INFEcrION AND IMMUNITY, Apr. 1992, p. 1613-1617 0019-9567/92/041613-05$02.00/0 Copyright X) 1992, American Society for Microbiology
Vol. 60, No. 4
Adherence of Enterohemorrhagic Escherichia coli Strains to
a
Human Colonic Epithelial Cell Line (T84) DONALD K. WINSOR, JR.,1* SHAI ASHKENAZI,2 ROBERT CHIOVETTI,3 AND THOMAS G. CLEARY4 Department of Microbiology and Molecular Genetics' and Department of Pediatrics, 4 University of Texas Medical School at Houston, Houston, Texas 77030; Department of Pediatrics, Beilinson Medical Center, Petah Tikva, Israel 491002; and Life Cell Corporation, The Woodlands, Te-xas 773813 Received 18 October 1991/Accepted 20 January 1992
Enterohemorrhagic Escherichia coli (EHEC) produce Shiga-like toxins and attach to certain tissue culture cells. T.4 cells are human colonic carcinoma cells. Unlike previously studied cell lines, T.4 cells grown on collagen-coated surfaces polarize and produce tight junctions and desmosomes, forming a colonic epithelial cell layer in vitro. The purpose of this study was to examine the attachment of EHEC strains to the T84 cell line as a possibly more relevant in vitro model of EHEC adherence. Twelve EHEC strains were grown overnight in Penassay broth, suspended in minimal essential medium with and without 0.5% mannose, and incubated for 1 to 3 h with 5- to 7-day-old T84 cell monolayers grown on collagen-coated coverslips. The bacteria were removed, and attachment was quantitated microscopically. For both E. coli 0157:H7 and other EHEC serotypes, there were marked differences in adherence between strains (range of 152 to 3 bacteria per oil immersion field). Mannose partially inhibited the adherence of some EHEC strains. Adherence to the T.4 cells appeared to be related to the amount of pili present and not to the serotype. Electron micrographs showed that a highly adherent strain (strain 43-12) tended to form microcolonies in the area of tight junctions on the T84 cell monolayers. In addition, the attachment of these EHEC strains to T84 cells correlated with their ability to adhere to isolated rabbit colonocytes (r = 0.91, P = 0.00004; without mannose) (r = 0.60, P = 0.04; with mannose). These data show that there are EHEC strain-related differences in adherence which can be demonstrated in a human-derived colonic epithelial cell line (T,4) and that these cells can be used to study EHEC adherence. Strains of enterohemorrhagic Eschenchia coli (EHEC) outbreaks of hemorrhagic colitis (HC) and of hemolytic uremic syndrome (HUS) (8, 11, 14, 17). These strains produce Shiga-like toxin (SLT) I and/or II and belong to a variety of serotypes in addition to 0157:H7 (20, 25). It has been shown in humans and in animal models that these organisms attach primarily to the colonic epithelium, are noninvasive, and produce large amounts of SLT, which is believed to be the primary mediator of both local and systemic pathology (16, 24, 25). These organisms adhere to the animal intestinal epithelium by an attaching-effacing mechanism similar to that described for enteropathogenic E. coli, although the actual means of attachment remains unclear (20, 25). Mannose-sensitive pili have been implicated in the adherence to isolated rabbit colonocytes and isolated human intestinal epithelial cells of some EHEC strains (1, 4), while outer membranes have been shown to inhibit the adherence of other EHEC strains to HEp-2 cells (4, 7, 18-22). Since EHEC attaches primarily to the colonic epithelium in vivo, the use of a colonic epithelial cell line which polarizes, forms tight junctions and microvilli, and maintains electrical resistance in vitro might be most appropriate for the study of EHEC adherence. The use of such a cell line could lead to a better understanding of the changes in the physiology of the colonic epithelium during infection. The T84 cell line was derived from a colonic carcinoma which, when grown on collagen-coated surfaces, polarizes and exhibits compartmentalization of organelles and formation of tight junctions, desmosomes, and a basolateral and apical
surface with microvilli in vitro and can be used to study changes in electrical conductivity of the epithelial cell layer (12). For these reasons and because the adherences of EHEC to these cells would more closely mimic the in vivo situation, we examined the ability of 12 EHEC strains (0157:H7 and non-0157:H7) to attach to T84 cells in the presence and absence of mannose, compared these results with binding to isolated rabbit colonocytes, and evaluated the adherence of EHEC to T84 cells morphologically by electron microscopy.
cause
*
MATERIALS AND METHODS Bacterial strains. The E. coli strains and their serotypes, amounts of piliation, and toxin types are listed in Table 1. Strain H30 is a strain that was originally described by Konowalchuk et al. (10). Strains 43-12, 212-2, and 306-8 were isolated from children in Argentina with diarrhea or HUS (11). The remainder of the E. coli strains were obtained from M. Osterholm, Minnesota Department of Public Health. For the adherence assays, bacteria were grown in Penassay broth overnight without shaking at 37°C. T.4 cell growth. T84 cells were grown in Dulbecco's minimal essential medium-Ham's F12 medium (DMEM/H) containing 5% newborn calf serum, 200 mM L-glutamine, 5,000 U of penicillin per ml, and 5,000 ,ug of streptomycin per ml in an atmosphere of 5% CO2. For the EHEC adherence assay, the T84 cells were seeded (106 cells per ml) into wells of a 24-well tissue culture plate, each of which contained a 12-mm glass coverslip coated with rat tail collagen (type 1; Sigma Chemical Co., St. Louis, Mo.). The cells were then grown for 5 to 7 days before use (12). Three hours before infection, the growth medium was removed
Corresponding author. 1613
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INFECT. IMMUN.
WINSOR ET AL.
TABLE 2. Adherence of EHEC strains to T84 cells and rabbit colonocytes
TABLE 1. Characteristics of the EHEC strains used in this study Strain
Serotype
Piliationa
306-8 942 E473 E773 987 1058 E670 E854 212-2 546 H30 43-12
0157:H7 0157:H7
NDb
0157:1-7 0157:H7 0157:H7 0157:H7 0157:H7 0157:H7 0157:H7 0157:H7 026:H11 Ount:H21
ND ND ND 12 ND
NPc 6 NP ND NP 20
Toxin type SLT-/II SLT-II SLT-II
SLT-I/II SLT-I/II SLT-I SLT-I/II SLT-I/II SLT-II SLT-II SLT-I SLT-II
Disease
HUS HUS HUS HUS HC HC HC HC HC None DIAd HUS
a Data from Ashkenazi et al. (1). b
ND, not determined. c NP, nonpiliated. d DIA, diarrhea.
