Báo cáo khoa học: Functional interaction of Escherichia coli heat-labile enterotoxin with blood group A-active glycoconjugates from differentiated HT29 cells docx

The presence of non-GM1 recep-tors of Escherichia coli heat-labile enterotoxin LT-I on both types of dif-ferentiated HT29 cells was indicated by the inability of cholera toxin B subunit

enterotoxin with blood group A-active glycoconjugates from differentiated HT29 cells

Estela M Galva´n, German A Roth and Clara G Monferran

Departamento de Quı´mica Biolo´gica – CIQUIBIC (CONICET), Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba, Argentina

The type I heat-labile toxin produced by

enterotoxi-genic Escherichia coli (LT-I), and cholera toxin (CT)

secreted by Vibrio cholerae, are responsible for the

diarrhea observed in traveller’s diarrhea and cholera,

respectively These enterotoxins are the closest

struc-tural and functionally related members of the CT

fam-ily [1,2] LT-I and CT are AB5 toxins, in which the

pentameric B subunit [B subunit of E coli heat labile

toxin (LT-B), B subunit of cholera toxin (CT-B)]

medi-ates toxin binding to membrane receptors on polarized

intestinal epithelial cells Upon binding, the holotoxin

enters the cell and moves by retrograde transport to the trans-Golgi and the endoplasmic reticulum [3,4] The A subunit, responsible for the toxic activity, undergoes controlled proteolytic cleavage and reduc-tion in the endoplasmic reticulum, giving rise to the fully active A1-peptide, which is translocated to the cytoplasm [5] ADP ribosylation of the a subunit of the heterotrimeric GTP-binding protein by the A1 -pep-tide renders adenylylate cyclase irreversibly activated and, consequently, increases cyclic AMP production, leading to net fluid secretion [6,7]

Keywords

ABH glycoconjugates; differentiated HT29

cells; Escherichia coli heat-labile toxin;

glycosphingolipids; toxin receptors

Correspondence

C G Monferran, Departamento de Quı´mica

Biolo´gica, Facultad de Ciencias Quı´micas,

Universidad Nacional de Co´rdoba, Ciudad

Universitaria, Co´rdoba X5000HUA, Argentina

Fax: +54 351 4334074

Tel: +54 351 4334168 ⁄ 4334171

E-mail: cmonfe@mail.fcq.unc.edu.ar

(Received 1 March 2006, revised 28 April

2006, accepted 22 May 2006)

doi:10.1111/j.1742-4658.2006.05368.x

Human colon adenocarcinoma cells (ATCC) and the clone HT29-5F7 were cultured under conditions that differentiate cells to a polarized intestinal phenotype Differentiated cells showed the presence of junctional complexes and intercellular lumina bordered by microvilli Intestinal brush border hydrolase activities (sucrase, aminopeptidase N, lactase and mal-tase) were detected mainly in differentiated HT29-ATCC cells compared with the differentiated clone, HT29-5F7 The presence of non-GM1 recep-tors of Escherichia coli heat-labile enterotoxin (LT-I) on both types of dif-ferentiated HT29 cells was indicated by the inability of cholera toxin B subunit to block LT-I binding to the cells Binding of LT-I to cells, when GM1 was blocked by the cholera toxin B subunit, was characterized by

an increased number of LT-I receptors with respect to undifferentiated control cells Moreover, both types of differentiated cells accumulated higher amounts of cyclic AMP in response to LT-I than undifferentiated cells Helix pomatia lectin inhibited the binding of LT-I to cells and the subsequent production of cyclic AMP LT-I recognized blood group A-active glycosphingolipids as functional receptors in both HT29 cell lines and the active pro-sucrase form of the glycoprotein carrying A-blood group activity present in HT29-ATCC cells These results strongly suggest that LT-I can elicit an enhanced functional response using blood group A-active glycoconjugates as additional receptors on polarized intestinal epithelial cells

Abbreviations

CT, cholera toxin; CT-B, B subunit of cholera toxin; LT-I, type I heat-labile toxin produced by enterotoxigenic Escherichia coli; LT-B, B subunit

of E coli heat labile toxin; TEM, transmission electron microscopy.

