Antisecretory factor peptide AF-16 inhibits the secreted autotransporter toxin-stimulated transcellular and paracellular passages of fluid in cultured human enterocyte-like cells.

Antisecretory Factor Peptide AF-16 Inhibits the Secreted Autotransporter Toxin-Stimulated Transcellular and Paracellular Passages of Fluid in Cultured Human Enterocyte-Like Cells Valérie Nicolas,c,d Vanessa Liévin-Le Moala,b,d

Both the endogenous antisecretory factor (AF) protein and peptide AF-16, which has a sequence that matches that of the active N-terminal region of AF, inhibit the increase in the epithelial transport of fluid and electrolytes induced by bacterial toxins in animal and ex vivo models. We conducted a study to investigate the inhibitory effect of peptide AF-16 against the increase of transcellular passage and paracellular permeability promoted by the secreted autotransporter toxin (Sat) in a cultured cellular model of the human intestinal epithelial barrier. Peptide AF-16 produced a concentration-dependent inhibition of the Sat-induced increase in the formation of fluid domes, in the mucosal-to-serosal passage of D-[1-14C]mannitol, and in the rearrangements in the distribution and protein expression of the tight junction (TJ)-associated proteins ZO-1 and occludin in cultured human enterocyte-like Caco-2/TC7 cell monolayers. In addition, we show that peptide AF-16 also inhibits the cholera toxin-induced increase of transcellular passage and the Clostridium difficile toxin-induced effects on paracellular permeability and TJ protein organization in Caco-2/TC7 cell monolayers. Treatment of cell monolayers by the lipid raft disorganizer methyl-␤-cyclodextrin abolished the inhibitory activity of peptide AF-16 at the transcellular passage level and did not modify the effect of the peptide at the paracellular level.

D

iarrheal diseases affect millions of people, and 2.5 million children under the age of 5 years die from these diseases every year (1). Diarrheal disease develops through a multifactorial process that counteracts the net absorption of water as the result of an increase in the secretion or a decrease in the absorption of water. In the intestinal tract, the passage of water across the epithelial barrier is tightly regulated by both the transcellular and paracellular movements of fluid and electrolytes. Transcellular passage develops through an asymmetric intracellular distribution of membrane-associated pumps and channels, whereas paracellular permeability is regulated by structural and functional proteins located at the tight junctions (TJs) (2). Enteric bacterial pathogens have developed sophisticated strategies to manipulate the host’s normal water balance by producing both structural and functional changes in the epithelial barrier (3). In particular, enterovirulent Escherichia coli strains alter the structural organization of polarized epithelial cells and/or deregulate the functional systems involved in the regulation of the transcellular or paracellular passage of fluids and electrolytes in the intestinal epithelial barrier by the production of deleterious cytotoxic or cytotonic toxins (3). The gastrointestinal system uses a variety of antisecretory or proabsorptive hormonal and protein agonists to balance the outflow of fluid and electrolytes. Those that have been more extensively studied are neuropeptide Y/peptide YY (NPY/PYY) (4) and antisecretory factor (AF) (5). AF is a 41-kDa endogenous protein which was originally purified from the pig pituitary gland by Lönnroth and Lange (6). Its gene has been cloned and sequenced (7). AF is phylogenetically well preserved, since it appears to be a single protein with several conformational variants (8), and no AF-like proteins have been reported. AF is present in most tissues, including the nasal, respiratory, urinary, and gastrointestinal mucosae (9, 10), and is secreted into plasma and other tissue fluids in mam-

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mals (11–13). AF appears in rat tissues after a challenge with cholera toxin (CT) or Clostridium difficile toxin (CDT) (14–16). A region of AF that supports its antisecretory activity has been identified between residues 36 and 51, located in the N-terminal part of the full-length protein (14, 17–20). Experimentally, AF inhibits the intestinal secretion of fluids induced by a variety of toxins, including CT (6, 7, 14, 17, 21–26), Campylobacter toxin (24, 27), Escherichia coli heat-labile enterotoxin (LT) (18, 23) and heatstable enterotoxins (ST) (23, 25, 28), CDT (14, 15, 21), and Dinophysis toxin (24). Moreover, AF and the AF peptide containing the active peptide sequence from residues 36 to 51 have been shown to block the out-in permeation of 36Cl in nerve cell membranes isolated from rabbit Dieter cells (20, 29, 30). Clinically, AF appears to be effective, since administration of a medicinal food containing AF-rich egg yolk powder (B221, Salovum) to children suffering from acute or chronic diarrhea reduced the frequency of passage of stools and solidified their consistency (31, 32). Peptide AF-16 (VCHSKTRSNPENNVGL) (7, 17) displays the

Received 7 October 2014 Returned for modification 19 November 2014 Accepted 16 December 2014 Accepted manuscript posted online 22 December 2014 Citation Nicolas V, Liévin-Le Moal V. 2015. Antisecretory factor peptide AF-16 inhibits the secreted autotransporter toxin-stimulated transcellular and paracellular passages of fluid in cultured human enterocyte-like cells. Infect Immun 83:907–922. doi:10.1128/IAI.02759-14. Editor: S. R. Blanke Address correspondence to Vanessa Liévin-Le Moal, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/IAI.02759-14

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Downloaded from http://iai.asm.org/ on March 14, 2015 by University of Pittsburgh HSLS

CNRS, UMR 8076 BioCIS, Faculté de Pharmacie, Châtenay-Malabry, Francea; LabEx LERMIT—Laboratory of Excellence in Research on Medication and Innovative Therapeutics, Châtenay-Malabry, Franceb; IFR 141 IPSIT, Plateforme Imagerie Cellulaire, Faculté de Pharmacie, Châtenay-Malabry, Francec; Université Paris-Sud, Faculté de Pharmacie, Châtenay-Malabry, Franced