from each well and replaced with medium which did not contain serum or antibiotics. Preparation of rabbit intestinal cells. Cells from the proximal colons of adult New Zealand White rabbits were obtained by treating intestinal loops of rabbits with solutions containing EDTA, citrate, and dithiothreitol as previously described (1). EHEC adherence assays. (i) T.4 Cells. The bacteria were washed and suspended to 6 x 109 organisms per ml in DMEM/H with or without 0.5% mannose. Each strain was assayed three to five times. In each assay, two or three coverslips per strain were each inoculated with 0.5 ml of the bacterial suspension. After 1 h of incubation at 37°C in 5% CO2, the inoculum was removed, the T84 cells were washed three times with phosphate-buffered saline (PBS), and the coverslips were Giemsa stained. Each coverslip was mounted on a microscope slide, and the number of bacteria attached to T84 cells in 10 randomly chosen oil immersion fields was determined. T84 cells seeded on collagen-coated surfaces grow in such a manner that distinguishing between individual cells is almost impossible since the individual cell membranes are indistinguishable. Only fields with confluent T84 cells were counted: thus, bacterial counts were done on a constant surface area (one oil immersion field at x1,000 magnification) and used as the unit of measure. (ii) Rabbit intestinal cells. A suspension of intestinal cells (200 ,ul of 107 cells per ml) was incubated with [3H]thymidine-labeled bacteria (100 ,ul of 6 x 109 bacterial cells per ml) at 37°C for 45 min. Intestinal cells with bound bacteria were separated from unbound bacteria by Percoll density gradient centrifugation, and the layer containing the intestinal cells with bound bacteria was counted in a scintillation counter (1). Each strain was assayed three to five times. Electron microscopy. T84 cells were grown on 12-mmdiameter, round Thermanox plastic coverslips (Nunc, Inc., Naperville, Ill.) coated with collagen. The confluent monolayers were infected as previously described. The inoculum was removed and the monolayers were washed three times with PBS and incubated with DMEM/H without serum or antibiotics for 8 h. These infected monolayers were then processed for electron microscopy by fixation for 1 to 2 h in 2.5% glutaraldehyde-0.1 M sodium cacodylate (pH 7.3) and then washed with cacodylate buffer and postfixed in 1% osmium tetroxide-0.1 M sodium cacodylate. The coverslips were transferred to cacodylate buffer, and they were cleaved
Strain
43-12 H30 942 306-8 987 E773 E854 E670 546 1058 E473 212-2
T84 cellsa Without With mannose mannose
152 87 50 41 32 30 ± 27 ± 22 ± 13 ± 12 ± 11 ± 3±
27c 32 24 16 10 3 5 7 2 5 4 0
74 ± 48 ± 32 ± 25 ± 60 ± 14 ± 27 ± 7±
8 23 6 5
± ± ± ±
27c 26 16 9 28 4 10 2 3 6 1 1
Rabbit colonocytesb Without With mannose mannose
32 ± 10 ± 9± 2± 8± 2± 6± 2± 1± 1± 6± 4±
3c 3 2 1 3 1 2 1 1 1 2 1
17 ± 2c 6±3 2± 1 3± 1 2±1 3± 1 13 ± 4 2± 1 3± 1 1± 1 3± 1 1±1
a Number of bacteria attached per oil immersion field. b Number of bacteria per colonocyte. c Mean of three to five independent assays ± standard error of the mean.
into rectangles (ca. 3 by 5 mm) for flat embedding. The Thermanox rectangles were dehydrated in an ascending ethanol series (30, 50, 70, and 95%, and two times 100%, 10 min each) and then in propylene oxide (two times, 10 min). The specimens were transferred to new sample vials containing mixtures of Spurr low-viscosity resin and propylene oxide for infiltration. The infiltration schedule consisted of incubations in Spurr resin-propylene oxide (1:2 for 1 h; 2:1 overnight) and then in fresh Spurr resin for 4 h. Individual Thermanox rectangles were placed in flat embedding molds with new Spurr resin, and the resin was polymerized at 60°C overnight. Silver to light-gold ultrathin sections (60 to 90 nm) were cut with a diamond knife on a Reichert Ultracut B ultramicrotome, and the sections were collected on naked 300-hexagonal-mesh nickel grids. The sections were stained for 45 min in saturated aqueous uranyl acetate and for 15 min in lead citrate. The specimens were examined, and electron micrographs were made by using a Philips CM12 electron microscope operating at 80 keV in the transmission mode. The amounts of piliation of six of these strains were determined by electron microscopy of negative-stained preparation (1). These strains were chosen because they represented high, moderate, and low levels of adherence. Statistical analysis. Comparison of EHEC binding to T84 cells in the presence and absence of mannose and comparison of the binding to T84 cells and rabbit colonocytes were done by linear regression. RESULTS Adherence to T.4 cells and rabbit colonocytes. Adherence of these EHEC strains to T84 cell monolayers and rabbit colonocytes in the presence and absence of mannose is shown in Table 2. Strain 43-12 was the most adherent in both systems, followed by strain H30. Both are non-0157:H7 strains, with 43-12 being isolated from a patient with HUS and H30 being isolated from a patient with diarrhea. The 0157:H7 strains adhered to these two cell types to various degrees, with no apparent relationship between adherence, toxin type produced, or associated disease. There appears to be a relationship between adherence to T84 cells and the presence of fimbriae. Of the six strains for which the amounts of piliation have been determined (strains 987, E670, E824, 212-2, H30, and 43-12 [1]), the most adherent
EHEC ADHERENCE TO EPITHELIAL CELL LINE
VOL. 60, 1992
0O
000-
Zo 0= 0
o
-I
Ua .* o
32
64
96
128
160
T84 cells
(bacteria/oll Immersion field) FIG. 1. Relations between adherence of EHEC strains to cells and that to ir ship solated rabbit colonocytes in the absence of mannose
(r
=
0.91; P = 0.00004). Each point represents a single
strain.
strain (strain 43-1 2) was also the most piliated, the strains with moderate pi Iliation (strains 987 and E854) were also moderately adher ent, and the strains with little or no (E670, 212-1) wer e only minimally adherent. Strain H30 IS the second most adherent strain but IS nonpiliated under these growth con ditions. To determine if the presence of mannose had a sinnilar effect on these EHEC strains, regardless of piliation, w e compared the adherence of these strains to T84 cell monc layers in the presence and absence of mannose by lineai regression. Mannose only partially inhibited the adherenl ce of some of these strains; it dld not completely block adherence of any strains to T cells. A significant cc Irrelation between the adherence of these EHEC strains to I th4 cells and that to isolated rabbit colonocytes was seen in the absence (r = 0.91, P = 0.00004; Fig. 1) and in the presencce (r = 0.60; P = 0.04) of mannose. These data strongly sugggest that these two systems are measuring closely related ph enomena. Electron micros4copy. Figure 2A is an electron micrograph of uninfected T84 4cells grown on collagen-coated coverslips. Microvilli, tight jiunctions, and desmosomes are visible in this micrograph. )An electron micrograph of the attachment of EHEC 43-12, tI ie most adherent of these EHEC strains, to T84 cells after 8 h of incubation is shown in Fig. 2B. Microcolonies of tthis strain were frequently seen around the areas containing tight junctions, with single bacterial cells being found on ot:;her parts of the T84 cell surface. Bacterial cells were seen b oth in close association with the T84 cell membrane and in close proximity to the membrane.
