Vol. 58, No. 11
INFECTION AND IMMUNITY, Nov. 1990, p. 3717-3723
0019-9567/90/113717-07$02.00/0 Copyright © 1990, American Society for Microbiology
Enterotoxin and Cytotoxin Production by Enteroinvasive Escherichia coli ALESSIO FASANO,'t BRADFORD A. KAY,' ROBERT G. RUSSELL' 2 DAVID R. MANEVAL, JR.,' AND MYRON M. LEVINE'* Center for Vaccine Development, Division of Geographic Medicine, Department of Medicine,' and Department of Pathology,2 University of Maryland School of Medicine, Baltimore, Maryland 21201 Received 29 May 1990/Accepted 30 August 1990
It has long been suspected that besides their ability to invade enterocytes, enteroinvasive Escherichia coli (EIEC) strains have the ability to elaborate an enterotoxin. We tested 35 EIEC strains for cytotoxins and 9 (1 per serogroup) for enterotoxins. All 35 strains exhibited low levels of Vero cell cytotoxins that are immunologically and genetically distinct from Shiga-like toxin I or II of enterohemorrhagic E. coli. Sterile supernatants and cell lysates of two EIEC strains were tested in rabbit ileal loops, and both stimulated moderate fluid accumulation (circa 0.5 ml/cm) without tissue damage; secretory activity was confirmed in Ussing chambers, where these two strains and the seven others tested significantly increased short circuit current without altering tissue conductance. Curing the 140-MDa invasiveness plasmid from an EIEC strain did not diminish enterotoxin production. Culture in minimal Fe2+ medium is necessary to detect expression of the enterotoxin which is circa 68 to 80 kDa in size and is distinct from the EIEC cytotoxin.
Enteroinvasive Escherichia coli (EIEC) strains are an important cause of diarrheal disease and dysentery in young children in less-developed countries (12, 29, 30) and of food-borne outbreaks of enteric disease in adults in industrialized countries (19, 28, 31). Like Shigella spp., with which they share antigens, the cardinal pathogenic feature of EIEC strains is their ability to invade and multiply within epithelial cells of the distal small intestine and the colon (12, 19, 28, 31). This invasive property depends on expression of several outer membrane proteins, structural genes for which are found on 140-MDa enteroinvasive plasmids (plnv) (10, 11) that are essentially identical to the plasmids found in Shigella flexneri. Two features of the clinical syndrome caused by EIEC suggest that cytotoxins and enterotoxins are involved in pathogenesis. Like shigellosis, EIEC infection can result in high fever, malaise, abdominal cramps, and overt dysentery characterized by the tenesmic passage of scanty stools containing blood and mucus (4). While epithelial cell invasiveness is clearly the predominant virulence property responsible for the dysenteric syndrome, by analogy with Shigella, spp. (26), cytotoxins may in part dictate the severity of the dysenteric symptoms. More importantly, watery diarrhea typically precedes dysentery in EIEC infection, and both in individual cases and in outbreaks due to EIEC (19, 28-31), watery diarrhea is often seen exclusively without dysentery. These clinical observations suggest that EIEC produces an enterotoxin that is responsible for the watery diarrhea. Accordingly, a series of EIEC strains were examined for the production of Vero cell cytotoxins and for enterotoxins. MATERIALS AND METHODS Bacterial strains. A total of 35 E. coli strains, representing diarrheal isolates of all major EIEC 0 serogroups from Corresponding author. t Present address: Department of Pediatrics, University of
*
Napoli, Naples, Italy.
diverse geographic locations, were initially cultured on blood agar to assess their purity. Isolated colonies were subcultured to Casamino Acids-yeast extract agar and incubated in ambient air overnight at 37°C. Epithelial cell invasiveness and virulence of the E. coli strains were confirmed by demonstrating the ability of the strains to cause purulent keratoconjunctivitis following inoculation into the conjunctival sac of guinea pigs (33), as originally described by Sereny for Shigella spp. (27). Each strain was also shown to hybridize at high stringency with a DNA probe that detects the EIEC virulence plasmid (7, 15, 33). Preparation of supernatants and sonic extracts. The 35 Sereny test-positive, EIEC probe-positive strains were inoculated into 5 ml of Chelex-treated syncase broth (23). The bacteria were harvested by centrifugation, and the culture supernatants were collected and filter sterilized. The bacterial cells were washed twice in phosphate-buffered saline (PBS), suspended in 1.5 ml of PBS (pH 7.4), and disrupted in a French pressure cell at 12,000 lb/in2 (23). Lysates were then mixed with 3.5 ml of PBS (final volume, 5 ml), clarified by centrifugation (18,000 x g for 20 min at 4°C), and filter sterilized. The supernatants and lysates prepared in this manner were used in assays to detect cytotoxins and enterotoxins. Vero cell assay for cytotoxicity. Cytotoxicity assays were performed with Vero cells by the method of Gentry and Dalrymple (6). Serial twofold dilutions (1:2 to 1:64) of culture supernatants and cell lysates were tested, and the cytotoxic dose required to kill 50% of Vero cells was estimated spectrophotometrically (6). Supernatants and lysates from enterohemorrhagic E. coli (EHEC) strain 933 (serotype 0157:H7), which elaborates Shiga-like toxins I (SLT-I) and SLT-II (22, 24), served as the positive control, while the negative controls were prepared from E. coli K-12 HB101; controls were included in each microtiter plate. Since the positive control killed more than 50% of Vero cells at a 1:64 dilution, a 10-fold dilution of this sample was tested. Cytotoxic titers were expressed as the reciprocal of the 50% cytotoxic dose of culture supernatant or cell lysate per 3717
3718
FASANO ET AL.