Nicolas and Liévin-Le Moal

MATERIALS AND METHODS Peptide AF-16, reagents, and antibodies. The peptide AF-16 (VCHSKT RSNPENNVGL; Lantmännen AS-Faktor AB, Stockholm, Sweden) and scrambled peptide (GRSNKVENCLPHNSTV) were from E. Johansson (Institute of Biomedicine, Department of Infectious Diseases, Section of Clinical Bacteriology, Göteborg University, Gothenburg, Sweden) (17). CT (catalog no. C8252) and methyl-␤-cyclodextrin (MBCD), a cholesterol-depleting agent which is commonly used to extract cholesterol from membrane lipid rafts, were purchased from Sigma-Aldrich Chimie SARL (L’Isle d’Abeau Chesnes, France). D-[1-14C]mannitol (specific activity, 45 to 60 mCi/mmol) was purchased from PerkinElmer (Courtaboeuf, France). The rabbit polyclonal anti-ZO1 antibody (clone Z-R1), mouse monoclonal antioccludin antibody (clone OC-3F10), and antihuman transferrin receptor polyclonal antibody (PAb) were from Zymed (Invitrogen, Cergy, France). Monoclonal antibody (MAb) 1H4, directed against human decay-accelerating factor (hDAF), was from D. M. Lublin (Department of Pathology, Washington University School of Medicine, St. Louis, MO). Sat was immunolocalized using an anti-plasmid-encoded toxin (anti-Pet) PAb (42), obtained from F. Navarro-Garcia (Department of Cell Biology, CINVESTAV-IPN, Mexico City, Mexico). Fluorescein isothiocyanate (FITC)-conjugated donkey antirabbit and antimouse antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), as was horseradish peroxidase-linked secondary antibody (Jackson ImmunoResearch Laboratories Inc., Newmarket, England). Bacterial strains and growth conditions. The wild-type E. coli strain IH11128 was kindly provided by B. Nowicki (Department of Microbiology and Immunology, Baylor College of Medicine, Houston, TX) (43). The sat gene isolated from wild-type strain IH11128 (satIH11128) and the gene mutated to replace the serine at residue 260 of the serine protease motif by an isoleucine were cloned into the pACYC184 vector and have been previously described (40). The recombinants E. coli AAEC185psat-IH11128 (AAEC185psat), which carries the psat gene from strain IH11128, and AAEC185psat-S256I, which carries the psat gene with an S-to-I mutation at residue 256 (S256I), were obtained by transforming E.

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coli strain AAEC185 (40). IH11128, AAEC185psat, and AAEC185psat-S256I were cultured in LB broth (Difco Laboratories, Detroit, MI) at 37°C for 24 h. C. difficile strain ATCC 9689 was cultured in brain heart infusion for 24 h under anaerobic conditions (Difco Laboratories) (44). Production of CFCSs. Broth filtrates (1,000 ml) of E. coli AAEC185, recombinant E. coli AAEC185psat, or C. difficile strain ATCC 9689 (optical density at 600 nm, 1) were used. Bacteria were removed by centrifugation at 12,000 ⫻ g for 10 min at 4°C. Cell-free culture supernatants (CFCSs) of AAEC185 (CFCS AAEC185), AAEC185psat (CFCS AAEC185psat), and ATCC 9689 (CFCS CD9689) were filtered through a 0.22-␮m-pore-size filter. Samples were concentrated by cross filtration to a volume of 1 ml. It is noteworthy that for the experiments the concentrated CFCS AAEC185psat was used in place of the highly purified toxin, considering that the enzyme activity of the purified toxin declined significantly without a change of the protein content during storage (⫺20°C), whereas the enzyme activity remained stable in the stored, concentrated CFCS AAEC185psat. Cell line and culture conditions. The TC7 clone (Caco-2/TC7) was established from the parental human colon Caco-2 cell line (35). Cells were routinely grown in Dulbecco-Vogt modified Eagle medium (DMEM; which contains 25 mM glucose) supplemented with 15% heatinactivated (30 min, 56°C) fetal calf serum (FCS; Invitrogen) and 1% nonessential amino acids. For maintenance purposes, cells were passaged weekly using 0.02% trypsin in Ca2⫹- and Mg2⫹-free phosphate-buffered saline (PBS) containing 3 mM EDTA. Experiments and cell maintenance were carried out at 37°C in an atmosphere of 10% CO2–90% air. The culture medium was changed daily. For the assays, fully differentiated cells were used at postconfluence after 15 days in culture. The T84 cell line (ATCC, Rockville, MD) is composed of colonic epithelial cells derived from a human colonic carcinoma (35). Cells were routinely grown in a 1:1 (vol/vol) mixture of DMEM and Ham’s F-12 medium supplemented with 15 mM HEPES, 14 mM NaHCO3, and 6% fetal calf serum (Invitrogen), pH 7.5, at 37°C in a 10% CO2–90% air atmosphere. The culture medium was changed daily. For the assays, fully differentiated cells were used at postconfluence after 15 days in culture. Bacterial cell infection. The cell monolayers were washed twice with PBS. Infecting bacteria were suspended in DMEM, and a total of 108 CFU/well of this suspension was added to each well of the tissue culture plate. The plates were incubated at 37°C in 10% CO2–90% air for 3 h. The monolayers were then washed three times with sterile PBS. Each assay was conducted in triplicate with three successive passages of cultured cells. Quantification of viability and adhesion of bacteria. The viability of strain AAEC185psat after treatment was determined by incubating 107 CFU/ml of the pathogen with or without peptide AF-16 at 37°C. Initially and after 3 h of contact, aliquots were removed, serially diluted, and plated on LB agar to determine the bacterial colony count of the pathogen. Each assay was conducted in triplicate. Results were expressed as the log number of CFU/ml. The association of strain AAEC185psat with Caco-2/TC7 cell monolayers was quantified by a bacterial colony count assay. After 3 h of infection (108 CFU/ml), infected cells were lysed with 1% sterile saponin. Appropriate dilutions were made in sterile PBS and then plated on LB agar to determine the number of viable cell-associated bacteria. Each assay was conducted in triplicate. Results were expressed as the log number of CFU/ml. Cytotoxicity assays. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays according to the manufacturer’s instructions (Sigma-Aldrich). Cell integrity was determined by measuring the level of released lactate dehydrogenase (LDH) according to the manufacturer’s instructions (Enzyline LDH kit; bioMérieux, Dardilly, France). Imaging and quantification of fluid domes. To quantify the fluidformed domes, the coverslips were examined by phase-contrast microscopy using an Aristoplan microscope (Leitz, Germany) with epifluorescence (Plan Aprochromat ⫻40/1.32 to 0.6 oil immersion objective). For