pisi
DISCUSSION E. coli strains Mvhich elaborate large quantities of SLT and belong to certain serotypes, primarily 0157:H7, are known to cause HC and HUS (8, 14). The extraintestinal manifestations of these dliseases are believed to be caused by the systemic effects o f SLT (16). However, the exact nature of the bacterial cell-c:olonic epithelial cell interaction is unclear. There is disagreeDment about to which cell lines EHEC adheres (7, 9). C urrent data have suggested both that the 60-MDa plasmid mediates a fimbrial adhesion important in Henle 407 (INT4017) cell adherence (7) and that fimbriae play no role in adherenIce (23). Loss of the plasmid has even been i
1615
associated with both increased Henle cell adherence (6) and decreased adherence (23). Additionally, plasmidless derivatives are still pathogenic in piglets (25). Published data on hemagglutination also disagree on the significance of the plasmid or the pili (9, 23). The variable and conflicting data reflect the inadequacy of current models to study EHEC adherence. Because of the lack of appropriate in vitro models, questions about how the bacteria attach to the colonic epithelium and how or when SLT is delivered across the epithelium still remain. Optimally, an in vitro model to determine the answer to these questions ought to include a colonic cell type of human origin which, when cultured, would have characteristics of the colonic epithelium, including the formation of tight junctions and desmosomes and transport properties similar to the in vivo situation. The T84 cell line fulfills these criteria. These cells form tight junctions and desmosomes and transport ions in response to pharmacological agents in a manner similar to that of the natural colonic epithelium (5, 12, 26). In this study, we have shown that both 0157:H7 and non-0157:H7 strains of EHEC adhere to T84 cells and that this adherence correlates with their adherence to rabbit colonocytes, one of the models used to study EHEC patho-
genesis (1, 4, 20). Additionally, E. coli HS, which is a commensal fecal E. coli strain, attaches to the T84 cells at a level similar to that of strain 212-2, the EHEC strain with the
lowest level of adherence (data not shown). Some EHEC strains will attach to other cell lines, such as HEp-2 and Henle 407 cells, but these cells may be less appropriate for the study of colonic pathogens. HEp-2 and Henle 407 cells do not differentiate into intact epithelial cell layers, thus not allowing the study of changes in epithelial cell function during infection (6, 19, 22, 23). The CaCo-2 cell line has been used to examine the adherence of enteropathogenic E. coli and a limited number of EHEC strains (9). The CaCo-2 cell line was also derived from a human colonic carcinoma, polarizes, forming tight junctions and desmosomes, and has been used to study epithelial cell transport (2). However,
CaCo-2 cells differentiate into an epithelial cell layer which resembles the small intestinal epithelium because the mature monolayer possesses levels of sucrase-isomaltase similar to those of the small intestine (15) and because small-intestinal pathogens, such as enterotoxigenic and enteropathogenic E. coli strains, adhere to these cells (3, 9) whereas a colonic pathogen, EHEC, does not (9). The amount of attachment to the polarized T84 cells varied depending on the EHEC strain and the amount of piliation. Strain 43-12 was highly piliated (1); the most adherent, strain 987, was moderately piliated (1) and only moderately adherent; and strain 212-2 was nonpiliated (1) and minimally adherent to the T84 cells. This is in accordance with other studies in which the highly piliated EHEC strains adhered in greater numbers than nonpiliated strains (1, 4). Since these pili are thought to be a form of type 1 pili and since mannose has been shown to at least partially inhibit the adherence of EHEC strains (1, 4, 9, 18), we examined the effect mannose had on the T84 cell adherence of these EHEC strains. In no instance did the presence of mannose eliminate the adherence of these strains. Mannose decreased the adherence of some EHEC strains (43-12, H30, 942, 306-8, E773, E670, 546, and E473), had no effect on some (E854 and 212-2), and appeared to increase the adherence of others (987 and 1058). Ashkenazi et al. (1) reported similar results, with mannose inhibiting the attachment of strain 43-12 by 40% and that of strain H30 by 10 to 20%. Binding of strains 987 and 212-2 to rabbit colonocytes was not significantly inhibited by the
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FIG. 2. Transmission electron micrograph of uninfected T84 cell monolayer (A) and T84 cell monolayer infected with EHEC 43-12 showing association of bacteria with T84 cell surface after 8 h of incubation (B). Magnification, x 8,595. Arrows indicate tight junctions, and arrowheads indicate desmosomes. MV, microvilli.
