence and other data indicate that STIa is synthesized as a 72 amino acid precursor molecule referred to as pre-pro-ST1 (Stieglitz et al. 1988; Okamoto and Takahara 1990). The 72 amino acid structure consists of a 19 amino acid signal peptide, followed by a 35 amino acid pro sequence, then the 18 amino acid STI. The function of the pro region is not known, but it does not appear to be involved in transport of the peptide into the extracellular environment. Manipulation of the amino acid sequence of ST1 has been used to alter its properties substantially. Synthetic ST1 peptides have been prepared with as much as a 15-fold increase in antigenicity and a two-thirds reduction in toxicity. Synthesis of ST1 by E. coli is subject to catabolite repression, and optimal yields are obtained in glucose-free media (Alderete and Robertson 1977; Stieglitz et al. 1988).

Physicochemical features ST1 has a molecular weight of approximately 2000. It is resistant to 100°C for 15 min, is soluble in water and organic solvents, and is resistant to proteolytic enzymes such as pronase, trypsin, and chymotrypsin (Smith and Halls 1967; Alderete and Robertson 1978). It is acid resistant but susceptible to alkaline pH. It is completely inactivated by reducing and oxidizing agents which disrupt disulfide bonds (Robertson et al. 1983). STIa and STIb share antigenic determinants and biologically active core sequences which reside in the carboxy-terminal amino acid residues. They differ in the amino terminal end of the molecular. STIa molecules have an amino acid sequence consisting of

Asn-Thr-Phe-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-A

Cys-Ala-Gly-Cys-Tyr, whereas STIb molecules have a sequence of Asn-Ser-Ser-Asn-Tyr-Cys-Cys-Glu-Leu-Cys Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr (Moseley et al. 1983a; Sekizaki et al. 1985; Thompson and Giannella 1985; Stieglitz et al. 1988). There is a characteristic central sequence of 13 amino acids with 6 cysteine residues which participate in disulfide bridge formation. The tertiary structure formed by the three disulfide bonds is critical, and all three disulfide bonds are required for full biological activities (Okamoto et al. 1987).

Mechanism of action ST1 activates particulate guanylate cyclase in the brush border of intestinal epithelial cells in the jejunum and ileum, thereby leading to elevation of the levels of cGMP (Field et al. 1978; Hughes et al. 1978). ST1 does not bind to particulate guanylate cyclase from tissues other than intestinal

REVIEW / SYNTHESE


tissue because other tissues lack the specific receptor. A high-affinity ST1 receptor is tightly coupled to particulate guanylate cyclase in membranes of rate intestinal epithelial cells (Waldman et al. 1986). Franz et al. (1984) and Dreyfus and Robertson (1984) showed that the ST1 receptor appeared to be a protein or glycoprotein (molecular weight 100 OOO), and there were only about 50 000 receptors per intestinal epithelial cell. ST1 could readily be dissociated from its receptor by treatment with dithiothreitol, suggesting that a disulfide bond in ST1 reacts with a sulfhydryl group in the receptor. Binding of ST1 to receptor was saturable, time and temperature dependent, and stimulated by monovalent cations. Kuno et al. (1986) used classical separation techniques to characterize the ST1 rat intestine receptor as a 160-kDa molecule. However, Ivens et al. (1990) recently demonstrated that several proteins in crude intestinal membranes bind ST1 specifically. Furthermore, they identified different molecular organization among the receptor proteins, which include proteins of a size similar to 57- and 75-kDa proteins previously identified as ST1 receptors by Gariepy and Schoolnik (1986). Robertson (1988) reported that, although younger pigs are more sensitive to ST1 than are older pigs, there were no differences in guanylate cyclase activity in STI-stimulated intestinal epithelial brush border membranes from 7-dayold pigs compared with cells from 7-week-old pigs. He concluded that sensitivity to ST1 was not the factor that determined age susceptibility to STI-positive ETEC in pigs. In contrast, Cohen et al. (1988) examined samples of small and large intestine of children of different ages and showed that the concentration of receptors for ST1 decreased rapidly with age, and that increased STI-mediated stimulation of guanyl cyclase was correlated with increased receptor density. The rate of guanylate cyclase stimulation by ST1 is rapid, with maximal levels of cGMP observed within 5 min. Elevated cGMP causes increased fluid secretion by a mechanism that is unknown. The end result of ST1 action is inhibition of Na +-coupled C1- absorption in villus tips, plus stimulation of electrogenic C1- secretion in crypt cells, which leads to excessive fluid in the lumen of the gut.