AF active sequence (14, 17–20). Considering that AF exerts antagonistic activity in vivo and ex vivo against both CT, deregulating transcellular passage (33), and CDT, deregulating paracellular permeability (34) in the intestinal epithelial barrier, we conducted a study to investigate in vitro the antagonistic activity of peptide AF-16 against the bacterial toxin-induced increase of transcellular passage and paracellular permeability in cultured, human enterocyte-like Caco-2/TC7 cell monolayers that structurally and functionally mimic the human intestinal epithelial barrier (35). As an agonist of transcellular passage and paracellular permeability, we used a bacterial toxin, the secreted autotransporter toxin (Sat) (36), which belongs to the family of serine protease autotransporters of Enterobacteriaceae (SPATEs) (37, 38). Sat produces the hypersecretion of water into the luminal compartment of rabbit intestinal loops (39). Sat activates the transcellular passage of fluids in Caco-2/TC7 cell monolayers, resulting in an increase in the formation of fluid-formed domes (referred to here as “fluid domes”) (40) and the disassembly of TJ-associated structural and functional proteins, which in turn increases the paracellular passage of fluids in Caco-2/TC7 cell monolayers without modifying the transepithelial resistance (TER) of cell monolayers or promoting cell cytotoxicity (40, 41). We report here that peptide AF-16 concentration dependently inhibits the Sat-induced increase in the formation of fluid domes, blocks the Sat-induced rearrangement of TJ-associated ZO-1 and occludin proteins, and inhibits the Sat-induced increase in the paracellular passage of D-[114 C]mannitol in cultured Caco-2/TC7 cell monolayers.

Peptide AF-16 Inhibits Effects of Sat


Western blot analysis of TJ-associated proteins. The cells were washed once with cold PBS and then treated for 15 min at 4°C with an extraction buffer (25 mM HEPES, 0.5% Triton X-100, 150 mM NaCl, 2 mM EDTA) containing protease and phosphatase inhibitors. Protein fractions were dissolved in the appropriate volume of Laemmli buffer and held at 100°C for 5 min. Proteins were immediately separated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; a 12% SDS-polyacrylamide gel was used for occludin, and a 7% SDS-polyacrylamide gel was used for ZO-1). For Western blot analysis, the gels were transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech Inc., Orsay, France) and examined using an ECL⫹ detection system under the conditions recommended by the manufacturer (Amersham Pharmacia Biotech). The membranes were incubated with primary anti-ZO-1, antioccludin, or antiactin antibody and then with horseradish peroxidase conjugated with antirabbit or antimouse antibody as the secondary antibody. Isolation of detergent-insoluble and detergent-soluble fractions. Untreated and MBCD-treated (5 mM, 2 h) cells were washed twice with ice-cold PBS and scraped into cold 10 mM EDTA–PBS. The cells were washed twice by centrifugation and then added to an equal volume of extraction buffer (50 mM Tris HCl, 300 mM NaCl, 10 mM EGTA, 10 mM NaVO4, 20 mM Na PPi, 20 mM NaF, protease inhibitors) for 30 min on ice. After centrifuging at 800 ⫻ g for 10 min, the supernatant was mixed with 1.6 ml of 60% sucrose prepared in 0.5⫻ extraction buffer without detergent, and the mixture was placed in the bottom of an ultracentrifuge tube. A 5 to 60% discontinuous sucrose gradient was established above the sample, and the mixture was centrifuged at 160,000 ⫻ g for 16 h in an SW41 rotor (Beckman Instruments). A total of 10 1-ml fractions were collected from the top of each gradient, and then the fractions were analyzed by means of SDS-PAGE and Western blot analysis. For immunoblotting, the proteins of the gels were transferred to a PVDF membrane (Amersham Pharmacia Biotech) and probed overnight with an anti-human transferrin receptor PAb or MAb 1H4, directed against hDAF. The blots were then incubated with horseradish peroxidase-linked secondary antibody, followed by chemiluminescence detection, according to the manufacturer’s instructions (Amersham Pharmacia Biotech). Statistical analysis. All experiments were repeated at least in triplicate. The statistical significance was determined using Student’s t test. Significance was established when P was ⬍0.01.

RESULTS

Effect of peptide AF-16 on the Sat-induced increase in formation of fluid domes in Caco-2/TC7 cell monolayers. Fluid-filled, blister-like structures known as fluid domes that are formed in Caco-2 cell monolayers result from the accumulation of fluid in randomly distributed areas that evolve permanently in cell monolayers (45, 46). Infection with wild-type E. coli strain IH11128 and recombinant E. coli AAEC185psat secreting Sat or CFCS AAEC185psat containing the secreted Sat (⬃20 ␮g/ml) dramatically increased the number of fluid domes in Caco-2/TC7 cell monolayers (40). As expected, counting of the number of fluid domes observed by DIC CLSM in Caco-2/TC7 cell monolayers (Fig. 1A) showed that the number of fluid domes was greater in cells infected with strain IH11128 and recombinant E. coli strain AAEC185psat than in uninfected cell monolayers or monolayers infected with the control E. coli strain AAEC185 (Fig. 1B). As expected (40), treatment with recombinant strain AAEC185psat-S256I expressing the Sat mutated in its serine protease motif, used as a control, failed to produce an increase in fluid dome formation (Fig. 1B). When the cell monolayers were treated with CFCS AAEC185psat containing the secreted Sat (40), there was an increase in the number of fluid domes (Fig. 1B), which developed in a concentration-dependent man-