milligram of protein; the protein content was measured by the method of Bradford (1). In toxin neutralization experiments, supernatants and lysates from 10 EIEC strains of different serogroups were incubated for 60 min at 37°C with an anti-SLT-I monoclonal antibody and with polyclonal monospecific anti-SLT-II antibodies (titers, 1:25) kindly provided by Alison O'Brien, Uniformed Services University of the Health Sciences, Bethesda, Md. The cytotoxicity assay was then performed as described above. DNA probes. Colonies of the various E. coli strains were transferred to Whatman 541 paper filters and treated by standard protocol with NaOH and heat to rupture the bacterial cells and denature and fix the DNA to the filter (15, 16, 29). Hybridization was then performed by standard protocol (15, 16, 29) with 32P-dATP-labeled DNA probes (18) that detect the structural genes for SLT-I and SLT-II (22) and the toxin-converting phages that carry SLT-I and SLT-II (933J and 933W, respectively) (22). Rabbit ileal loop. Adult New Zealand White rabbits were starved for 24 h but allowed water ad libitum and then anesthetized with ketamine (50 mg/kg of body weight) and acepromazine (1 mg/kg), followed by xylazine (7 mg/kg) intramuscularly. The supernatants and lysates of test EIEC strains and of the positive control (EHEC strain 933, 0157: H7) and the negative control (normal flora strain E. coli HS, 09:H8) (13, 14) (1 ml each) were injected into the lumen of the intestine proximal to a tie placed near the mesoappendix (21); a second tie isolated the site of inoculation. Proceeding proximally along the ileum, a series of six loops, 7 to 8 cm long, separated by double ties, were isolated and inoculated (21). The sequence of inoculation of loops with the different preparations was randomized so that it varied from rabbit to rabbit. After 18 h, the animals were sacrificed, the fluid volume and length of the loops were measured, and sections of intestine from each loop were fixed in 10% formalinized saline to be examined by light microscopy. Enterotoxin detection with Ussing chambers. The Ussing chamber experiments were performed as previously described (8). Briefly, New Zealand White male adult rabbits weighing 2 to 3 kg were anesthesized by methoxyflurane inhalation and then sacrificed by air embolism. A 20-cm segment of distal ileum was excised, opened along the mesenteric border, and rinsed free of intestinal contents. Four pieces of intestine stripped of the serosal and muscular layers were then mounted in Ussing chambers (1.12-cm2 opening), bathed in Ringer's solution at 37°C, and gassed with 95% 02-5% CO2- Once the tissue reached a steadystate condition, 300 ,ul of EIEC strain CVD/EI-34 (0136: H-) supernatant or lysate was added to the mucosal side. Three hundred microliters was also added to the serosal side to preserve osmotic balance. Chelex-treated syncase broth and PBS were also tested as supernatant and lysate negative controls, respectively. Variation in transepithelial electrical potential difference (PD), total tissue conductance, and short circuit current (Isc) were recorded. PD is the difference in electrical charges between the mucosal and serosal sides of the tissue mounted in an Ussing chamber that is due to differences in concentrations of ions. Isc is the amount of continuous current necessary to nullify the PD. Total tissue conductance, representing the reciprocal of tissue resistance (i.e., 1/tissue resistance), denotes the permeability of an intact tissue to electrical charges carried on ions due to both transcellular and paracellular fluxes. Preliminary sizing of EIEC enterotoxin by exclusion filtra-
INFECT. IMMUN.