S TII The genes that encode ST11 are on plasmids, which are heterogeneous and may also determine other properties, including LT, ST1, colonization factors, drug resistance, colicin production, and transfer functions (Gyles et al. 1974; Harnett and Gyles 1985). The nucleotide sequence of the gene for ST11 has been reported (Lee et al. 1983; Picken et al. 1983). The structural gene encodes a mature protein of 48 amino acids and a signal peptide of 23 amino acids. The ST11 gene is part of an approximately 9-kb transposon designated Tn4.521 (Lee et al. 1985; Hu and Lee 1988). Production of ST11 appears to be restricted to porcine ETEC, but genes for ST11 have been detected in E. coli from feces of normal humans on farms in Thailand (Echeverria et al. 1985). When DNA probes are used to sample ETEC from pigs, ST11 is the enterotoxin gene that is most frequently detected (Moon et al. 1986). For some considerable time ST11 appeared to be active in the intestine of pigs but not of other animals tested. However, recent reports have demonstrated that ST11 is susceptible to inactivation in the gut by trypsin, and that if it is protected from proteolysis,

74 1

it is active in the intestine of rats and mice (Whipp 1987, 1990). Nothing is known of the mechanism of action of STII. It does not cause elevation of cGMP. Unlike ST1 and LT, ST11 appears to induce bicarbonate rather than chloride secretion. ST1 has recently been associated with histologic damage to intestinal epithelium, and it is conceivable that such damage could be responsible for impaired absorption of fluids (Whipp et al. 1987).

Summary Vero cell cytotoxins and cytotonic enterotoxins produced by E. coli are toxic proteins, which have been implicated in a number of specific diseases in humans and animals. Nomenclature for these toxins is complicated by the existence of different names for the same toxin. The Vero cell cytotoxins are called verotoxins because they are lethal for Vero cells in culture; they are also known as Shiga-like toxins (SLTs) because they are clearly related to Shiga toxin in structure, amino acid sequence, mechanism of action, and biological activity. SLTs belong to two classes. SLT-I is identical with Shiga toxin and is in a class by itself (class I). The other SLTs are closely related to each other and form a second class (class 11). Class I1 SLTs include SLT-11, SLTIIv, SLT-IIvha, SLT-IIvhb, and SLT-IIva. All SLTs that have been investigated are A-B subunit protein toxins, whose A subunits possess N-glycosidase activity against 28s rRNA and cause inhibition of protein synthesis in eukaryotic cells. These toxins are enterotoxic as well as cytotoxic. SLTs produced in the intestine are absorbed into the blood stream and affect vascular endothelial cells in target organs. They may also have a direct toxic effect on enterocytes. Diseases in which E. coli SLTs have been implicated include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans and edema disease in pigs. Variation in receptor specificities among SLTs may be the reason for different disease syndromes in different host species. The E. coli enterotoxins belong to three distinct classes: heat-labile enterotoxin (L T), heat-stable enterotoxin type I or type a (STI, STa), and heat-stable enterotoxin type 11 or type b (STII, STb). There is clear evidence that these cytotonic enterotoxins play an essential role in diarrheal disease. LT is an A-B subunit protein toxin, closely related to cholera toxin. Following binding of LT to receptors in enterocytes the A subunit is internalized. The enzymatically active A subunit transfers ADP-ribose from NAD to a GTPdependent adenylate cyclase regulatory protein, thereby elevating intracellular levels of adenylate cyclase. The increased levels of cyclic AMP cause stimulation of A kinase and lead to hypersecretion of electrolytes and fluid. ST1 is a small peptide of 18 or 19 amino acids. It binds to receptors in enterocytes and stimulates particulate guanyl cyclase . Elevated intracellular cyclic GMP stimulates G kinase, resulting in increased C1- secretion and impaired absorption of Na +C1- . ST11 is a peptide toxin whose mechanism of action is unknown. The enterotoxins are implicated in diarrheal disease in humans and animals. The role of enterotoxins in disease has been more definitively established than has the role of Shiga-like toxins. The existence of counterpart diseases in animals in the case of enterotoxigenic E. coli and the absence of such diseases in the most serious type of SLT-associated disease has contributed to this situation.