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determination of the height of the fluid-formed domes and the area of disorganized cells around fluid-formed domes in a single-cell monolayer, the projections obtained by differential interferential contrast (DIC) using LSM510 confocal laser-scanning microscopy (CLSM) and the 488-nm laser wavelength were analyzed using ImageJ (version 1.42) software (NIH, USA). Projections obtained by DIC CLSM were also analyzed using the DepthCod function of Zeiss software (LSM510, version 2.5). For each sample exposed to control or infectious conditions, more than 30 randomly selected, fluid-formed domes were examined. Duplicate samples were used for each determination. Each sample was examined by two investigators to eliminate the possibility of any bias. Photographic images were resized, organized, and labeled using Adobe Photoshop software (San Jose, CA). TER measurement. Monolayers of Caco-2/TC7 cells were grown in filters mounted in culture chambers (Costar culture plate inserts; 0.4-mm pore size; 4.7 cm2; 3 ⫻ 104 cells per cm2), an arrangement which delineates an apical (luminal) and a basolateral (serosal) reservoir. TER was measured using a volt ohmmeter (Millicel ERS; Millipore, Saint Quentin, France). TER (in units of ohms times square centimeters) was calculated as the measured electrical resistance times the surface area of the filter. The background reading for a culture-free control filter was subtracted. Paracellular permeability measurements. The permeability of the Caco-2/TC7 cell monolayers was determined by measuring the paracellular passage of D-[1-14C]mannitol from the apical to the basolateral compartments of the culture chamber (Costar culture plate inserts; 0.4-mm pore size; 4.7 cm2; 3 ⫻ 104 cells per filter). D-[1-14C]mannitol was dissolved in the culture medium. To measure the flux in the apical-to-basolateral direction, the tracer solution (2.5 mCi/ml) was loaded on the apical side of the monolayer, and the cells were incubated for 1 h at 37°C. After the incubation period, the tracer concentrations in the apical and basolateral compartments were assayed. The concentrations of D-[1-14C]mannitol were determined by measurement in a beta scintillation counter. The values were corrected for the background radioactivity of the medium. Immunofluorescence and quantification of TJ-associated proteins. For the indirect immunofluorescence labeling of TJ-associated ZO-1 and occludin proteins, cultured cells were prepared on glass coverslips, which were then placed in 24-well TPP tissue culture plates (ATGC, Marne la Vallée, France). To detect the TJ-associated proteins, the cells were fixed in 3% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature and then treated with 50 mM NH4Cl for 10 min. The cells in the monolayers were permeabilized using 0.1% Triton X-100 in PBS for 4 min at room temperature and then washed twice with PBS and once with PBS containing 0.1% gelatin and 10% FCS. Primary antibodies (anti-ZO-1 and anti-occludin; dilution, 1:100) were diluted in 0.1% gelatin in PBS. The coverslips were incubated with primary antibodies for at least 2 h, washed three times in PBS, and then incubated with the appropriate secondary conjugated antibodies (dilution, 1:200) for at least 30 min. For CLSM examination of immunofluorescence labeling of TJ-associated proteins, the samples were visualized using a model LSM510 Meta microscope (Zeiss, Germany) equipped with an air-cooled 488-nm argon ion laser and a 543-nm helium neon laser and configured with an Axiovert 100M microscope using a Plan Apochromat 63⫻/1.4 oil-immersion objective lens. The green and red fluorescence emissions were collected with a 505- to 550-nm bandpass and a 560-nm long pass emission filter, respectively, under a sequential mode. The pinhole was set at 1.0 Airy unit. The interval between each z-section was 0.50 ␮m. For each microscopic image of a random region, 60 to 90 z-section images were collected and stacked to form one two-dimensional image by maximum intensity projection using the accompanying Zeiss software (LSM510, version 2.5) on a Windows NT 4 workstation. To quantify the number of cells displaying the normal expression of TJ-associated proteins at the cell-to-cell contacts, the CLSM images were analyzed using Imaris software (version 6.21; Bitplane, Zurich, Switzerland). For each sample, images of at least 10 randomly selected fields, each representing ⬃100 cells, were recorded.


ner, compared with the number of fluid domes obtained after treatment with concentrated CFCS AAEC185 (Fig. 1C). We wanted to find out whether peptide AF-16 (VCHSKTRSN PENNVGL) would inhibit the AAEC185psat-induced increase in the number of fluid domes formed. When the Caco-2/TC7 cell monolayers were infected with recombinant E. coli AAEC1845psat in the presence of increasing concentrations of AF-16, the data showed that as the concentration of AF-16 increased, there were fewer fluid domes than the number of fluid domes in cells infected with recombinant E. coli AAEC185psat alone (Fig. 1D). As a control, the scrambled peptide (GRSNKVENCLPHNSTV) displayed no inhibitory activity against the AAEC185psat-induced increase in the number of fluid domes (Fig. 1D). We checked that the observed inhibitory effect of peptide AF-16 on recombinant E. coli AAEC185psat-infected cells did not result from a bactericidal effect of the peptide on bacteria or an inhibition of bacterial adhesion to the apical domain of Caco-2/ TC7 cells forming monolayers. There was no change in the viability of recombinant E. coli AAEC185psat bacteria after 3 h of direct contact between peptide AF-16 and the bacteria (viabilities of un-

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treated AAEC185psat and AAEC185psat treated with AF-16 at 50 ␮M, 6.8 ⫾ 0.3 and 6.6 ⫾ 0.4 log CFU/ml, respectively). Moreover, infection of cell monolayers with untreated or peptide AF-16treated recombinant E. coli AAEC185psat bacteria for 3 h resulted in identical levels of adhering bacteria (adherence of AAEC185psat and AAEC185psat treated with AF-16 at 50 ␮M to Caco-2/TC7 cells, 6.3 ⫾ 0.2 and 6.1 ⫾ 0.4 log CFU/ml, respectively). We quantified the amount of fluid accumulating within the fluid domes by measuring the height and the relative surface area of the fluid domes observed by DIC CLSM under the various different experimental conditions investigated, as described above (Fig. 2A). We found that the height and the relative surface area of the fluid domes were significantly greater in recombinant E. coli AAEC1845psat-infected Caco-2/TC7 cell monolayers than in control cell monolayers (Fig. 2B). We also observed that in CT-exposed cell monolayers, the height and relative surface area of the fluid domes were greater than those in control cell monolayers (Fig. 2B). In contrast to the previously observed Sat-induced cytotoxicity that developed in human nonpolarized bladder, kidney, and cervix epithelial cells (42, 47), there was no sign of cell cyto-