LT-I and CT bind with high affinity to the

ganglio-side GM1 in cell membranes and other systems [1]

Despite the high amino acid sequence and structural

homology, LT-B and CT-B are bacterial lectins that

also recognize non-GM1 carbohydrate structures with

different specificity Numerous studies have shown that

LT-I binds glycosphingolipids and glycoproteins from

intestinal mucosal cells of several animal species [8–14],

although most of these interactions have no recognized

biological function We have previously reported a

dif-ferential ability of glycosphingolipids and glycoproteins

obtained from pig and rabbit gastrointestinal tract

tis-sue to interact with LT-I, depending on the type of

ABH blood group determinant carried by these

glyco-conjugates Conversely, CT showed almost no

inter-action with either blood group-active glycolipids or

glycoproteins [12–14] Furthermore, LT-I recognized

ABH glycoconjugates among the abundant non-GM1

receptor population on rabbit intestinal brush border

membranes and was demonstrated to activate adenylate

cyclase, suggesting that ABH glycolipids and

glycopro-teins are LT-I functional receptors in rabbit intestine

[15] Recently, we have demonstrated that LT-I binding

to blood group A-active glycosphingolipids from the

plasma membrane of human adenocarcinoma HT29

cells elicits a signal transduction pathway, resulting in

an increase of the cellular cyclic AMP levels [16]

HT29 cells and other few intestinal cell lines

undergo morphological and functional differentiation

in vitro Under standard culture conditions, HT29 cells

are covered by irregular microvilli and devoid of tight

junctions When HT29 cells are cultured under specific

conditions [17–21], they develop some features of

distinct pathways of enterocyte differentiation,

charac-terized basically by cell polarization The plasma

mem-brane of enterocyte-like cells differentiated in vitro

exhibits two structural and functionally different

domains - apical and basolateral - separated by tight

junctions The apical membrane is characterized by the

presence of microvilli containing peptidase and

glyco-hydrolase digestive enzymes, whereas the basolateral

membrane displays distinct surface protein markers

[22–25] It is well known that enterocyte-like

differenti-ation overcomes the impaired glycosylation and

rapid degradation of the glycoprotein observed in the

undifferentiated stage, allowing the expression of

sucrase-isomaltase, which carries ABH blood group

determinants [26,27]

Because the natural target of LT-I is a polarized

intestinal cell, the purpose of this study was to

investi-gate the interaction of LT-I with non-GM1 receptors

of polarized HT29 cells Toxic activity, triggered by

LT-I binding to additional receptors, was measured as

intracellular cyclic AMP accumulation We also inves-tigated the nature of alternate LT-I receptors in differ-entiated cells

Results

Characterization of differentiated HT29 cells

In order to analyze the interaction of LT-I with cells that resemble the polarized enterocyte, HT29 cells from American type culture collection (ATCC) (HT29-ATCC), and the clone HT29-5F7, were grown under conditions appropriate for stimulating intestinal differ-entiation Some structural and biochemical features of the differentiated cells have been studied Contrary to that observed in undifferentiated HT29-ATCC cells, cells at late confluence clearly showed, by phase-con-trast microscopy, intercellular lumina that were visible

as vesicles or cysts between cells (Fig 1A) By trans-mission electron microscopy (TEM), it was clearly evident that intercellular lumina were bordered by abundant microvilli provided by surrounding cells facing the medium, and junctional complexes between cells were frequently observed (Fig 1B–D) TEM sec-tions also showed that confluent differentiated HT29-ATCC cells were formed by three to four cell layers, while undifferentiated cells at confluence had five to

Fig 1 Morphological studies of differentiated HT29 cells Phase contrast micrograph (A) and thin sections (B–D) of postconfluent cultures of HT29-ATCC cells (day 21) grown in RPMI-1640 contain-ing 10% fetal bovine serum Note the presence of intercellular lumina (ICL) (A and B), apical brush border (arrows in C) and junc-tional complexes between adjacent cells facing the lumen (arrows

in D) Magnification: A, ·40; B, ·4000; C, ·12 000; and D, ·20 000.