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Conclusion Genetic studies and classical protein separation techniques have contributed to our understanding of structure-function relationships for SLTs and LTs. These groups of toxins have similar structural organization, but differ in .their enzymatic activities and in the receptors to which they bind. The end result is that the tissues that are affected and the kinds of disease that are produced are quite different for the two types of toxins. There is a reasonably good understanding of the roles of both groups of toxins in disease, but the picture is clearer for LTs than for SLTs. The existence of good animal models of LT-mediated disease and the absence of a good model for SLT-mediated disease have contributed to this situation. There is a considerable body of information on the STs but knowledge on ST11 lags behind that on STI. Once again, this is largely due to the paucity of biological systems for investigation of STII. Abaas, S., Franklin, A., Kuhn, I., et al. 1989. Cytotoxin activity on Vero cells among Escherichia coli strains associated with diarrhea in cats. Am. J . Vet. Res. 50: 1294-1296. Aimoto, S., Takao, T., Shimonishi, Y., et al. 1982. Amino acid sequence of heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli. Eur. J. Biochem. 129: 257-263. Alderete, J.F., and Robertson, D.C. 1977. Repression of heatstable enterotoxin synthesis in enterotoxigenic Escherichia coli. Infect. Immun. 17: 629-633. Alderete, J.F, and Robertson, D.C. 1978. Purification and chemical characterization of the heat-stable enterotoxin produced by porcine strains of enterotoxigenic Escherichia coli. Infect. Immun. 19: 1021-1030. Alonso, P., Blanco, J., Blanco, M., and Gonzalez, E.A. 1987. Frequent production of toxins by Escherichia coli strains isolated from human urinary tract infections. FEMS Microbiol. Lett. 48: 391-396. Ashkenazi, A.U., Cleary, T.G., Lopez, E., and Pickering, L.K. 1988. Anticytotoxin-neutralizing antibodies in immune globulin preparations: potential use in hemolytic-uremic syndrome. J. Pediatr. 113: 1008-1014. Barrett, T.J., Potter, M.E., and Wachsmuth, I.K. 1989. Continuous peritoneal infusion of Shiga-like toxin I1 (SLT 11) as a model for SLT 11-induced diseases. J. Infect. Dis. 159: 774-777. Bettelheim, K.A., Goldwater, P.N., Dwyer, B.W., et al. 1990. Toxigenic Escherichia coli associated with sudden infant death syndrome. Scand. J. Infect. Dis. 22: 467-476. Blanco, J., Alonso, M .P., Gonzalez, E. A., et al. 1990. Virulence factors of bacteraemic Escherichia coli with particular reference to production of cytotoxic necrotising factor (CNF) by P-fimbriate strains. J . Med. Microbial. 31: 175-183. Borczyk, A.A., Karmali, M.A., Lior, H., and Duncan, L.M.C. 1987. Bovine reservoir for verotoxin producing Escherichia coli 0157:H7. Lancet, i: 98. Burgess, M.N., Bywater, R.J., Cowley, C.M., et al. 1978. Biological evaluation of a methanol-soluble, heat-stable Escherichia coli enterotoxin in infant mice, pigs, rabbits, and calves. Infect. Immun. 21: 526-531. Calderwood, S.B., and Mekalanos, J . J. 1987. Iron regulation of Shiga-like toxin expression in Escherichia coli is mediated by the fur locus. J . Bacteriol. 169: 4759-4764. Calderwood, S.B., Auclair, F., Donohue-Rolfe, A., et al. 1987. Nucleotide sequence of the Shiga-like toxin genes of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 84: 4364-4368. Caprioli, A., Donneli, G., and Falbo, L. 1984. A cell division-active protein from Escherichia coli. Biochem. Biophys. Res. Commun. 118: 587-593. Caprioli, A., Falbo, V., and Ruggeri, F.M. 1987. Cytotoxic necrotising factor production by hemolytic strains of Escherichia

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