tion. To obtain a preliminary estimate of the molecular weight of the enterotoxin, supernatant of EIEC strain CVD/ EI-34 (0136:H-) was serially passed through Diaflo ultrafilters (Amicon, Danvers, Mass.) YM100 (100,000-molecularweight cutoff) and YM30 (30,000-molecular-weight cutoff). The retentates were washed with PBS (Oxoid) by two successive 10:1 volume restorations with PBS, reconcentration, and reconstitution to the original volume. The filtrates and retentates were then tested in Ussing chambers and in rabbit ileal loops for enterotoxic activity and in Vero cells for
cytotoxicity. Partial purification of EIEC enterotoxin. Exclusion chromatography was performed with a Protein Pak 125 column (Waters, Milford, Mass; 7.8 by 300 mm; fractionation range, 10 to 80 kDa). One hundred microliters of 250-fold-concentrated 30- to 100-kDa fraction obtained from supernatant of EIEC strain CVD/EI-34 (0136:H-) was applied and eluted with PBS at 0.5 ml/min. The A280 was monitored, and 0.5-ml fractions were collected and tested in Ussing chambers. RESULTS Cytotoxin production. Both supernatant and lysate of the positive control strain, EHEC 0157:H7 strain 933, showed a high level of cytotoxicity, while the supernatant and lysate of HB 101, the negative control, showed none (Table 1). Against these two extremes, all 35 of the EIEC strains exhibited low levels of cytotoxic activity. This activity was not neutralized by anti-SLT-I or anti-SLT-II antibodies (data not shown). These data corroborate the findings of Marques et al. (20) and Cleary and Murray (3), who earlier reported that EIEC strains elaborate cytotoxins that are not neutralized by anti-SLT-I or anti-SLT-II antibodies. We further observed that our 35 strains elaborating cytotoxins failed to hybridize with DNA probes for SLT-I and SLT-II or with probes for the toxin-converting bacteriophages (933J and 933W) that carry the SLT-I and SLT-II genes in EHEC (Table 1, footnote a). Thus, with 35 EIEC strains examined both genetically and phenotypically, we demonstrate that EIEC cytotoxins are genetically as well as immunologically distinct from SLT-I and SLT-II. Rabbit ileal loops. In the initial experiment, lysate and supernatant of prototype EIEC strain CVD/EI-34 (0136: H-) were inoculated into isolated ileal loops of rabbit intestine, while adjacent loops were inoculated with lysates and supernatants from the positive- and negative-control strains. The positive control, EHEC strain 933, elicited pronounced fluid accumulation in the loops (Table 2) and caused severe mucosal damage (Fig. 1A). In contrast, the ileal loops inoculated with material from the negative control, strain HS, caused no significant fluid accumulation (Table 2) and no epithelial damage (Fig. 1B). The loops injected with either EIEC supernatant or EIEC lysate showed intermediate results characterized by moderate fluid accumulation at 18 h postinoculation (Table 2). The mean fluid accumulation in loops inoculated with material from the 0136:H- EIEC strain was significantly greater than in the negative-control loops (P < 0.05) but significantly less than the fluid accumulation in the EHEC-treated loops (P < 0.05). No tissue damage in loops inoculated with EIEC lysates (Fig. 1C and D) or EIEC supernatants (data not shown) was observed. A second experiment in rabbit loops was carried out with supernatant and lysate of 0143:H- strain 4608-58, its plasmid-cured derivative 4608-58A4, and negative control E. coli HS. The 0143:H- strain also caused significant fluid accu-
ENTEROINVASIVE E. COLI ENTEROTOXIN
VOL. 58, 1990
TABLE 2. Fluid accumulation in rabbit ileal loops
TABLE 1. Results of testing 35 enteroinvasiveness probepositive, Sereny test-positive EIEC strains for Vero cell cytotoxins and for hybridization with DNA probes that detect SLT-I, SLT-II, and the toxin-converting phages that carry SLT-I and SLT-11'
Serogroup
Source
CVD/EI-2 CVD/EI-4 CVD/EI-5 CVD/EI-10 CVD/EI-11
CVD/EI-15 CVD/EI-19 CVD/EI-20 CVD/EI-22 CVD/EI-23 CVD/EI-24
CVD/EI-25 CVD/EI-32 CVD/EI-33 CVD/EI-34 CVD/EI-37 CVD/EI-39 CVD/EI-42 CVD/EI-43 4608-58c CVD/EI-48
CVD/EI-50 CVD/EI-51
CVD/EI-54 CVD/EI-55 CVD/EI-57 CVD/EI-58 CVD/EI-59
CVD/EI-65 CVD/EI-66 CVD/EI-68
CVD/EI-75 CVD/EI-76 CVD/EI-77
028ac:H028ac:H028ac:H028ac:H029:H029:H029:H-
Japan
Brazil Brazil Brazil Brazil Chile 0112ac:H- Brazil 0112ac:H- Brazil 0124:H- Japan 0124:H30 Brazil 0124:H30 United States 0124:H- France 0136:H- Japan 0136:H- Brazil 0136:H- Brazil 0136:H- Japan 0136:H- United States 0143:H- Chile 0143:H- Brazil 0143:H- United States 0144:H0144:H0144:H0144:H0152:H0152:H0152:H0152:H0164:H0164:H0164:H0167:H0167:H0167:H-
Controls HB 101 (negative control) EHEC 933 0157:H7
3.5 3.2 4.3 3.9 4.1
4.4 3.5 3.2 4.5 4.6 3.3 4.0 5.7 4.3 3.6 3.5 4.1 3.9 5.7 4.0 5.7
NDd
ND
Japan Brazil Brazil United States Brazil Brazil Brazil
Japan Brazil
Japan Japan Brazil Brazil Brazil
0.5
x
0.65 ± 0.13c 1.52 ± 0.50d 0.22 ± 0.09
0.48 ± 0.12c 1.28 ± 0.35d 0.03 ± 0.06
EIEC 0143:H_e EIEC 0143:H_e plasmid cured HS
0.40 ± 0.OSC 0.49 ± 0.14c
0.41 ± 0.09c 0.43 ± 0.10c
0.09 ± 0.03
0.06 ± 0.04
2 5.7 5.0 4.8 5.0 4.4 3.7 4.5 3.8 3.6 4.4 4.4 4.1 3.2 5.8 3.9 4.0 5.3 3.8 3.5
4.5 4.3 3.9 4.2 4.1 3.3 4.0 3.2 2.8 2.7 2.9 3.0 2.4 4.0 3.4 3.6 4.2 3.3 3.2 4.0 2.8 2.4 3.8 4.1 2.9 2.9 4.8 3.5 2.9 2.6
Chile
EIEC 0136:HEHEC 0157:H7 HS
Lysate
natant
CVD/El-1
Lysateb (ml/cm + SE)
1
(102/mg of protein) of:
Super-
Supernatantb (ml/cm + SE)
Expt no.a and materials
CD50b Strain
3719
103 3.4
x
3
EIEC 0136:HL broth, 24 h L broth, 72 h Minimal Fe2' broth, 24 h Minimal Fe2' broth, 72 h Negative control strain HS
0.01 0.015 0.77 0.53 0.01
± 0.003 ± 0.005 ± 0.20c ± 0.11' ± 0.005
a 1, Initial detection of EIEC enterotoxin (six animals); 2, effect of 140-MDa plasmid on enterotoxin expression (five animals); 3, effect of [Fe2] on enterotoxin expression (three animals). b Defived from Chelex-treated (low-[Fe2+]) syncase broth cultures, except in experiment 3, in which L broth was also used. ' P < 0.05 compared with HS. d p < 0.01 compared with HS. The significance of the differences was calculated using Student's t test for paired variates. e Isogenic strains.