FIG 1 Effect of peptide AF-16 and scrambled peptide on the increase in the formation of fluid domes induced by wild-type IH11128, recombinant E. coli AAEC185psat, or concentrated CFCS of recombinant E. coli AAEC185psat (which contains Sat) in fully differentiated Caco-2/TC7 cell monolayers. (A) Highmagnification micrographs (x-y optical sections) showing fluid domes observed by DIC CLMS at three levels in cell monolayers infected with AAEC185 or recombinant E. coli AAEC185psat (108 CFU/ml, 3 h of infection). The micrographs are representative of those from three separate experiments. (B) The number of fluid domes in control untreated cell monolayers; E. coli AAEC185-, wild-type IH11128-, recombinant E. coli AAEC185psat-, and recombinant E. coli AAEC185psat-S256I-infected cell monolayers (108 CFU/ml, 3 h of infection) or cell monolayers exposed to concentrated CFCS of E. coli AAEC185psat (⬃20 ␮g/ml, 3 h). (C) Concentration-dependent increase in the number of fluid domes in cell monolayers subjected to increased volumes of concentrated CFCS AAEC185psat. (D) Effect of increasing concentrations of peptide AF-16 and scrambled peptide on the number of fluid domes in recombinant E. coli AAEC185psat-infected cell monolayers. The number of fluid domes per monolayer was determined by DIC light microscopy counting. (B to D) Each value shown is the mean ⫾ SD from three experiments (three successive passages of Caco-2/TC7 cells). *, P ⬍ 0.01 versus AAEC185 (B) and P ⬍ 0.01 versus AAEC185psat (D); **, P ⬍ 0.01 versus AAEC185psat.


toxicity in recombinant E. coli AAEC1845psat-infected fully polarized Caco-2/TC7 cells (not shown). However, it is noteworthy that at the tip of the fluid domes observed in E. coli AAEC185psatinfected cell monolayers, the cells were enlarged as a result of the mechanotension due to the presence of a large volume of fluid at the basal domain of the fluid domes, but there was no disruption of cell-to-cell contacts (Fig. 2C). This phenomenon develops without modifying cell integrity, since there was an absence of LDH release (not shown). There was a concentration-dependent reduction in the heights and relative surface areas of the fluid domes in cell monolayers infected with recombinant E. coli AAEC185psat in the presence of increasing concentrations of peptide AF-16 compared to those of fluid domes in infected but untreated cell monolayers (Fig. 2D). Examination of the fluid domes in cell monolayers infected with E. coli AAEC185psat in the presence of peptide AF-16 reveals that the cells at the tip of the fluid domes were not enlarged and formed normal cell-to-cell junctions (Fig. 2C).


Effect of peptide AF-16 on the Sat-induced increase of paracellular permeability in filter-grown Caco-2/TC7 cell monolayers. Using paracellular markers of different sizes {[14C]- or [3H]mannitol, 182 Da; fluorescein-5 sulfonic acid (FS), 478 Da; [3H]polyethylene glycol ([3H]PEG), 900 Da}, it has previously been demonstrated that Sat in Caco-2 cell monolayers induces an increase in the level of paracellular passage of mannitol and does not induce the passage of FS or PEG (40, 41). The paracellular permeability in filter-grown Caco-2/TC7 cell monolayers was measured using D-[1-14C]mannitol. The mucosal-to-serosal passage of D-[1-14C]mannitol across the control or E. coli AAEC1845infected Caco-2/TC7 cell monolayers was negligible (Table 1). As expected (40), infection of filter-grown Caco-2/TC7 cell monolayers with recombinant E. coli AAEC184psat resulted in a highly significant increase in the mucosal-to-serosal passage of D-[114 C]mannitol compared to that in control or E. coli AAEC1845infected Caco-2/TC7 cell monolayers (Table 1). As expected (40), there was an absence of an increase in the mucosal-to-serosal pas-


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FIG 2 Quantification of the height and relative surface area of fluid domes. (A) A high-magnification micrograph (x-z optical section) showing a fluid dome observed by DIC CLMS in a cell monolayer infected with E. coli AAEC185 (3 h). Arrows, measures of height and relative surface area of the fluid domes. The micrograph is representative of the micrographs from three separate experiments. (B) Quantification of the height and relative surface area of fluid domes in control untreated and E. coli AAEC185- and recombinant E. coli AAEC185psat-infected (3 h) cell monolayers. (C) Observation by DIC CLSM of cells at the tip of fluid domes. Arrows, cell-to-cell junctions. (B and C) Each value shown is the mean ⫾ SD from three experiments (three successive passages of Caco-2/TC7 cells). (D) Effect of increasing concentrations of peptide AF-16 on the height (left) and relative surface area (right) of fluid domes in recombinant E. coli AAEC185psat-infected cell monolayers. The samples were analyzed by DIC CLSM (horizontal x-y optical sections from the plane to the tops of the domes, 0.5-␮m section). To determine the height of the fluid domes, the projections obtained by DIC CLSM were analyzed using ImageJ (version 1.42) software (NIH, USA). *, P ⬍ 0.01 versus the results for the control (B) and P ⬍ 0.01 versus the results for AAEC185psat (D).