seven cell layers (data not shown) Similar structural

characteristics were also observed on HT29-5F7 cells

at late confluence (data not shown)

In order to characterize biochemically the

differen-tiated stage of HT29-ATCC and HT29-5F7 cells,

intestinal enzyme activities were measured in brush

border-rich membrane fractions Table 1 shows that

maltase (EC.3.2.1.20), lactase (EC.3.2.1.23), sucrase

(EC.3.2.1.48) and aminopeptidase N (EC.3.4.11.2)

were active in differentiated HT29-ATCC cells, and

that lower amounts of maltase and aminopeptidase N

activity were present in polarized HT29-5F7 Together,

these results indicated that HT29 cells, when growing

under appropriate conditions, could acquire some

mor-phological and biochemical characteristics of

entero-cytes

LT-I binding to differentiated HT29 cells and

cyclic AMP-induced production

Several concentrations of nontoxic LT-B and CT-B

were assayed for competitive inhibition on125I-labelled

LT-I binding to differentiated HT29 cells Figure 2

shows that complete inhibition of LT-I binding to

HT29-ATCC cells was dependent on the LT-B

concen-tration, indicating the specificity of the 125I-labelled

LT-I preparation When CT-B was assayed at

concen-trations similar to those used for LT-B, toxin binding

was not blocked From these results it is apparent that

most of the LT-I receptors are not shared with CT-B

CT-B was able to block 125I-labelled CT binding to

both differentiated HT29 cell types in a

concentration-dependent manner (results not shown)

In order to determine the number of LT-I receptors, additional to GM1, on the cell membrane of polarized and nonpolarized HT29-ATCC and HT29-5F7 cells,

we measured the binding of 125I-labelled LT-I in the absence and in the presence of CT-B Saturation curves performed at steady state showed that in polar-ized and nonpolarpolar-ized cells, there was little difference

in the binding of 125I-labelled LT in the presence and

in the absence of CT-B (Fig 3A,B) Moreover, Fig 3 shows that the binding capacity for non-GM1 recep-tors was approximately four times higher on differenti-ated HT29-ATCC cells than on undifferentiated control cells (Fig 3A) Differentiated HT29-5F7 cells also exhibited a significantly higher number of addi-tional LT-I receptor sites with respect to the undiffer-entiated stage (1600 versus 800 fmolÆ10)6 cells) (Fig 3B) Helix pomatia lectin, which recognizes the carbohydrate structure of blood group A, inhibited

125I-labelled LT-I binding to differentiated HT29 and HT29-5F7 cells in a dose-dependent manner (Fig 4) These results indicate that the differentiation process increased the expression of non-GM1 receptors for LT-I and that blood group A-active glycoconjugates may be alternate LT-I receptors in both cell lines The functional response of differentiated cells to LT-I was determined in terms of the cyclic AMP

Table 1 Activity of brush border membrane-associated enzymes

(mUÆmg)1 protein) Sucrase (EC.3.2.1.48), maltase (EC.3.2.1.20),

lactase (EC.3.2.1.23) and aminopeptidase N (EC.3.4.11.2) activities

were measured in brush border membranes (P2 fractions) from

un-differentiated and un-differentiated HT29-ATCC and HT29-5F7 cells, as

described in the Experimental procedures Values are the mean ±

SD of two experiments ND, not detected.

Undifferentiated Differentiated HT29-ATCC

HT29-5F7

Fig 2 Effect of B subunits of cholera toxin (CT-B) and Escheri-chia coli heat-labile toxin (LT-B) on the binding of 125I-labelled heat-labile enterotoxin (LT-I) to cells HT29-ATCC cells grown at confluence for 18 days were incubated with different concentra-tions of CT-B or LT-B for 30 min at 4
C and then further incubated with 125 I-labeled LT-I (5.0 n M ) for 60 min at 4
C Binding of 125 I-labelled LT-I was determined as indicated in the Experimental pro-cedures Each point is the mean of triplicate determinations, with a

standard deviation 610%.