104
Only EHEC 933 exhibited hybridization with any of the probes tested; it hybridized with probes for SLT-I and 933J but not with probes SLT-II and 933W. CD50, 50% cytotoxic dose. c Strain from Walter Reed Army Institute of Research. ND, Nondetectable. a
b
d
mulation (Table 2) compared with that in the negative control. Supernatant and lysate of the plasmid-cured variant of the 0143:H- strain elicited a degree of fluid accumulation virtually identical to that of the parent 0143:H- strain, showing that (at least in this strain) expression of the enterotoxin does not depend on the presence of the plasmid. In a third experiment (Table 2), we demonstrated that culture of EIEC under conditions of Fe2+ deprivation is necessary to achieve expression of the enterotoxin. Prototype EIEC strain CVD/EI-34 (0136:H-) was cultured for 24 and 72 h both in Fe2'-containing medium (L broth) and in Fe2'-deprived medium (Chelex-treated syncase broth) (23). Enterotoxin was detectable only in supernatants and sonic
extracts made from cultures in the Fe2+-deprived syncase broth. Ussing chamber experiments. To affirm that the fluid accumulation elicited by EIEC in ileal loops was due to an enterotoxic activity, EIEC 0136:H- supernatant and lysate were tested in rabbit small intestine mounted in Ussing chambers (8). Both supernatants and lysates evoked, after a lag of almost 80 min, a significant increase in PD and Isc when compared with the negative controls (Table 3; Fig. 2), peaking after 3 h. In contrast, no significant variations in tissue conductance were observed. Electrical responses to 5 mM theophylline (an inhibitor of intracellular phosphodiesterases [2]), added to the serosal side of the tissues at the end of the experiments, were similar in EIEC-exposed tissue and control-exposed tissues (Isc, 111.1 + 18.4 and 104.7 + 22.9, respectively), suggesting that cyclic AMP is not the intracellular messenger of the EIEC enterotoxic effect. No histological damage was observed by light microscopy of either EIEC supernatant- or lysate-exposed tissues fixed at
the end of the experiment (4-h exposure). Supernatants from eight additional EIEC serotypes were then tested in Ussing chambers; extracts from all significantly altered transepithelial PD and Isc without significantly altering tissue conductance (Table 3). Supernatant from the plasmid-cured variant of the 0143:H- EIEC strain (strain 4608-58A4) retained enterotoxic activity in the Ussing chamber (Table 3), corroborating the findings made with rabbit ileal loops (Table 2) that, at least in this strain, neither structural nor regulatory genes for enterotoxin are located on the plasmid. Experiments in Ussing chambers confirmed that detection of enterotoxin required growth conditions with minimal [Fe2+]. The change in Isc for supernatant of EIEC strain CVD/EI-34 (0136:H-) tested in triplicate was 35.1 + 4.1 RA/cm2 when grown in minimal Fe2+ medium and 18.4 ± 3.1 p.A/cm2 when grown in Fe2+-rich medium (L broth) (P < 0.05).
3720
INFECT. IMMUN.
FASANO ET AL.
A
-
N%.
C
D
TU p
44
I.