TABLE 1 Effect of peptide AF-16 on the E. coli AAEC185psat-induced increase in paracellular permeability in filter-grown Caco-2/TC7 cell monolayers

Control AAEC185 AAEC185psat AAEC185psat-S256I AAEC185psat ⫹ AF-16 at 10 ␮M AAEC185psat ⫹ AF-16 at 25 ␮M AAEC185psat ⫹ AF-16 at 50 ␮M AAEC185psat ⫹ AF-16 at 75 ␮M AAEC185psat ⫹ AF-16 at 100 ␮M AAEC185psat ⫹ scrambled peptide at 75 ␮M

2.75 ⫾ 0.85 3.06 ⫾ 0.77 31.88 ⫾ 2.14* 5.11 ⫾ 0.97** 27.35 ⫾ 3.18 22.25 ⫾ 2.05** 14.85 ⫾ 1.12** 8.12 ⫾ 0.32** 4.85 ⫾ 0.21** 29.12 ⫾ 4.45***

a Paracellular passage of D-[1-14C]mannitol was measured in the mucosal-to-serosal direction with and without infection. The results are expressed as the percentage of 14 D-[1- C]mannitol that passed into the mucosal compartment. *, P ⬍ 0.01 compared to the results for AAEC185; **, P ⬍ 0.01 compared to the results for AAEC185psat; ***, P ⬍ 0.01 compared to the results for AAEC185psat plus AF-16 at 75 ␮M.

FIG 3 Distribution of protein ZO-1 in uninfected control or recombinant E. coli AAEC185psat-infected, fully differentiated Caco-2/TC7 cell monolayers in the presence or absence of increasing concentrations of peptide AF-16. Cells were indirectly immunostained with an antibody to ZO-1. Cell monolayers were infected with AAEC185 or recombinant E. coli AAEC185psat (108 CFU/ml, 3 h of infection). The samples were analyzed by CLSM. (A) Low-magnification (left) and high-magnification (right) micrographs (x-z optical sections) showing the presence of ZO-1 at the upper part of the junctional domain (TJs) of cells forming cell monolayers. Hatched lines, lateral and basal domains of polarized cells in the cell monolayer. (B) En face micrographs show the ZO-1 immunofluorescence labeling obtained on the maximum intensity projection (MIP) of optical sections. The micrographs are representative of those from three independent experiments. Boxed areas, areas of higher magnification shown in the adjacent panels.

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Treatment

% D-[1-14C]mannitol passagea

sage of D-[1-14C]mannitol in cell monolayers infected with recombinant strain AAEC185psat-S256I expressing Sat mutated in its serine protease motif (Table 1). When the filter-grown Caco-2/ TC7 cell monolayers were infected with the recombinant E. coli AAEC1845psat in the presence of increasing concentrations of AF16, the results showed that the increase in the mucosal-to-serosal passage of D-[1-14C]mannitol was concentration dependently inhibited (Table 1). In contrast, the scrambled peptide failed to inhibit the AAEC185psat-induced increase in the mucosal-to-serosal passage of D-[1-14C]mannitol (Table 1). Effect of peptide AF-16 on Sat-induced rearrangement of the TJ-associated protein distribution in Caco-2/TC7 cell monolayers. Sat induces dramatic rearrangements of the TJ-associated proteins ZO-1, ZO-3, and occludin and, to a lesser extent, rearrangement of claudin-1 (40, 41). By means of indirect immunofluorescence labeling coupled to CLSM analysis, we investigated the distribution of ZO-1 and occludin in control and infected Caco-2/TC7 cell monolayers. Examination of the immunolabeling of ZO-1 (Fig. 3) and occludin (Fig. 4) showed that these proteins localized at cell-to-cell contacts in uninfected Caco-2/TC7 cells displaying the typical honeycomb-like pattern of differentiated confluent intestinal cells. As expected (40), apical infection


of Caco-2/TC7 cell monolayers with recombinant E. coli AAEC1845psat resulted in a dramatic loss of both ZO-1 (Fig. 3) and occludin (Fig. 4) immunolabeling at the TJs. There was a progressive reappearance of both ZO-1 (Fig. 3 and 5A) and occludin (Fig. 4 and 5B) immunofluorescence labeling in cell monolayers infected in the presence of increased concentrations of peptide AF16. In contrast, the scrambled peptide failed to inhibit the AAEC185psat-induced delocalization of ZO-1 (Fig. 5A). The decrease in the distribution of ZO-1 and occludin at TJs observed in AAEC185psat-infected cells and the normal distribution observed after peptide AF-16 treatment prompted us to conduct an analysis at the protein level (Fig. 5C). Western blot analysis of isolated uninfected control cell membranes showed that ZO-1 and occludin were expressed as a single band at 220 kDa in the case of ZO-1 and as two bands at 65 and 72 kDa for occludin, corresponding to the nonphosphorylated and hyperphosphorylated forms of this protein, respectively. Consistent with the findings of Guignot et al. (40), the protein levels of these TJ-associated proteins were ⬃90% lower in the membranes of AAEC185psatinfected cells than in those of uninfected control cells. In the cells infected with AAEC185psat in the presence of peptide AF-16, the levels of ZO-1 and of the nonphosphorylated and hyperphosphorylated forms of occludin were the same as those in uninfected control cells (Fig. 5C). The inhibitory activity of peptide AF-16 is not cell or Sat specific. In order to ensure that the findings with recombinant E. coli AAEC185psat and peptide AF-16 are not cell type specific, we examined the effect of peptide AF-16 on cultured human colonic T84 cell monolayers forming fluid domes (48). As reported above


with Caco-2/TC7 cells, peptide AF-16 treatment resulted in a decrease in the recombinant E. coli AAEC185psat-induced increase in the level of formation of fluid domes and delocalization of the ZO-1 protein (Table 2). We then investigated whether peptide AF-16 is able to antagonize the increased formation of fluid domes by another bacterial toxin activating the transcellular passage of fluid and electrolytes. To do this, we chose to use CT, which is known to induce a dramatic increase in the transcellular passage of fluid and electrolytes in the intestine (33). Consistent with the finding that CT induced an increase in fluid dome formation in polarized epithelial cells forming monolayers (49), we observed an increase in the number of fluid domes formed after exposing Caco-2/TC7 cell monolayers to CT (20 ng/ml) (Fig. 6A). As a control, there was no increase in the number of fluid domes formed in the presence of concentrated CFCS CD9689 containing CDTs (44) (Fig. 6A). Fewer fluid domes were formed in cells exposed to CT in the presence of peptide AF-16 (Fig. 6A). In contrast, there was an absence of an inhibitory effect against the CT-induced increase in the number of fluid domes formed in the presence of the scrambled peptide (Fig. 6A). In order to examine whether the antagonistic activity of peptide AF-16 on the increase in paracellular permeability at the TJs is Sat specific or not, we chose to use CDTs known to produce structural and functional changes at the TJs (34). Consistent with previous observations (50–53), we observed a dramatic decrease in the amount of TER (Fig. 6B), an increase in the mucosal-to-serosal passage of D-[1-14C]mannitol (Fig. 6C), and the disappearance of ZO-1 from TJs in Caco-2/TC7 cells exposed to the concentrated CFCS CD9686 containing CDTs (44) (Fig. 6D). As a control, the