content, measured after incubation with increasing

concentrations of toxin Figure 5 shows that

differenti-ated HT29-ATCC cells increased the cyclic AMP

con-tent compared with control cells, reaching a maximum

at a toxin concentration of 12 nm

Table 2 shows that both differentiated HT29-ATCC

and HT29-5F7 cell lines accumulated twice the

con-tent of cyclic AMP, with respect to control cells, in

response to 10 nm LT-I acting on either the total or

the non-GM1 receptor population When cells were pre-incubated with H pomatia lectin before the addi-tion of LT-I, the cyclic AMP level was significantly diminished These results strongly suggest that LT-I alternate receptors can elicit a functional response

in both polarized HT29-ATCC and HT29-5F7 cells and, furthermore, that blood group A-active glyco-conjugates could represent a major proportion of the functional additional receptors to LT-I in both cell lines

Fig 3 Binding of Escherichia coli heat-labile enterotoxin (LT-I) to

HT29 cells in culture Differentiated and control HT29-ATCC (A)

and HT29-5F7 (B) cells were incubated with increasing

concentra-tions of125I-labelled LT-I for 60 min at 4
C in the absence or in

the presence of 1.0 l M unlabelled cholera toxin B subunit (CT-B).

The bound 125 I-labelled toxin was determined as described in the

Experimental procedures Results have been corrected for the

nonspecific binding of 125 I-labelled LT-I The levels of nonspecific

binding were not greater than 10% of total binding for each toxin

concentration In all panels, each point represents the mean ± SD

of three experiments.

Fig 4 Concentration-dependent effect of Helix pomatia lectin on

125 I-labelled LT-I binding to differentiated cells Lectin was pre-incu-bated with differentiated HT29-ATCC and HT29-5F7 cells for

30 min at 4
C before the addition of 125

I-labelled LT-I (10 n M ) and then further incubated for 60 min at 4
C Bound toxin was meas-ured as indicated in the Experimental procedures Each point repre-sents the mean of triplicate determinations ± SD.

Fig 5 Intracellular cyclic AMP stimulated by Escherichia coli heat-labile enterotoxin (LT-I) in HT29-ATCC cells Undifferentiated and differentiated HT29-ATCC cell monolayers were incubated with increasing concentrations of LT-I in the presence of 1.0 l M CTB for

90 min at 37
C Cyclic AMP was assayed as described in the Experimental procedures Each point is the mean of triplicate deter-minations.

Presence of blood group A-active

glycoconjugates on differentiated HT29 cells

and interaction with LT-I

Because cell polarization involves marked changes in

the cell architecture as well as in the expression and

sorting of new membrane molecules, we investigated

the nature of ABH glycoconjugates able to bind LT-I

in the HT29-ATCC and HT29-5F7 differentiated cells Brush border membrane preparations (P2) from differ-entiated cells were examined by western blotting for the interaction with LT-I and for the presence of blood group A activity Figure 6A shows that LT-I only recognized one blood group A-active glycoprotein, which was identified as pro-sucrase-isomaltase by reac-tion with the corresponding antibody and the expected relative migration after SDS⁄ PAGE In the P2 frac-tions from differentiated HT29-5F7, no glycoprotein with the ability to bind LT-I (data not shown), and no glycoprotein carrying the blood group A determinant, were detected

Total lipid extracts from both differentiated HT29-ATCC and HT29-5F7 cells were separated by HPTLC and assayed for binding of the blood group A mAb and LT-I by the TLC-overlay technique Figure 6B shows that LT-I recognized GM1 and several blood group A-active glycosphingolipids, migrating more slowly than GM1, from lipid extracts of both differen-tiated cells The ability of LT-I to interact more effi-ciently with the complex glycosphingolipids carrying the blood group A determinant from polarized cells is similar to that previously observed with lipid extracts from undifferentiated HT29 cells [16]