B
,V.,
ENTEROINVASIVE E. COLI ENTEROTOXIN
VOL. 58, 1990
3721
TABLE 3. Isc and PD variations in Ussing chambers induced by supernatants of EIEC strains cultured in Chelex-treated (low-[Fe2"]) syncase medium Strain Strain of animals) (no.~~Serotype
CVD/EI-4 CVD/EI-10 CVD/EI-20 CVD/EI-23 CVD/EI-34 4608-58b
4608-58A4b,c CVD/EI-48 CVD/EI-59 CVD/EI-66
028ac:H-(2) 029:H-(3) 0112ac:H-(3) 0124:H-(3) 0136:H-(5) 0143:H-(4) 0143:H-(4) 0144:H-(2) 0152:H-(2) 0164:H-(2)
EIEC
0.80 1.25 0.95 0.70 0.98 0.74 0.65 0.90 0.70 1.40
± ± ± ±
± ±
±
± ± ±
0.1 0.1 0.3 0.1 0.1 0.1 0.1 0.2 0.1 0.3
PDO Negative control 0.1 -0.35 0.05 0.05 0.17 -0.25 0.01 0.2 0.2 0.2
± ± ± ±
± ± ± ± ± ±
0.2 0.1 0.2 0.2 0.2 0.2 0.2 0.1 0.1 0.1
sc
P
INFECTION AND IMMUNITY, Nov. 1990, p. 3717-3723
0019-9567/90/113717-07$02.00/0 Copyright © 1990, American Society for Microbiology
Enterotoxin and Cytotoxin Production by Enteroinvasive Escherichia coli ALESSIO FASANO,'t BRADFORD A. KAY,' ROBERT G. RUSSELL' 2 DAVID R. MANEVAL, JR.,' AND MYRON M. LEVINE'* Center for Vaccine Development, Division of Geographic Medicine, Department of Medicine,' and Department of Pathology,2 University of Maryland School of Medicine, Baltimore, Maryland 21201 Received 29 May 1990/Accepted 30 August 1990
It has long been suspected that besides their ability to invade enterocytes, enteroinvasive Escherichia coli (EIEC) strains have the ability to elaborate an enterotoxin. We tested 35 EIEC strains for cytotoxins and 9 (1 per serogroup) for enterotoxins. All 35 strains exhibited low levels of Vero cell cytotoxins that are immunologically and genetically distinct from Shiga-like toxin I or II of enterohemorrhagic E. coli. Sterile supernatants and cell lysates of two EIEC strains were tested in rabbit ileal loops, and both stimulated moderate fluid accumulation (circa 0.5 ml/cm) without tissue damage; secretory activity was confirmed in Ussing chambers, where these two strains and the seven others tested significantly increased short circuit current without altering tissue conductance. Curing the 140-MDa invasiveness plasmid from an EIEC strain did not diminish enterotoxin production. Culture in minimal Fe2+ medium is necessary to detect expression of the enterotoxin which is circa 68 to 80 kDa in size and is distinct from the EIEC cytotoxin.
Enteroinvasive Escherichia coli (EIEC) strains are an important cause of diarrheal disease and dysentery in young children in less-developed countries (12, 29, 30) and of food-borne outbreaks of enteric disease in adults in industrialized countries (19, 28, 31). Like Shigella spp., with which they share antigens, the cardinal pathogenic feature of EIEC strains is their ability to invade and multiply within epithelial cells of the distal small intestine and the colon (12, 19, 28, 31). This invasive property depends on expression of several outer membrane proteins, structural genes for which are found on 140-MDa enteroinvasive plasmids (plnv) (10, 11) that are essentially identical to the plasmids found in Shigella flexneri. Two features of the clinical syndrome caused by EIEC suggest that cytotoxins and enterotoxins are involved in pathogenesis. Like shigellosis, EIEC infection can result in high fever, malaise, abdominal cramps, and overt dysentery characterized by the tenesmic passage of scanty stools containing blood and mucus (4). While epithelial cell invasiveness is clearly the predominant virulence property responsible for the dysenteric syndrome, by analogy with Shigella, spp. (26), cytotoxins may in part dictate the severity of the dysenteric symptoms. More importantly, watery diarrhea typically precedes dysentery in EIEC infection, and both in individual cases and in outbreaks due to EIEC (19, 28-31), watery diarrhea is often seen exclusively without dysentery. These clinical observations suggest that EIEC produces an enterotoxin that is responsible for the watery diarrhea. Accordingly, a series of EIEC strains were examined for the production of Vero cell cytotoxins and for enterotoxins. MATERIALS AND METHODS Bacterial strains. A total of 35 E. coli strains, representing diarrheal isolates of all major EIEC 0 serogroups from Corresponding author. t Present address: Department of Pediatrics, University of
*
Napoli, Naples, Italy.
diverse geographic locations, were initially cultured on blood agar to assess their purity. Isolated colonies were subcultured to Casamino Acids-yeast extract agar and incubated in ambient air overnight at 37°C. Epithelial cell invasiveness and virulence of the E. coli strains were confirmed by demonstrating the ability of the strains to cause purulent keratoconjunctivitis following inoculation into the conjunctival sac of guinea pigs (33), as originally described by Sereny for Shigella spp. (27). Each strain was also shown to hybridize at high stringency with a DNA probe that detects the EIEC virulence plasmid (7, 15, 33). Preparation of supernatants and sonic extracts. The 35 Sereny test-positive, EIEC probe-positive strains were inoculated into 5 ml of Chelex-treated syncase broth (23). The bacteria were harvested by centrifugation, and the culture supernatants were collected and filter sterilized. The bacterial cells were washed twice in phosphate-buffered saline (PBS), suspended in 1.5 ml of PBS (pH 7.4), and disrupted in a French pressure cell at 12,000 lb/in2 (23). Lysates were then mixed with 3.5 ml of PBS (final volume, 5 ml), clarified by centrifugation (18,000 x g for 20 min at 4°C), and filter sterilized. The supernatants and lysates prepared in this manner were used in assays to detect cytotoxins and enterotoxins. Vero cell assay for cytotoxicity. Cytotoxicity assays were performed with Vero cells by the method of Gentry and Dalrymple (6). Serial twofold dilutions (1:2 to 1:64) of culture supernatants and cell lysates were tested, and the cytotoxic dose required to kill 50% of Vero cells was estimated spectrophotometrically (6). Supernatants and lysates from enterohemorrhagic E. coli (EHEC) strain 933 (serotype 0157:H7), which elaborates Shiga-like toxins I (SLT-I) and SLT-II (22, 24), served as the positive control, while the negative controls were prepared from E. coli K-12 HB101; controls were included in each microtiter plate. Since the positive control killed more than 50% of Vero cells at a 1:64 dilution, a 10-fold dilution of this sample was tested. Cytotoxic titers were expressed as the reciprocal of the 50% cytotoxic dose of culture supernatant or cell lysate per 3717
3718
FASANO ET AL.