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VOL. 60, 1992
EHEC ADHERENCE TO EPITHELIAL CELL LINE
presence of mannose (1). The similarities of the two systems were further confirmed by the correlation of adherences, both in the presence and absence of mannose, to T84 cells
9. Knutton, S., T. Baldwin, P. H. Williams, and A. S. McNeish. 1989. Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infect. Immun. 57: 1290-1298. 10. Konowalchuk, J., J. I. Speirs, and S. Stavric. 1977. Vero response to a cytotoxin of Escherichia coli. Infect. Immun. 18:775-779. 11. Lopez, E. L., M. Diaz, S. Grinstein, S. Devoto, F. Mendilaharzu, B. E. Murray, S. Ashkenazi, E. Rubeglio, M. Woloj, M. Vasquez, M. Turco, L. K. Pickering, and T. G. Cleary. 1989. HUS and diarrhea in Argentine children: the role of shiga-like toxins. J. Infect. Dis. 160:469-475. 12. Madara, J. L., J. Stafford, K. Dharmsathaphorn, and S. Carlson. 1987. Structural analysis of human intestinal epithelial cell line. Gastroenterology 92:1133-1145. 13. Marcom, M. A., D. McCool, J. Forstner, and G. Forstner. 1990. Inhibition of mucin secretion in a colonic adenocarcinoma cell line of DIDS and potassium channel blockers. Biochim. Biophys. Acta 1052:17-23. 14. Pai, C. H., R. Gordon, H. V. Sime, and L. E. Bryan. 1984. Sporadic cases of hemorrhagic colitis associated with Escherichia coli 0157:H7: clinical, epidemiologic and bacteriologic features. Ann. Intern. Med. 101:783-742. 15. Pinto, M., S. Robine-Leon, M. D. Appay, M. Kedinger, N. Triadou, E. Dussaulx, B. Lacroix, P. Simon-Assmann, K. Haffen, J. Fogh, and A. Zweibaum. 1983. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line CaCo-2 in culture. Biol. Cell 47:323-330. 16. Richardson, S. E., M. A. Karmali, L. E. Becker, and C. R. Smith. 1988. The histopathology of the hemolytic uremic syndrome associated with verocytotoxin-producing Escherichia coli infections. Hum. Pathol. 19:1102-1108. 17. Riley, L. W., R. S. Remis, D. D. Hegerson, H. B. McGee, J. G. Well, B. R. Davis, R. J. Hebert, E. S. Olcott, L. M. Johnson, N. T. Hargrett, P. A. Blake, and M. L. Cohen. 1983. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med. 308:681-685. 18. Sajjan, S. U., and J. F. Forstner. 1990. Characteristics of binding of Escherichia coli serotype 0157:H7 strain CL-49 to purified intestinal mucin. Infect. Immun. 58:860-867. 19. Sherman, P., F. Cockerill, F., R. Soni, and J. Brunton. 1991. Outer membranes are competitive inhibitors of Escherichia coli 0157:H7 adherence to epithelial cells. Infect. Immun. 59:890899. 20. Sherman, P., R. Soni, and M. Karmali. 1988. Attaching and effacing adherence of vero cytotoxin-producing Escherichia coli to rabbit intestinal epithelium in vivo. Infect. Immun. 56:756-761. 21. Sherman, P., R. Soni, and H. Yeger. 1988. Characterization of flagella purified from enterohemorrhagic, vero-cytotoxin-producing Escherichia coli serotype 0157:H7. J. Clin. Microbiol. 26:1367-1372. 22. Sherman, P. M., and R. Soni. 1988. Adherence of vero cytotoxin-producing Escherichia coli of serotype 0157:H7 to human epithelial cells in tissue culture: role of outer membranes as bacterial adhesins. J. Med. Microbiol. 26:11-17. 23. Toth, I., M. L. Cohen, H. S. Rumschlag, L. W. Riley, E. H. White, J. H. Carr, W. W. Bond, and I. K. Wachsmuth. 1990. Influence of the 60-megadalton plasmid on adherence of Escherichia coli 0157:H7 and genetic derivatives. Infect. Immun. 58:1223-1231. 24. Tzipori, S., I. K. Wachsmuth, C. Chapman, R. Birner, J. Brittingham, C. Jackson, and J. Hogg. 1986. The pathogenesis of hemorrhagic colitis caused by Escherichia coli 0157:H7 in gnotobiotic piglets. J. Infect. Dis. 154:712-716. 25. Tzipori, S., I. K. Wachsmuth, J. Smithers, and C. Jackson. 1988. Studies in gnotobiotic piglets on non-0157:H7 Escherichia coli serotypes isolated from patients with hemorrhagic colitis. Gastroenterology 94:590-597. 26. Wasserman, S. I., K. E. Barrett, P. A. Huott, G. Beverlum, M. F. Kagnoff, and K. Darmsathaphorn. 1988. Immune-related intestinal C1- secretion. I. Effect of histamine on the T84 cell line. Am. J. Physiol. 254:C53-C62.
and rabbit colonocytes, suggesting that the mannose-sensitive binding and mannose-resistant binding are related to each other. Examination of electron micrographs showing adherence of strain 43-12 revealed the growth of microcolonies in the area of tight junctions. Some bacterial cells were seen in close association with the T84 cell membrane; however, many bacterial cells appeared to be only in close proximity to the T84 cell membrane. In studies of Henle 407 cell adherence of E. coli 7785, Toth et al. (23) have described an electron-translucent area separating the eucaryotic cell membrane from closely associated bacteria without attaching-effacing lesions. The spacing between adjacent bacterial cells or bacterial cells in close proximity to the T84 membrane appeared consistent, suggesting that the bacterial cells were possibly being held in mucus, since T84 cells have been shown to produce mucus (13). The nature of the embedding procedure used in this study would not preserve the mucus overlying these cells. In conclusion, we have shown that the EHEC strains adhere to the T84 colonic carcinoma cell line in a manner similar to that of other, more cumbersome, model systems. The advantage of using the T84 cell is that these cells are of human origin and can polarize with tight junctions and desmosomes. This system will allow the study of changes in the physiology of the colonic epithelium exposed to EHEC infection. Preliminary experiments in our laboratories indicate that these cells may not be killed by SLT. Studies using T84 cell monolayers in Ussing chambers examining the effect of SLT on changes in electrical conductance, ion transport, and potential SLT transport across the polarized monolayers are currently under way. REFERENCES 1. Ashkenazi, S., L. May, M. LaRocco, E. L. Lopez, and T. G. Cleary. 1991. The effect of postnatal age on the adherence of enterohemorrhagic Escherichia coli to rabbit intestinal cells. Pediatr. Res. 29:14-19. 2. Audus, K. L., R. L. Bartel, I. J. Hidalgo, and R. T. Borchardt. 1990. The use of cultured epithelial and endothelial cells for drug transport and metabolism studies. Pharm. Res. 7:435-451. 3. Darfeville-Michaud, A., D. Aubel, G. Chauviere, C. Rich, M. Bourges, A. Servin, and B. Joly. 1990. Adhesion enterotoxigenic Eschenichia coli to the human colon carcinoma cell line CaCo-2 in culture. Infect. Immun. 58:893-902. 4. Durno, C. R., R. Soni, and P. Sherman. 1989. Adherence of vero cytotoxin-producing Escherichia coli serotype 0157:H7 to isolated epithelial cells and brush border membranes in vitro: role of type 1 fimbriae (pili) as bacterial adhesin expressed by strain CL-49. Clin. Invest. Med. 12:194-200. 5. Holmgren, J., J. Fryklund, and H. Larsson. 1989. Gammainterferon-mediated down-regulation of electrolyte secretion by intestinal epithelial cell: a local immune mechanism? Scand. J. Immunol. 30:499-503. 6. Junkins, A. D., and M. P. Doyle. 1989. Comparison of adherence properties of Escherichia coli 0157:H7 and a 60-megadalton plasmid-cured derivative. Curr. Microbiol. 19:21-27. 7. Karch, H., J. Heesemann, R. Laufs, A. D. O'Brien, C. 0. Tacket, and M. M. Levine. 1987. A plasmid of enterohemorrhagic Escherichia coli 0157:H7 is required for expression of a new fimbrial antigen for adhesion to epithelial cells. Infect. Immun. 55:455-461. 8. Karmali, M. A., M. Petric, C. Lim, P. C. Fleming, G. S. Arbus, and H. Lior. 1985. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J. Infect. Dis. 151:775-782.