Discussion

LT-I is a major virulence factor of enterotoxigenic

E coli, which colonizes human and animal intestines The toxic activity of LT-I on the target cell is mediated

by permanent activation of adenylate cyclase, which increases the cyclic AMP level in intestinal mucosa cells Consequently, alteration in Na+ and Cl– fluxes

in villus and crypt cells has been involved in the char-acteristic symptoms of diarrhea

The polarized HT29 cell model was used, in this work, to study the interaction of LT-I with non-GM1 receptors The undifferentiated HT29 parental cell line contains a very small proportion of differentiated cell types, which, under a pressure selection process, emerge as one of mainly two differentiated polarized enterocyte-like or mucus-secreting phenotypes [17] The mechanisms by which biochemical conditions or drug pressure induce survival of colon carcinoma cells are currently under study [28–31] In the present work, enterocytic differentiation was induced in HT29-ATCC parental cells and clone HT29-5F7, as detected by ultrastructural and functional studies At late conflu-ence, cells were polarized, had well developed brush border at the apical membrane and expressed several intestinal enzymes from the mature enterocyte The

Table 2 Cyclic AMP production elicited by Escherichia coli

heat-labile enterotoxin (LT-I) on HT29 cells Effect of CT-B and Helix

po-matia lectin Undifferentiated and differentiated HT29-ATCC and

HT29-5F7 cells were pre-incubated at 4
C with 1.0 l M CTB or

10 l M H pomatia lectin and then cells were further incubated with

10 n M LT-I at 37
C for 90 min Intracellular cyclic AMP was

meas-ured as described in the Experimental procedures Values are the

mean ± SD of two experiments.

Cells

Cyclic AMP (pmol ⁄ 10 6 cells) LT-I LT-I + CT-B LT-I + HP

HT29 differentiated 1260 ± 130 915 ± 90 112.0 ± 28

HT29-5F7 differentiated 2270 ± 120 1600 ± 130 2.1 ± 0.3

Fig 6 Blood group A-active glycoconjugates from differentiated

HT29 cells and their ability to interact with Escherichia coli

heat-labile enterotoxin (I) (A) Blood group antigenic activity and

LT-I-binding properties of brush border-enriched P2 fractions from

dif-ferentiated HT29-ATCC-P2 fractions were separated by SDS ⁄ PAGE

and electrotransferred to nitrocellulose Nitrocellulose strips were

incubated with mouse monoclonal anti-(blood group A) or

anti-(suc-rase-isomaltase) (SI) Ig and then with horseradish peroxidase

(HRP)-conjugated monoclonal anti-mouse Ig For LT-I binding,

nitro-cellulose strips were incubated with 5.0 n M LT-I followed by

incu-bation with rabbit anti-LT-I Ig and HRP-conjugated Protein A In all

cases, peroxidase was revealed by a chemiluminescent reaction.

(B) Blood group A activity and LT-I binding to glycosphingolipids

from HT29-ATCC and HT29-5F7 differentiated cells HPTLC plates

were overlaid with anti-(blood group A) IgM and then with a

secon-dary HRP-conjugated antibody Peroxidase was revealed with

0.05% 4-chloro-1-naphtol and 0.01% hydrogen peroxide as

sub-strate solution.

morphological features of differentiated HT29-ATCC

and HT29-5F7 cells observed in this work closely

resembled that previously reported [21,22,29,30]

Func-tionally, differentiation was accompanied by the

expression of aminopeptidase N, lactase, maltase and

sucrase activities Sucrase-isomaltase is localized at the

apical brush border membranes of HT29 cells

differen-tiated in RPMI [22] and by glucose deprivation [26,27]

LT-I binds to the high-affinity receptor, GM1, and

to alternate receptors (glycosphingolipid and

gycopro-teins) from several cell membranes [8–10,12–16]