milligram of protein; the protein content was measured by the method of Bradford (1). In toxin neutralization experiments, supernatants and lysates from 10 EIEC strains of different serogroups were incubated for 60 min at 37°C with an anti-SLT-I monoclonal antibody and with polyclonal monospecific anti-SLT-II antibodies (titers, 1:25) kindly provided by Alison O'Brien, Uniformed Services University of the Health Sciences, Bethesda, Md. The cytotoxicity assay was then performed as described above. DNA probes. Colonies of the various E. coli strains were transferred to Whatman 541 paper filters and treated by standard protocol with NaOH and heat to rupture the bacterial cells and denature and fix the DNA to the filter (15, 16, 29). Hybridization was then performed by standard protocol (15, 16, 29) with 32P-dATP-labeled DNA probes (18) that detect the structural genes for SLT-I and SLT-II (22) and the toxin-converting phages that carry SLT-I and SLT-II (933J and 933W, respectively) (22). Rabbit ileal loop. Adult New Zealand White rabbits were starved for 24 h but allowed water ad libitum and then anesthetized with ketamine (50 mg/kg of body weight) and acepromazine (1 mg/kg), followed by xylazine (7 mg/kg) intramuscularly. The supernatants and lysates of test EIEC strains and of the positive control (EHEC strain 933, 0157: H7) and the negative control (normal flora strain E. coli HS, 09:H8) (13, 14) (1 ml each) were injected into the lumen of the intestine proximal to a tie placed near the mesoappendix (21); a second tie isolated the site of inoculation. Proceeding proximally along the ileum, a series of six loops, 7 to 8 cm long, separated by double ties, were isolated and inoculated (21). The sequence of inoculation of loops with the different preparations was randomized so that it varied from rabbit to rabbit. After 18 h, the animals were sacrificed, the fluid volume and length of the loops were measured, and sections of intestine from each loop were fixed in 10% formalinized saline to be examined by light microscopy. Enterotoxin detection with Ussing chambers. The Ussing chamber experiments were performed as previously described (8). Briefly, New Zealand White male adult rabbits weighing 2 to 3 kg were anesthesized by methoxyflurane inhalation and then sacrificed by air embolism. A 20-cm segment of distal ileum was excised, opened along the mesenteric border, and rinsed free of intestinal contents. Four pieces of intestine stripped of the serosal and muscular layers were then mounted in Ussing chambers (1.12-cm2 opening), bathed in Ringer's solution at 37°C, and gassed with 95% 02-5% CO2- Once the tissue reached a steadystate condition, 300 ,ul of EIEC strain CVD/EI-34 (0136: H-) supernatant or lysate was added to the mucosal side. Three hundred microliters was also added to the serosal side to preserve osmotic balance. Chelex-treated syncase broth and PBS were also tested as supernatant and lysate negative controls, respectively. Variation in transepithelial electrical potential difference (PD), total tissue conductance, and short circuit current (Isc) were recorded. PD is the difference in electrical charges between the mucosal and serosal sides of the tissue mounted in an Ussing chamber that is due to differences in concentrations of ions. Isc is the amount of continuous current necessary to nullify the PD. Total tissue conductance, representing the reciprocal of tissue resistance (i.e., 1/tissue resistance), denotes the permeability of an intact tissue to electrical charges carried on ions due to both transcellular and paracellular fluxes. Preliminary sizing of EIEC enterotoxin by exclusion filtra-
INFECT. IMMUN.