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Vol. 60, No. 4
Adherence of Enterohemorrhagic Escherichia coli Strains to
a
Human Colonic Epithelial Cell Line (T84) DONALD K. WINSOR, JR.,1* SHAI ASHKENAZI,2 ROBERT CHIOVETTI,3 AND THOMAS G. CLEARY4 Department of Microbiology and Molecular Genetics' and Department of Pediatrics, 4 University of Texas Medical School at Houston, Houston, Texas 77030; Department of Pediatrics, Beilinson Medical Center, Petah Tikva, Israel 491002; and Life Cell Corporation, The Woodlands, Te-xas 773813 Received 18 October 1991/Accepted 20 January 1992
Enterohemorrhagic Escherichia coli (EHEC) produce Shiga-like toxins and attach to certain tissue culture cells. T.4 cells are human colonic carcinoma cells. Unlike previously studied cell lines, T.4 cells grown on collagen-coated surfaces polarize and produce tight junctions and desmosomes, forming a colonic epithelial cell layer in vitro. The purpose of this study was to examine the attachment of EHEC strains to the T84 cell line as a possibly more relevant in vitro model of EHEC adherence. Twelve EHEC strains were grown overnight in Penassay broth, suspended in minimal essential medium with and without 0.5% mannose, and incubated for 1 to 3 h with 5- to 7-day-old T84 cell monolayers grown on collagen-coated coverslips. The bacteria were removed, and attachment was quantitated microscopically. For both E. coli 0157:H7 and other EHEC serotypes, there were marked differences in adherence between strains (range of 152 to 3 bacteria per oil immersion field). Mannose partially inhibited the adherence of some EHEC strains. Adherence to the T.4 cells appeared to be related to the amount of pili present and not to the serotype. Electron micrographs showed that a highly adherent strain (strain 43-12) tended to form microcolonies in the area of tight junctions on the T84 cell monolayers. In addition, the attachment of these EHEC strains to T84 cells correlated with their ability to adhere to isolated rabbit colonocytes (r = 0.91, P = 0.00004; without mannose) (r = 0.60, P = 0.04; with mannose). These data show that there are EHEC strain-related differences in adherence which can be demonstrated in a human-derived colonic epithelial cell line (T,4) and that these cells can be used to study EHEC adherence. Strains of enterohemorrhagic Eschenchia coli (EHEC) outbreaks of hemorrhagic colitis (HC) and of hemolytic uremic syndrome (HUS) (8, 11, 14, 17). These strains produce Shiga-like toxin (SLT) I and/or II and belong to a variety of serotypes in addition to 0157:H7 (20, 25). It has been shown in humans and in animal models that these organisms attach primarily to the colonic epithelium, are noninvasive, and produce large amounts of SLT, which is believed to be the primary mediator of both local and systemic pathology (16, 24, 25). These organisms adhere to the animal intestinal epithelium by an attaching-effacing mechanism similar to that described for enteropathogenic E. coli, although the actual means of attachment remains unclear (20, 25). Mannose-sensitive pili have been implicated in the adherence to isolated rabbit colonocytes and isolated human intestinal epithelial cells of some EHEC strains (1, 4), while outer membranes have been shown to inhibit the adherence of other EHEC strains to HEp-2 cells (4, 7, 18-22). Since EHEC attaches primarily to the colonic epithelium in vivo, the use of a colonic epithelial cell line which polarizes, forms tight junctions and microvilli, and maintains electrical resistance in vitro might be most appropriate for the study of EHEC adherence. The use of such a cell line could lead to a better understanding of the changes in the physiology of the colonic epithelium during infection. The T84 cell line was derived from a colonic carcinoma which, when grown on collagen-coated surfaces, polarizes and exhibits compartmentalization of organelles and formation of tight junctions, desmosomes, and a basolateral and apical
surface with microvilli in vitro and can be used to study changes in electrical conductivity of the epithelial cell layer (12). For these reasons and because the adherences of EHEC to these cells would more closely mimic the in vivo situation, we examined the ability of 12 EHEC strains (0157:H7 and non-0157:H7) to attach to T84 cells in the presence and absence of mannose, compared these results with binding to isolated rabbit colonocytes, and evaluated the adherence of EHEC to T84 cells morphologically by electron microscopy.
cause
*
MATERIALS AND METHODS Bacterial strains. The E. coli strains and their serotypes, amounts of piliation, and toxin types are listed in Table 1. Strain H30 is a strain that was originally described by Konowalchuk et al. (10). Strains 43-12, 212-2, and 306-8 were isolated from children in Argentina with diarrhea or HUS (11). The remainder of the E. coli strains were obtained from M. Osterholm, Minnesota Department of Public Health. For the adherence assays, bacteria were grown in Penassay broth overnight without shaking at 37°C. T.4 cell growth. T84 cells were grown in Dulbecco's minimal essential medium-Ham's F12 medium (DMEM/H) containing 5% newborn calf serum, 200 mM L-glutamine, 5,000 U of penicillin per ml, and 5,000 ,ug of streptomycin per ml in an atmosphere of 5% CO2. For the EHEC adherence assay, the T84 cells were seeded (106 cells per ml) into wells of a 24-well tissue culture plate, each of which contained a 12-mm glass coverslip coated with rat tail collagen (type 1; Sigma Chemical Co., St. Louis, Mo.). The cells were then grown for 5 to 7 days before use (12). Three hours before infection, the growth medium was removed
Corresponding author. 1613
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INFECT. IMMUN.
WINSOR ET AL.
TABLE 2. Adherence of EHEC strains to T84 cells and rabbit colonocytes
TABLE 1. Characteristics of the EHEC strains used in this study Strain
Serotype
Piliationa
306-8 942 E473 E773 987 1058 E670 E854 212-2 546 H30 43-12
0157:H7 0157:H7
NDb
0157:1-7 0157:H7 0157:H7 0157:H7 0157:H7 0157:H7 0157:H7 0157:H7 026:H11 Ount:H21
ND ND ND 12 ND
NPc 6 NP ND NP 20
Toxin type SLT-/II SLT-II SLT-II
SLT-I/II SLT-I/II SLT-I SLT-I/II SLT-I/II SLT-II SLT-II SLT-I SLT-II
Disease
HUS HUS HUS HUS HC HC HC HC HC None DIAd HUS
a Data from Ashkenazi et al. (1). b
ND, not determined. c NP, nonpiliated. d DIA, diarrhea.