We have previously described that ABH-active

glycoconjugates could act as alternate LT-I receptors

on intestinal brush border membranes from pig and

rabbits and in undifferentiated HT29-ATCC cells

[12–16] In the present work, we found that specific

LT-I binding to differentiated intestinal cells is not

sig-nificantly diminished in the presence of a molar excess

of CT-B (Fig 3), which may reflect a very low

contri-bution of GM1 to LT-I binding on cells Saturation

curves performed on differentiated HT29-ATCC and

HT29-5F7 cells showed that125I-labelled CT maximally

bound 74 and 28 fmolÆ10)6cells, respectively (data not

shown), supporting the idea of the existence of an

unbalanced ratio between alternate⁄ GM1 receptor sites

in HT29 cells By using the polarized HT29-ATCC cell

line and the HT29-5F7 clone we demonstrated an

increased expression (two- to four-fold) of non-GM1

LT-I receptor sites with respect to the undifferentiated

control cells

The dose-dependent inhibition of LT-I binding by

H pomatialectin clearly indicates that LT-I recognized

blood group A-active glycoconjugates on the cell

sur-face of undifferentiated [16] and differentiated HT29

cells (Fig 4) Although no direct quantification of

blood group A-active glycoconjugates on the cell

sur-face was performed, we assumed that the higher

num-ber of LT-I receptor sites on differentiated cells should

result from a greater number of blood group A-active

glycoconjugates on the cell surface Differentiation of

adenocarcinoma cell lines (e.g HT29, Caco-2) to an

enterocyte like-status involves a change in

morphologi-cal features, such as the development of brush border

membranes A great increase of the brush border

membrane surface in differentiated cells (Fig 1) may

increase the number of receptor sites provided by

blood group A-glycosphingolipid and blood group

A-glycoprotein sucrase-isomaltase (the latter on the

HT29-ATCC plasma membrane)

Polarized cells were also capable of inducing an

increase in the intracellular cyclic AMP level in

response to LT-I concentrations higher than 6 nm

This effect was observed, even at 10 nm toxin, when

the number of occupied binding sites of differentiated and control cells were similar We have no clear explanation for this observation and further studies are necessary to add new insight into the mechanism of the toxin action on these cells However, we speculate that the enhanced cyclic AMP production can be rela-ted to the polarized status of cells, which may allow a more efficient coupling of the secondary signal path-ways triggered by the toxin in respect to nonpolarized HT29 cells For CT, it has been shown that a small percentage of the cell-bound toxin is converted to A1 peptide over a period of time during which the full activation of adenylate cyclase is reached [6] Because

CT binding to differentiated cells was completely blocked by 100 nm CT-B in the present work (results not shown), we attributed cyclic AMP accumulation in polarized cells to the action of LT-I on low-affinity non-GM1 LT-I receptors Apparently, these alternate receptors account for 70% of the total cyclic AMP response to LT-I in both polarized cell lines (Table 2) Using toxin overlay assays, we found that several blood group A-glycosphingolipids from HT29-ATCC and HT29-5F7 cell lines, migrating more slowly than GM1, efficiently bound LT-I These results, together with the inhibitory effect of H pomatia on toxin action, indicated that glycoconjugates bearing the blood group A determinant are additional receptors to LT-I in HT29-ATCC and HT29-5F7 cells We have recently reported that blood group A-active glycosp-hingolipids, migrating more slowly than GM1, are additional LT-I receptors in parental HT29 cells and that these non-GM1 receptors may account for 50%

of the cyclic AMP response elicited by the toxin in these cells [16] The results from this work indicate that blood group A-active glycosphingolipids are major functional LT-I alternate receptors in HT29-ATCC and HT29-57 cells

Even though glycosphingolipid distribution in polarized cells was not investigated in this work, we speculate that polarized HT29 cells have glycosphingo-lipid-enriched brush border membranes resembling the mature enterocyte [32] Interestingly, a glycoprotein band present in the brush border-enriched membrane preparation from HT29-ATCC cells bound LT-I This glycoprotein was identified as the glycosylated blood group A-active pro-sucrase-isomaltase by western blot assays (Fig 5) The glycosylated pro-form of sucrase-isomaltase has been clearly detected in the enterocytic differentiated HT29 cells carrying the A blood group of the human donor [26,27] Furthermore, the results of the present work suggest that this glycoprotein may function as an LT-I receptor on human intestinal brush border membranes Sucrase-isomaltase has already

been postulated as a glycoprotein receptor of LT-I on

intestinal brush border membranes from several animal

species, but we have detected this interaction between

LT-I and blood group-A active-sucrase-isomaltase in

porcine and rabbit intestines [13,14] The present

results, together with our earlier findings, support the

idea that the blood group A determinant (mostly of the

type 2 oligosaccharide chain) from glycosphingolipids

and glycoproteins may actually be involved in the

car-bohydrate structure recognized by LT-I Recently, the

fine structural basis of the interaction of a hybrid

between CT and LT-I and a type 2 blood group A

pen-tasaccharide, which involves a novel binding site at the

toxin molecule, was established [33]