tion. To obtain a preliminary estimate of the molecular weight of the enterotoxin, supernatant of EIEC strain CVD/ EI-34 (0136:H-) was serially passed through Diaflo ultrafilters (Amicon, Danvers, Mass.) YM100 (100,000-molecularweight cutoff) and YM30 (30,000-molecular-weight cutoff). The retentates were washed with PBS (Oxoid) by two successive 10:1 volume restorations with PBS, reconcentration, and reconstitution to the original volume. The filtrates and retentates were then tested in Ussing chambers and in rabbit ileal loops for enterotoxic activity and in Vero cells for
cytotoxicity. Partial purification of EIEC enterotoxin. Exclusion chromatography was performed with a Protein Pak 125 column (Waters, Milford, Mass; 7.8 by 300 mm; fractionation range, 10 to 80 kDa). One hundred microliters of 250-fold-concentrated 30- to 100-kDa fraction obtained from supernatant of EIEC strain CVD/EI-34 (0136:H-) was applied and eluted with PBS at 0.5 ml/min. The A280 was monitored, and 0.5-ml fractions were collected and tested in Ussing chambers. RESULTS Cytotoxin production. Both supernatant and lysate of the positive control strain, EHEC 0157:H7 strain 933, showed a high level of cytotoxicity, while the supernatant and lysate of HB 101, the negative control, showed none (Table 1). Against these two extremes, all 35 of the EIEC strains exhibited low levels of cytotoxic activity. This activity was not neutralized by anti-SLT-I or anti-SLT-II antibodies (data not shown). These data corroborate the findings of Marques et al. (20) and Cleary and Murray (3), who earlier reported that EIEC strains elaborate cytotoxins that are not neutralized by anti-SLT-I or anti-SLT-II antibodies. We further observed that our 35 strains elaborating cytotoxins failed to hybridize with DNA probes for SLT-I and SLT-II or with probes for the toxin-converting bacteriophages (933J and 933W) that carry the SLT-I and SLT-II genes in EHEC (Table 1, footnote a). Thus, with 35 EIEC strains examined both genetically and phenotypically, we demonstrate that EIEC cytotoxins are genetically as well as immunologically distinct from SLT-I and SLT-II. Rabbit ileal loops. In the initial experiment, lysate and supernatant of prototype EIEC strain CVD/EI-34 (0136: H-) were inoculated into isolated ileal loops of rabbit intestine, while adjacent loops were inoculated with lysates and supernatants from the positive- and negative-control strains. The positive control, EHEC strain 933, elicited pronounced fluid accumulation in the loops (Table 2) and caused severe mucosal damage (Fig. 1A). In contrast, the ileal loops inoculated with material from the negative control, strain HS, caused no significant fluid accumulation (Table 2) and no epithelial damage (Fig. 1B). The loops injected with either EIEC supernatant or EIEC lysate showed intermediate results characterized by moderate fluid accumulation at 18 h postinoculation (Table 2). The mean fluid accumulation in loops inoculated with material from the 0136:H- EIEC strain was significantly greater than in the negative-control loops (P < 0.05) but significantly less than the fluid accumulation in the EHEC-treated loops (P < 0.05). No tissue damage in loops inoculated with EIEC lysates (Fig. 1C and D) or EIEC supernatants (data not shown) was observed. A second experiment in rabbit loops was carried out with supernatant and lysate of 0143:H- strain 4608-58, its plasmid-cured derivative 4608-58A4, and negative control E. coli HS. The 0143:H- strain also caused significant fluid accu-
ENTEROINVASIVE E. COLI ENTEROTOXIN
VOL. 58, 1990
TABLE 2. Fluid accumulation in rabbit ileal loops
TABLE 1. Results of testing 35 enteroinvasiveness probepositive, Sereny test-positive EIEC strains for Vero cell cytotoxins and for hybridization with DNA probes that detect SLT-I, SLT-II, and the toxin-converting phages that carry SLT-I and SLT-11'
Serogroup
Source
CVD/EI-2 CVD/EI-4 CVD/EI-5 CVD/EI-10 CVD/EI-11
CVD/EI-15 CVD/EI-19 CVD/EI-20 CVD/EI-22 CVD/EI-23 CVD/EI-24
CVD/EI-25 CVD/EI-32 CVD/EI-33 CVD/EI-34 CVD/EI-37 CVD/EI-39 CVD/EI-42 CVD/EI-43 4608-58c CVD/EI-48
CVD/EI-50 CVD/EI-51
CVD/EI-54 CVD/EI-55 CVD/EI-57 CVD/EI-58 CVD/EI-59
CVD/EI-65 CVD/EI-66 CVD/EI-68
CVD/EI-75 CVD/EI-76 CVD/EI-77
028ac:H028ac:H028ac:H028ac:H029:H029:H029:H-
Japan
Brazil Brazil Brazil Brazil Chile 0112ac:H- Brazil 0112ac:H- Brazil 0124:H- Japan 0124:H30 Brazil 0124:H30 United States 0124:H- France 0136:H- Japan 0136:H- Brazil 0136:H- Brazil 0136:H- Japan 0136:H- United States 0143:H- Chile 0143:H- Brazil 0143:H- United States 0144:H0144:H0144:H0144:H0152:H0152:H0152:H0152:H0164:H0164:H0164:H0167:H0167:H0167:H-
Controls HB 101 (negative control) EHEC 933 0157:H7
3.5 3.2 4.3 3.9 4.1
4.4 3.5 3.2 4.5 4.6 3.3 4.0 5.7 4.3 3.6 3.5 4.1 3.9 5.7 4.0 5.7
NDd
ND
Japan Brazil Brazil United States Brazil Brazil Brazil
Japan Brazil
Japan Japan Brazil Brazil Brazil
0.5
x
0.65 ± 0.13c 1.52 ± 0.50d 0.22 ± 0.09
0.48 ± 0.12c 1.28 ± 0.35d 0.03 ± 0.06
EIEC 0143:H_e EIEC 0143:H_e plasmid cured HS
0.40 ± 0.OSC 0.49 ± 0.14c
0.41 ± 0.09c 0.43 ± 0.10c
0.09 ± 0.03
0.06 ± 0.04
2 5.7 5.0 4.8 5.0 4.4 3.7 4.5 3.8 3.6 4.4 4.4 4.1 3.2 5.8 3.9 4.0 5.3 3.8 3.5
4.5 4.3 3.9 4.2 4.1 3.3 4.0 3.2 2.8 2.7 2.9 3.0 2.4 4.0 3.4 3.6 4.2 3.3 3.2 4.0 2.8 2.4 3.8 4.1 2.9 2.9 4.8 3.5 2.9 2.6
Chile
EIEC 0136:HEHEC 0157:H7 HS
Lysate
natant
CVD/El-1
Lysateb (ml/cm + SE)
1
(102/mg of protein) of:
Super-
Supernatantb (ml/cm + SE)
Expt no.a and materials
CD50b Strain
3719
103 3.4
x
3
EIEC 0136:HL broth, 24 h L broth, 72 h Minimal Fe2' broth, 24 h Minimal Fe2' broth, 72 h Negative control strain HS
0.01 0.015 0.77 0.53 0.01
± 0.003 ± 0.005 ± 0.20c ± 0.11' ± 0.005
a 1, Initial detection of EIEC enterotoxin (six animals); 2, effect of 140-MDa plasmid on enterotoxin expression (five animals); 3, effect of [Fe2] on enterotoxin expression (three animals). b Defived from Chelex-treated (low-[Fe2+]) syncase broth cultures, except in experiment 3, in which L broth was also used. ' P < 0.05 compared with HS. d p < 0.01 compared with HS. The significance of the differences was calculated using Student's t test for paired variates. e Isogenic strains.