from each well and replaced with medium which did not contain serum or antibiotics. Preparation of rabbit intestinal cells. Cells from the proximal colons of adult New Zealand White rabbits were obtained by treating intestinal loops of rabbits with solutions containing EDTA, citrate, and dithiothreitol as previously described (1). EHEC adherence assays. (i) T.4 Cells. The bacteria were washed and suspended to 6 x 109 organisms per ml in DMEM/H with or without 0.5% mannose. Each strain was assayed three to five times. In each assay, two or three coverslips per strain were each inoculated with 0.5 ml of the bacterial suspension. After 1 h of incubation at 37°C in 5% CO2, the inoculum was removed, the T84 cells were washed three times with phosphate-buffered saline (PBS), and the coverslips were Giemsa stained. Each coverslip was mounted on a microscope slide, and the number of bacteria attached to T84 cells in 10 randomly chosen oil immersion fields was determined. T84 cells seeded on collagen-coated surfaces grow in such a manner that distinguishing between individual cells is almost impossible since the individual cell membranes are indistinguishable. Only fields with confluent T84 cells were counted: thus, bacterial counts were done on a constant surface area (one oil immersion field at x1,000 magnification) and used as the unit of measure. (ii) Rabbit intestinal cells. A suspension of intestinal cells (200 ,ul of 107 cells per ml) was incubated with [3H]thymidine-labeled bacteria (100 ,ul of 6 x 109 bacterial cells per ml) at 37°C for 45 min. Intestinal cells with bound bacteria were separated from unbound bacteria by Percoll density gradient centrifugation, and the layer containing the intestinal cells with bound bacteria was counted in a scintillation counter (1). Each strain was assayed three to five times. Electron microscopy. T84 cells were grown on 12-mmdiameter, round Thermanox plastic coverslips (Nunc, Inc., Naperville, Ill.) coated with collagen. The confluent monolayers were infected as previously described. The inoculum was removed and the monolayers were washed three times with PBS and incubated with DMEM/H without serum or antibiotics for 8 h. These infected monolayers were then processed for electron microscopy by fixation for 1 to 2 h in 2.5% glutaraldehyde-0.1 M sodium cacodylate (pH 7.3) and then washed with cacodylate buffer and postfixed in 1% osmium tetroxide-0.1 M sodium cacodylate. The coverslips were transferred to cacodylate buffer, and they were cleaved
Strain
43-12 H30 942 306-8 987 E773 E854 E670 546 1058 E473 212-2
T84 cellsa Without With mannose mannose
152 87 50 41 32 30 ± 27 ± 22 ± 13 ± 12 ± 11 ± 3±
27c 32 24 16 10 3 5 7 2 5 4 0
74 ± 48 ± 32 ± 25 ± 60 ± 14 ± 27 ± 7±
8 23 6 5
± ± ± ±
27c 26 16 9 28 4 10 2 3 6 1 1
Rabbit colonocytesb Without With mannose mannose
32 ± 10 ± 9± 2± 8± 2± 6± 2± 1± 1± 6± 4±
3c 3 2 1 3 1 2 1 1 1 2 1
17 ± 2c 6±3 2± 1 3± 1 2±1 3± 1 13 ± 4 2± 1 3± 1 1± 1 3± 1 1±1
a Number of bacteria attached per oil immersion field. b Number of bacteria per colonocyte. c Mean of three to five independent assays ± standard error of the mean.
into rectangles (ca. 3 by 5 mm) for flat embedding. The Thermanox rectangles were dehydrated in an ascending ethanol series (30, 50, 70, and 95%, and two times 100%, 10 min each) and then in propylene oxide (two times, 10 min). The specimens were transferred to new sample vials containing mixtures of Spurr low-viscosity resin and propylene oxide for infiltration. The infiltration schedule consisted of incubations in Spurr resin-propylene oxide (1:2 for 1 h; 2:1 overnight) and then in fresh Spurr resin for 4 h. Individual Thermanox rectangles were placed in flat embedding molds with new Spurr resin, and the resin was polymerized at 60°C overnight. Silver to light-gold ultrathin sections (60 to 90 nm) were cut with a diamond knife on a Reichert Ultracut B ultramicrotome, and the sections were collected on naked 300-hexagonal-mesh nickel grids. The sections were stained for 45 min in saturated aqueous uranyl acetate and for 15 min in lead citrate. The specimens were examined, and electron micrographs were made by using a Philips CM12 electron microscope operating at 80 keV in the transmission mode. The amounts of piliation of six of these strains were determined by electron microscopy of negative-stained preparation (1). These strains were chosen because they represented high, moderate, and low levels of adherence. Statistical analysis. Comparison of EHEC binding to T84 cells in the presence and absence of mannose and comparison of the binding to T84 cells and rabbit colonocytes were done by linear regression. RESULTS Adherence to T.4 cells and rabbit colonocytes. Adherence of these EHEC strains to T84 cell monolayers and rabbit colonocytes in the presence and absence of mannose is shown in Table 2. Strain 43-12 was the most adherent in both systems, followed by strain H30. Both are non-0157:H7 strains, with 43-12 being isolated from a patient with HUS and H30 being isolated from a patient with diarrhea. The 0157:H7 strains adhered to these two cell types to various degrees, with no apparent relationship between adherence, toxin type produced, or associated disease. There appears to be a relationship between adherence to T84 cells and the presence of fimbriae. Of the six strains for which the amounts of piliation have been determined (strains 987, E670, E824, 212-2, H30, and 43-12 [1]), the most adherent
EHEC ADHERENCE TO EPITHELIAL CELL LINE
VOL. 60, 1992
0O
000-
Zo 0= 0
o
-I
Ua .* o
32
64
96
128
160
T84 cells
(bacteria/oll Immersion field) FIG. 1. Relations between adherence of EHEC strains to cells and that to ir ship solated rabbit colonocytes in the absence of mannose
(r
=
0.91; P = 0.00004). Each point represents a single
strain.
strain (strain 43-1 2) was also the most piliated, the strains with moderate pi Iliation (strains 987 and E854) were also moderately adher ent, and the strains with little or no (E670, 212-1) wer e only minimally adherent. Strain H30 IS the second most adherent strain but IS nonpiliated under these growth con ditions. To determine if the presence of mannose had a sinnilar effect on these EHEC strains, regardless of piliation, w e compared the adherence of these strains to T84 cell monc layers in the presence and absence of mannose by lineai regression. Mannose only partially inhibited the adherenl ce of some of these strains; it dld not completely block adherence of any strains to T cells. A significant cc Irrelation between the adherence of these EHEC strains to I th4 cells and that to isolated rabbit colonocytes was seen in the absence (r = 0.91, P = 0.00004; Fig. 1) and in the presencce (r = 0.60; P = 0.04) of mannose. These data strongly sugggest that these two systems are measuring closely related ph enomena. Electron micros4copy. Figure 2A is an electron micrograph of uninfected T84 4cells grown on collagen-coated coverslips. Microvilli, tight jiunctions, and desmosomes are visible in this micrograph. )An electron micrograph of the attachment of EHEC 43-12, tI ie most adherent of these EHEC strains, to T84 cells after 8 h of incubation is shown in Fig. 2B. Microcolonies of tthis strain were frequently seen around the areas containing tight junctions, with single bacterial cells being found on ot:;her parts of the T84 cell surface. Bacterial cells were seen b oth in close association with the T84 cell membrane and in close proximity to the membrane.