Several epidemiological studies have demonstrated a

relationship between ABH blood group status and

high risk of developing cholera [34–37] Recently, a

study was carried out to eilucidate the relationship of

the ABH blood group, immunity and susceptibility

to symptomatic and asymptomatic infections with

V cholerae[38] An association has also been observed

in the occurrence of diarrhea after ingestion of E

coli-producing LT-I in volunteers [39] LT-I and, to a

much lesser degree, CT, interacted with ABH

glyco-conjugates from human and animal intestinal mucosa

[12–14], and furthermore, some of these interactions

have proved to be functional [15,16] These

interac-tions may have relevance in the clinical outcome of

diarrhea caused by LT-I and CT in relation to the

blood group of the patient

Regarding differentiated HT29 cells as intestinal

model system, it is apparent that enterocyte-like

differ-entiated HT29 cells provide a useful in vitro model to

evaluate the functional role of interactions between

bacterial virulence factors and intestinal polarized cells

Experimental procedures

Cell culture

The human colon adenocarcinoma HT29 parental cell line

(HT29-ATCC) was grown in Dulbecco’s modified Eagle’s

medium (D-MEM) containing 10% heat-inactivated fetal

bovine serum Enterocytic differentiation was performed as

described by Hekmati et al [21] Briefly, cells were switched

to RPMI-1640 containing 10% inactivated fetal bovine

serum, replated four times in this medium and then

exam-ined at late confluence (18–21 days) After HT29-ATCC

cells reached confluence, RPMI-1640 was changed every

day Clone HT29-5F7, which was selected by resistance to

5-fluoruracil (kindly donated by Dr T Lesuffleur, INSERM

U560, Lille, France) was usually grown in D-MEM,

con-taining 10% inactivated fetal bovine serum, and examined

at early confluence (undifferentiated) or at late confluence (12 days) when the cells exhibit a polarized phenotype [23,30] Antibiotics (100 UÆmL)1 penicillin, 100 lgÆmL)1 streptomycin) were added to both D-MEM and

RPMI-1640 Cell lines were maintained at 37
C in a humidified atmosphere containing 5% CO2 Cell number was deter-mined by Trypan blue exclusion in a hemocytometer

Toxin-binding assay LT-I was iodinated by a stoichiometric method with chlor-amine T [40], as described previously [11], and the specific activity for the iodinated LT-I was 3.0 lCiÆlg)1 Biological activity of the 125I-labelled LT-I preparation was 90%, measured as the percentage of 125I-labelled LT-I total pro-tein able to specifically bind to GM1-containing membranes (rat red blood cells or NHI 3T3 fibroblasts)

Toxin binding to cells in culture was assayed as previ-ously described [16] Briefly, cells were incubated in serum-free D-MEM buffered with 25 mm Hepes or RPMI-1640 containing 0.01% BSA without (total binding) or with unlabeled LT-B (1.0 lm) before the addition of125I-labelled toxin (3.0 lCiÆlg)1) After 60 min at 4
C, cells were washed, solubilized with NaOH and the radioactivity was counted Nonspecific binding was measured as the binding

of125I-labelled toxin in the presence of an excess of unlabe-led LT-B

To assay nonspecific binding of 125I-labelled LT-I or competitive inhibition by unlabelled LT-B or CT-B, the B subunits of toxin were incubated with cells for 30 min at