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FIG 4 Distribution of occludin protein in uninfected control or recombinant E. coli AAEC185psat-infected, fully differentiated Caco-2/TC7 cell monolayers in the presence or absence of increasing concentrations of peptide AF-16. Cell monolayers were infected with AAEC185 or recombinant E. coli AAEC185psat (108 CFU/ml, 3 h of infection). Cells were indirectly immunostained using an antibody to occludin. The samples were analyzed by CLSM. En face micrographs show the occludin immunofluorescence labeling obtained on the maximum intensity projection (MIP) of optical sections. The micrographs are representative of those from three independent experiments. Boxed areas, areas of higher magnification shown in the adjacent panels.


proteins in Caco-2/TC7 cells. The samples were analyzed by CLSM. (A) Quantification of cells showing normal ZO-1 immunolabeling at TJs. (B) Quantification of cells showing normal occludin immunolabeling at TJs. In panels A and B, each value shown is the mean ⫾ SD from three experiments (three successive passages of Caco-2/TC7 cells). (C) Western blot analysis of changes in ZO-1 and occludin protein levels in membranes of uninfected control, AAEC185psat-infected, and AAEC185psat-infected and peptide AF-16 (100 ␮M)-treated Caco-2/TC7 cells. The bands for the nonphosphorylated (65 kDa) and hyperphosphorylated (72 kDa) forms of occludin are indicated. The blots are representative of those from two independent Western blot analyses. *, P ⬍ 0.01 versus the results for AAEC185; **, P ⬍ 0.01 versus the results for recombinant AAEC185psat; ***, P ⬍ 0.01 versus the results for recombinant AAEC185psat treated with peptide AF-16 at 75 ␮M.

mucosal-to-serosal passage of D-[1-14C]mannitol and the ZO-1 distribution at TJs were unchanged in cell monolayers exposed to CT, and consistent with the findings described in previous reports (54, 55), TER was decreased after CT treatment (Fig. 6B to D). The decrease in TER, the increase in mucosal-to-serosal passage of 14 D-[1- C]mannitol, and the delocalization of the ZO-1 protein from TJs in CFCS CD9686-intoxicated Caco-2/TC7 cells were all antagonized in the presence of peptide AF-16 (Fig. 6B to D). In TABLE 2 Effect of peptide AF-16 on the AAEC185psat-induced increase in fluid dome formation and delocalization of TJ-associated ZO-1 protein in T84 cell monolayersa

Treatment AAEC185 AAEC185psat AAEC185psat ⫹ AF-16 at 75 ␮M AAEC185psat ⫹ scrambled peptide at 75 ␮M

No. of fluid domes

% cells showing normal expression of ZO-1 immunolabeling at TJs

15 ⫾ 4 115 ⫾ 14* 20 ⫾ 7** 102 ⫾ 17**

100 5 ⫾ 2* 72 ⫾ 12** 8 ⫾ 2**

a *, P ⬍ 0.01 compared to the results for AAEC185; **, P ⬍ 0.01 compared to the results for AAEC185psat..

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contrast, the scrambled peptide failed to inhibit the CFCS CD9686-induced changes (Fig. 6B to D). The inhibitory activity of peptide AF-16 does not result from an action on the production of Sat by AAEC185psat or from inhibition of the cell internalization of Sat. We investigated whether peptide AF-16 affects the production of Sat by AAEC185psat. When AAEC185psat bacteria were subjected to a preliminary incubation with peptide AF-16 (100 ␮M) for 1 h, washed, and then applied to Caco-2/TC7 cell monolayers, the results in Fig. 7A show that the treated bacteria induced an increase in the level of formation of fluid domes similar to that in untreated AAEC185psat. After Sat interacts with an unknown cell membrane-associated receptor, it had previously been demonstrated that Sat activity requires internalization of the toxin within epithelial cells (42). We first investigated whether the treatment of the Caco-2/TC7 cell monolayers with the traffic blocker brefeldin A modified the Satinduced increase in the level of formation of fluid domes. As shown in Fig. 7B, the CFCS AAEC185psat-induced increase in the formation of fluid domes in Caco-2/TC7 cell monolayers was abolished in the presence of brefeldin A. We then determined whether brefeldin A affects the entry of Sat into epithelial cells.




FIG 5 Effect of peptide AF-16 and scrambled peptide against the recombinant E. coli AAEC185psat-induced rearrangement of TJ-associated ZO-1 and occludin


the TJs in Caco-2/TC7 cell monolayers. (A) Effect on the CT-induced increase in the number of fluid domes. (B) Effect on the CDT-induced decrease in TER. (C) Effect on the CDT-induced increase in the paracellular passage of D-[1-14C]mannitol. (D) Effect on the CDT-induced delocalization of ZO-1 from TJs. The micrographs are representative of those from two separate experiments. (A to D) Each value shown is the mean ⫾ SD from two experiments (three successive passages of Caco-2/TC7 cells). CT, cholera toxin (20 ng/ml, 3 h); CDTs, the concentrated CFCS of CD9689 containing toxins A and B (50 ␮l, 3 h). *, P ⬍ 0.01 versus the results for the control; **, P ⬍ 0.01 versus the results for CT or CDTs; ***, P ⬍ 0.01 versus the results for CT plus peptide AF-16 or CDTs plus peptide AF-16.