pisi
DISCUSSION E. coli strains Mvhich elaborate large quantities of SLT and belong to certain serotypes, primarily 0157:H7, are known to cause HC and HUS (8, 14). The extraintestinal manifestations of these dliseases are believed to be caused by the systemic effects o f SLT (16). However, the exact nature of the bacterial cell-c:olonic epithelial cell interaction is unclear. There is disagreeDment about to which cell lines EHEC adheres (7, 9). C urrent data have suggested both that the 60-MDa plasmid mediates a fimbrial adhesion important in Henle 407 (INT4017) cell adherence (7) and that fimbriae play no role in adherenIce (23). Loss of the plasmid has even been i
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associated with both increased Henle cell adherence (6) and decreased adherence (23). Additionally, plasmidless derivatives are still pathogenic in piglets (25). Published data on hemagglutination also disagree on the significance of the plasmid or the pili (9, 23). The variable and conflicting data reflect the inadequacy of current models to study EHEC adherence. Because of the lack of appropriate in vitro models, questions about how the bacteria attach to the colonic epithelium and how or when SLT is delivered across the epithelium still remain. Optimally, an in vitro model to determine the answer to these questions ought to include a colonic cell type of human origin which, when cultured, would have characteristics of the colonic epithelium, including the formation of tight junctions and desmosomes and transport properties similar to the in vivo situation. The T84 cell line fulfills these criteria. These cells form tight junctions and desmosomes and transport ions in response to pharmacological agents in a manner similar to that of the natural colonic epithelium (5, 12, 26). In this study, we have shown that both 0157:H7 and non-0157:H7 strains of EHEC adhere to T84 cells and that this adherence correlates with their adherence to rabbit colonocytes, one of the models used to study EHEC patho-
genesis (1, 4, 20). Additionally, E. coli HS, which is a commensal fecal E. coli strain, attaches to the T84 cells at a level similar to that of strain 212-2, the EHEC strain with the
lowest level of adherence (data not shown). Some EHEC strains will attach to other cell lines, such as HEp-2 and Henle 407 cells, but these cells may be less appropriate for the study of colonic pathogens. HEp-2 and Henle 407 cells do not differentiate into intact epithelial cell layers, thus not allowing the study of changes in epithelial cell function during infection (6, 19, 22, 23). The CaCo-2 cell line has been used to examine the adherence of enteropathogenic E. coli and a limited number of EHEC strains (9). The CaCo-2 cell line was also derived from a human colonic carcinoma, polarizes, forming tight junctions and desmosomes, and has been used to study epithelial cell transport (2). However,
CaCo-2 cells differentiate into an epithelial cell layer which resembles the small intestinal epithelium because the mature monolayer possesses levels of sucrase-isomaltase similar to those of the small intestine (15) and because small-intestinal pathogens, such as enterotoxigenic and enteropathogenic E. coli strains, adhere to these cells (3, 9) whereas a colonic pathogen, EHEC, does not (9). The amount of attachment to the polarized T84 cells varied depending on the EHEC strain and the amount of piliation. Strain 43-12 was highly piliated (1); the most adherent, strain 987, was moderately piliated (1) and only moderately adherent; and strain 212-2 was nonpiliated (1) and minimally adherent to the T84 cells. This is in accordance with other studies in which the highly piliated EHEC strains adhered in greater numbers than nonpiliated strains (1, 4). Since these pili are thought to be a form of type 1 pili and since mannose has been shown to at least partially inhibit the adherence of EHEC strains (1, 4, 9, 18), we examined the effect mannose had on the T84 cell adherence of these EHEC strains. In no instance did the presence of mannose eliminate the adherence of these strains. Mannose decreased the adherence of some EHEC strains (43-12, H30, 942, 306-8, E773, E670, 546, and E473), had no effect on some (E854 and 212-2), and appeared to increase the adherence of others (987 and 1058). Ashkenazi et al. (1) reported similar results, with mannose inhibiting the attachment of strain 43-12 by 40% and that of strain H30 by 10 to 20%. Binding of strains 987 and 212-2 to rabbit colonocytes was not significantly inhibited by the
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FIG. 2. Transmission electron micrograph of uninfected T84 cell monolayer (A) and T84 cell monolayer infected with EHEC 43-12 showing association of bacteria with T84 cell surface after 8 h of incubation (B). Magnification, x 8,595. Arrows indicate tight junctions, and arrowheads indicate desmosomes. MV, microvilli.