4
C and then further incubated with125I-labelled LT-I for

60 min at 4
C The blocking effect of 125I-labelled LT binding by H pomatia lectin was also determined by pre-incubation of cells with lectin, as indicated for B subunits

of toxins

Toxin-stimulated accumulation of intracellular cyclic AMP

The toxin-stimulated accumulation of intracellular cyclic AMP was determined as described previously [16] Briefly, cells were pre-incubated without or with CT-B (1.0 lm), or

H pomatia lectin (10 lm), at 4
C LT-I was then added for 90 min at 37
C Finally, cells were treated with 0.1 m HCl and the dried acid extracts were assayed for cyclic AMP by RIA (Immunotech SA, Marseille, France), accord-ing to instructions of the manufacturer

Electron microscopy TEM was performed as follows Cell monolayers were fixed

in 2% glutaraldehyde and then postfixed in 1% OsO4 After dehydration in graded ethanol solutions, the cells were embedded in Epon Ultrathin sections were contrasted

with uranyl acetate and lead citrate Thin sections were

examined in a Jeol EX 1220 transmission electron

micro-scope (Jeol, Tokyo, Japan)

Hydrolase assays

Brush border-enriched membrane fractions (P2) were

pre-pared according to Trugnan et al [27] Briefly, cells were

scraped in Tris-mannitol buffer, pH 7.1, containing

prote-ase inhibitors (1.0 lgÆmL)1antipain, 17.5 lgÆmL)1

benzami-dine, 1.0 mm phenylmethylsulfonyl fluoride, 1.0 lgÆmL)1

pepstatin, 10 lgÆmL)1 aprotinin and 1.0 lgÆmL)1

leupep-tin) Cells were disrupted by sonication and then CaCl2was

added (to 18 mm) The homogenate was centrifuged (950 g,

10 min; Rotor Type 50, Beckman Instruments, Fullerton,

CA, USA) and the supernatant was centrifuged again

(33 500 g, 30 min) to yield the P2 fraction Proteins were

measured by the method of Lowry et al [41]

Glycohydrolases (sucrase, maltase and lactase) and

ami-nopeptidase N activities were determined in P2 fractions

according to Messer and Dalqvist [42] and Maroux et al

[43], respectively The enzyme activities are expressed as

milli-units (mU) per mg of protein One unit is defined as the

acti-vity that hydrolyzes 1.0 lmol of substrate per min at 37
C

ABH phenotyping of cellular glycoconjugates and

toxin-binding assays

To detect blood group-active and toxin-binding

glycopro-teins, P2 fractions were separated by 7.5% SDS⁄ PAGE,

electrotransferred to nitrocellulose sheets, and

immuno-stained as previously described [16] The sucrase–isomaltase

complex was identified using a mouse anti-(human

sucrase-isomaltase) IgG (kindly donated by Dr A Quaroni, Ithaca,

NY, USA) followed by a horseradish

peroxidase-conju-gated secondary antibody Peroxidase was detected with

an enhanced chemiluminiscence immunodetection system

(Amersham Biosciences, Uppsala, Sweden)

Total lipids from cells were extracted and separated using

HPTLC Glycolipids that bind either the toxins or the

anti-(blood group) IgM were immunodetected, essentially as

previously described [16]

Acknowledgements

We thank Dr W S Dallas (Glaxo Wellcome Research,

NC, USA) for providing the LT-I producing- bacterial

strains, Dr J D Clements (Tulane University, New

Orleans, LA, USA) for kindly donating LT-B, Dr

The`cla Lesuffleur (INSERM U560, France) for

provi-ding the HT29-5F7 clone and Dr Andrea Quaroni

(Cornell University, NY, USA) for providing mouse

monoclonal anti-human intestinal hydrolases This

work was supported partly by grants from Consejo

Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Agencia Nacional de Promocio´n

Cientı´fi-ca y Tecnolo´giCientı´fi-ca (BID 1201⁄ OC-AR, PICT 05–10607) and Secretarı´a de Ciencia y Te´cnica de la Universidad Nacional de Co´rdoba (SeCyT-UNC), Argentina EMG was a fellow from CONICET and GAR and CGM are senior career investigators from CONICET

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