104
Only EHEC 933 exhibited hybridization with any of the probes tested; it hybridized with probes for SLT-I and 933J but not with probes SLT-II and 933W. CD50, 50% cytotoxic dose. c Strain from Walter Reed Army Institute of Research. ND, Nondetectable. a
b
d
mulation (Table 2) compared with that in the negative control. Supernatant and lysate of the plasmid-cured variant of the 0143:H- strain elicited a degree of fluid accumulation virtually identical to that of the parent 0143:H- strain, showing that (at least in this strain) expression of the enterotoxin does not depend on the presence of the plasmid. In a third experiment (Table 2), we demonstrated that culture of EIEC under conditions of Fe2+ deprivation is necessary to achieve expression of the enterotoxin. Prototype EIEC strain CVD/EI-34 (0136:H-) was cultured for 24 and 72 h both in Fe2'-containing medium (L broth) and in Fe2'-deprived medium (Chelex-treated syncase broth) (23). Enterotoxin was detectable only in supernatants and sonic
extracts made from cultures in the Fe2+-deprived syncase broth. Ussing chamber experiments. To affirm that the fluid accumulation elicited by EIEC in ileal loops was due to an enterotoxic activity, EIEC 0136:H- supernatant and lysate were tested in rabbit small intestine mounted in Ussing chambers (8). Both supernatants and lysates evoked, after a lag of almost 80 min, a significant increase in PD and Isc when compared with the negative controls (Table 3; Fig. 2), peaking after 3 h. In contrast, no significant variations in tissue conductance were observed. Electrical responses to 5 mM theophylline (an inhibitor of intracellular phosphodiesterases [2]), added to the serosal side of the tissues at the end of the experiments, were similar in EIEC-exposed tissue and control-exposed tissues (Isc, 111.1 + 18.4 and 104.7 + 22.9, respectively), suggesting that cyclic AMP is not the intracellular messenger of the EIEC enterotoxic effect. No histological damage was observed by light microscopy of either EIEC supernatant- or lysate-exposed tissues fixed at
the end of the experiment (4-h exposure). Supernatants from eight additional EIEC serotypes were then tested in Ussing chambers; extracts from all significantly altered transepithelial PD and Isc without significantly altering tissue conductance (Table 3). Supernatant from the plasmid-cured variant of the 0143:H- EIEC strain (strain 4608-58A4) retained enterotoxic activity in the Ussing chamber (Table 3), corroborating the findings made with rabbit ileal loops (Table 2) that, at least in this strain, neither structural nor regulatory genes for enterotoxin are located on the plasmid. Experiments in Ussing chambers confirmed that detection of enterotoxin required growth conditions with minimal [Fe2+]. The change in Isc for supernatant of EIEC strain CVD/EI-34 (0136:H-) tested in triplicate was 35.1 + 4.1 RA/cm2 when grown in minimal Fe2+ medium and 18.4 ± 3.1 p.A/cm2 when grown in Fe2+-rich medium (L broth) (P < 0.05).
3720
INFECT. IMMUN.
FASANO ET AL.
A
-
N%.
C
D
TU p
44
I.
B
,V.,
ENTEROINVASIVE E. COLI ENTEROTOXIN
VOL. 58, 1990
3721
TABLE 3. Isc and PD variations in Ussing chambers induced by supernatants of EIEC strains cultured in Chelex-treated (low-[Fe2"]) syncase medium Strain Strain of animals) (no.~~Serotype
CVD/EI-4 CVD/EI-10 CVD/EI-20 CVD/EI-23 CVD/EI-34 4608-58b
4608-58A4b,c CVD/EI-48 CVD/EI-59 CVD/EI-66
028ac:H-(2) 029:H-(3) 0112ac:H-(3) 0124:H-(3) 0136:H-(5) 0143:H-(4) 0143:H-(4) 0144:H-(2) 0152:H-(2) 0164:H-(2)
EIEC
0.80 1.25 0.95 0.70 0.98 0.74 0.65 0.90 0.70 1.40
± ± ± ±
± ±
±
± ± ±
0.1 0.1 0.3 0.1 0.1 0.1 0.1 0.2 0.1 0.3
PDO Negative control 0.1 -0.35 0.05 0.05 0.17 -0.25 0.01 0.2 0.2 0.2
± ± ± ±
± ± ± ± ± ±
0.2 0.1 0.2 0.2 0.2 0.2 0.2 0.1 0.1 0.1
sc
P