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VOL. 60, 1992
EHEC ADHERENCE TO EPITHELIAL CELL LINE
presence of mannose (1). The similarities of the two systems were further confirmed by the correlation of adherences, both in the presence and absence of mannose, to T84 cells
9. Knutton, S., T. Baldwin, P. H. Williams, and A. S. McNeish. 1989. Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infect. Immun. 57: 1290-1298. 10. Konowalchuk, J., J. I. Speirs, and S. Stavric. 1977. Vero response to a cytotoxin of Escherichia coli. Infect. Immun. 18:775-779. 11. Lopez, E. L., M. Diaz, S. Grinstein, S. Devoto, F. Mendilaharzu, B. E. Murray, S. Ashkenazi, E. Rubeglio, M. Woloj, M. Vasquez, M. Turco, L. K. Pickering, and T. G. Cleary. 1989. HUS and diarrhea in Argentine children: the role of shiga-like toxins. J. Infect. Dis. 160:469-475. 12. Madara, J. L., J. Stafford, K. Dharmsathaphorn, and S. Carlson. 1987. Structural analysis of human intestinal epithelial cell line. Gastroenterology 92:1133-1145. 13. Marcom, M. A., D. McCool, J. Forstner, and G. Forstner. 1990. Inhibition of mucin secretion in a colonic adenocarcinoma cell line of DIDS and potassium channel blockers. Biochim. Biophys. Acta 1052:17-23. 14. Pai, C. H., R. Gordon, H. V. Sime, and L. E. Bryan. 1984. Sporadic cases of hemorrhagic colitis associated with Escherichia coli 0157:H7: clinical, epidemiologic and bacteriologic features. Ann. Intern. Med. 101:783-742. 15. Pinto, M., S. Robine-Leon, M. D. Appay, M. Kedinger, N. Triadou, E. Dussaulx, B. Lacroix, P. Simon-Assmann, K. Haffen, J. Fogh, and A. Zweibaum. 1983. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line CaCo-2 in culture. Biol. Cell 47:323-330. 16. Richardson, S. E., M. A. Karmali, L. E. Becker, and C. R. Smith. 1988. The histopathology of the hemolytic uremic syndrome associated with verocytotoxin-producing Escherichia coli infections. Hum. Pathol. 19:1102-1108. 17. Riley, L. W., R. S. Remis, D. D. Hegerson, H. B. McGee, J. G. Well, B. R. Davis, R. J. Hebert, E. S. Olcott, L. M. Johnson, N. T. Hargrett, P. A. Blake, and M. L. Cohen. 1983. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med. 308:681-685. 18. Sajjan, S. U., and J. F. Forstner. 1990. Characteristics of binding of Escherichia coli serotype 0157:H7 strain CL-49 to purified intestinal mucin. Infect. Immun. 58:860-867. 19. Sherman, P., F. Cockerill, F., R. Soni, and J. Brunton. 1991. Outer membranes are competitive inhibitors of Escherichia coli 0157:H7 adherence to epithelial cells. Infect. Immun. 59:890899. 20. Sherman, P., R. Soni, and M. Karmali. 1988. Attaching and effacing adherence of vero cytotoxin-producing Escherichia coli to rabbit intestinal epithelium in vivo. Infect. Immun. 56:756-761. 21. Sherman, P., R. Soni, and H. Yeger. 1988. Characterization of flagella purified from enterohemorrhagic, vero-cytotoxin-producing Escherichia coli serotype 0157:H7. J. Clin. Microbiol. 26:1367-1372. 22. Sherman, P. M., and R. Soni. 1988. Adherence of vero cytotoxin-producing Escherichia coli of serotype 0157:H7 to human epithelial cells in tissue culture: role of outer membranes as bacterial adhesins. J. Med. Microbiol. 26:11-17. 23. Toth, I., M. L. Cohen, H. S. Rumschlag, L. W. Riley, E. H. White, J. H. Carr, W. W. Bond, and I. K. Wachsmuth. 1990. Influence of the 60-megadalton plasmid on adherence of Escherichia coli 0157:H7 and genetic derivatives. Infect. Immun. 58:1223-1231. 24. Tzipori, S., I. K. Wachsmuth, C. Chapman, R. Birner, J. Brittingham, C. Jackson, and J. Hogg. 1986. The pathogenesis of hemorrhagic colitis caused by Escherichia coli 0157:H7 in gnotobiotic piglets. J. Infect. Dis. 154:712-716. 25. Tzipori, S., I. K. Wachsmuth, J. Smithers, and C. Jackson. 1988. Studies in gnotobiotic piglets on non-0157:H7 Escherichia coli serotypes isolated from patients with hemorrhagic colitis. Gastroenterology 94:590-597. 26. Wasserman, S. I., K. E. Barrett, P. A. Huott, G. Beverlum, M. F. Kagnoff, and K. Darmsathaphorn. 1988. Immune-related intestinal C1- secretion. I. Effect of histamine on the T84 cell line. Am. J. Physiol. 254:C53-C62.
and rabbit colonocytes, suggesting that the mannose-sensitive binding and mannose-resistant binding are related to each other. Examination of electron micrographs showing adherence of strain 43-12 revealed the growth of microcolonies in the area of tight junctions. Some bacterial cells were seen in close association with the T84 cell membrane; however, many bacterial cells appeared to be only in close proximity to the T84 cell membrane. In studies of Henle 407 cell adherence of E. coli 7785, Toth et al. (23) have described an electron-translucent area separating the eucaryotic cell membrane from closely associated bacteria without attaching-effacing lesions. The spacing between adjacent bacterial cells or bacterial cells in close proximity to the T84 membrane appeared consistent, suggesting that the bacterial cells were possibly being held in mucus, since T84 cells have been shown to produce mucus (13). The nature of the embedding procedure used in this study would not preserve the mucus overlying these cells. In conclusion, we have shown that the EHEC strains adhere to the T84 colonic carcinoma cell line in a manner similar to that of other, more cumbersome, model systems. The advantage of using the T84 cell is that these cells are of human origin and can polarize with tight junctions and desmosomes. This system will allow the study of changes in the physiology of the colonic epithelium exposed to EHEC infection. Preliminary experiments in our laboratories indicate that these cells may not be killed by SLT. Studies using T84 cell monolayers in Ussing chambers examining the effect of SLT on changes in electrical conductance, ion transport, and potential SLT transport across the polarized monolayers are currently under way. REFERENCES 1. Ashkenazi, S., L. May, M. LaRocco, E. L. Lopez, and T. G. Cleary. 1991. The effect of postnatal age on the adherence of enterohemorrhagic Escherichia coli to rabbit intestinal cells. Pediatr. Res. 29:14-19. 2. Audus, K. L., R. L. Bartel, I. J. Hidalgo, and R. T. Borchardt. 1990. The use of cultured epithelial and endothelial cells for drug transport and metabolism studies. Pharm. Res. 7:435-451. 3. Darfeville-Michaud, A., D. Aubel, G. Chauviere, C. Rich, M. Bourges, A. Servin, and B. Joly. 1990. Adhesion enterotoxigenic Eschenichia coli to the human colon carcinoma cell line CaCo-2 in culture. Infect. Immun. 58:893-902. 4. Durno, C. R., R. Soni, and P. Sherman. 1989. Adherence of vero cytotoxin-producing Escherichia coli serotype 0157:H7 to isolated epithelial cells and brush border membranes in vitro: role of type 1 fimbriae (pili) as bacterial adhesin expressed by strain CL-49. Clin. Invest. Med. 12:194-200. 5. Holmgren, J., J. Fryklund, and H. Larsson. 1989. Gammainterferon-mediated down-regulation of electrolyte secretion by intestinal epithelial cell: a local immune mechanism? Scand. J. Immunol. 30:499-503. 6. Junkins, A. D., and M. P. Doyle. 1989. Comparison of adherence properties of Escherichia coli 0157:H7 and a 60-megadalton plasmid-cured derivative. Curr. Microbiol. 19:21-27. 7. Karch, H., J. Heesemann, R. Laufs, A. D. O'Brien, C. 0. Tacket, and M. M. Levine. 1987. A plasmid of enterohemorrhagic Escherichia coli 0157:H7 is required for expression of a new fimbrial antigen for adhesion to epithelial cells. Infect. Immun. 55:455-461. 8. Karmali, M. A., M. Petric, C. Lim, P. C. Fleming, G. S. Arbus, and H. Lior. 1985. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J. Infect. Dis. 151:775-782.
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