To do this, we visualized the Sat internalized within CFCS AAEC185psat-treated HeLa cells by means of indirect immunolabeling with anti-Pet PAb, which cross-reacts with Sat (42), and CLMS examination and quantification. The punctate presence of Sat-positive immunofluorescence was observed throughout the cytoplasm of the untreated cells (Fig. 7C). When the cells were pretreated with brefeldin A, no Sat-positive immunolabeling was observed within the cell cytoplasm (Fig. 7C). In contrast, after the cells had been treated with peptide AF-16, there was no change in the intracellular presence of Sat, indicating that the peptide does not affect the entry of the toxin into epithelial cells (Fig. 7C). Roles of membrane-associated lipid rafts in inhibitory activities of peptide AF-16. Caco-2/TC7 cells were exposed to the lipid raft disorganizer methyl-␤-cyclodextrin (MBCD; 5 mM, 2 h) (Fig. 8). Disorganization of the lipid rafts after MBCD treatment was controlled by isolating the detergent-insoluble and detergent-soluble membranes by sucrose gradient ultracentrifugation and then revealing the presence or absence of lipid raft-associated human decay-accelerating factor (hDAF) and non-lipid raft-associated transferrin receptor (TfR) by Western blotting (Fig. 8A). As expected, hDAF was found only in low-density fractions (fractions 3 and 4), whereas TfR was present only in high-density fractions (fractions 8 and 9) in untreated cells. After the cells had been


pretreated with MBCD, hDAF remained present in the detergentinsoluble fractions and appeared within the detergent-soluble fractions, suggesting a partial disruption of lipid rafts (Fig. 8A). In contrast, TfR was still found only in the detergent-soluble fractions in both untreated and MBCD-treated cells (Fig. 8A). We investigated whether the MCBD treatment modified the abovedescribed inhibitory activity of peptide AF-16 against the AAEC1845psat-induced increase in fluid dome formation and the disappearance of ZO-1 from the TJs in Caco-2/TC7 cells (Fig. 8B and C, respectively). Pretreating the cells with MBCD did not modify the AAEC1845psat-induced increase in fluid dome formation or the disappearance of ZO-1 from the TJs. The antagonistic effect of peptide AF-16 against the AAEC1845psat-induced increase in dome formation was abolished in cells pretreated with MBCD (Fig. 8B). In contrast, pretreating the cells with MBCD did not abolish the inhibitory effect of peptide AF-16 against the AAEC1845psat-induced disappearance of ZO-1 from the TJs (Fig. 8C). We observed that the treatment of Caco-2/TC7 cells with MBCD abolished the ability of CT to induce an increase in fluid domes (not shown), a finding that is consistent with the role of the membrane lipid rafts in the entry of CT into cells (33). This made it impossible to assess the role of membrane lipid rafts in the inhibitory action of peptide AF-16 against the


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FIG 6 Effect of peptide AF-16 and scrambled peptide on the CT-induced increase in the number of fluid domes and CDT-induced delocalization of ZO-1 from


cells. (A) Quantification of fluid domes in Caco-2/TC7 cell monolayers infected with AAEC185psat pretreated or not with peptide AF-16 before cell infection. (B) Quantification of fluid domes in Caco-2/TC7 cell monolayers exposed to the concentrated CFCS of AAEC185psat with or without the presence of the endocytosis blocker brefeldin A. In panels A and B, each value shown is the mean ⫾ SD from three experiments (three successive passages of Caco-2/TC7 cells). (C) Low-magnification micrographs (left) and quantification of the intensity of Sat immunofluorescence (right) show the effects of peptide AF-16 or brefeldin A treatment on the entry of Sat into HeLa cells. Cells were exposed to the concentrated CFCS of AAEC185psat for 3 h. Internalized Sat was visualized by indirect immunofluorescence using anti-Pet PAb cross-reacting with Sat (42) and CLMS observation. (Inset) A high-magnification CLMS observation showing the vesicular presence of Sat in cells subjected to the concentrated CFCS of AAEC185psat. The micrographs are representative of those from two independent experiments. *, P ⬍ 0.01 versus the results for AAEC185 or CFCS AAEC185; **, P ⬍ 0.01 versus the results for CFCS AAEC185psat.

CT-induced formation of fluid domes. Since treatment with MBCD had no impact on the effect of CDTs at the paracellular level (not shown), we looked at whether the MBCD-induced disorganization of lipid rafts affected the inhibitory effect of peptide AF-16 on the rearrangement of ZO-1 induced by CDTs. We found that pretreating the cells with MBCD did not abolish the inhibitory effect of peptide AF-16 on the CDTinduced disappearance of ZO-1 from the TJs (not shown) (Fig. 8C). Overall, our findings indicate that the antagonistic effect of peptide AF-16 at the transcellular level occurs in a lipid raft-dependent manner, whereas, in contrast, the antagonistic activity at the paracellular level develops in a lipid raft-independent manner. DISCUSSION

The results reported here demonstrate that peptide AF-16 is able to induce a concentration-dependent inhibition of the Sat-induced increase in the transcellular passage of fluid, since it diminished the toxin-induced increase in the number and height of the fluid domes. Peptide AF-16 also produced a concentration-dependent inhibition of the Sat-induced disassem-

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bly of the TJ-associated ZO-1 and occludin proteins and abolished the toxin-induced increase in the paracellular passage of 14 D-[1- C]mannitol. In contrast, a scrambled peptide failed to inhibit the Sat-induced increase in the transcellular passage of fluid, the disassembly of the TJ-associated ZO-1, and the increase in the paracellular passage of D-[1-14C]mannitol. When the concentration-dependent effects of peptide AF-16 were compared to the Sat-induced increase in fluid dome formation, the disappearance of ZO-1 and occludin, and the increase in the paracellular passage of D-[1-14C]mannitol, it was noteworthy that peptide AF-16 could be seen to be active against the effect of Sat on transcellular passage at concentrations lower than those that are active against the effects of Sat on paracellular passage (50% inhibitory concentrations, ⬃21 ␮M and ⬃60 ␮M, respectively) (Fig. 9). In addition, the results showed the inhibitory activity of peptide AF-16 against the changes induced by CT at the transcellular passage level and CDTs at the paracellular permeability level. Fluid domes formed in polarized epithelial cells forming monolayers are highly dynamic structures resulting from the transcellular movement of water that results in the accumulation of liquid between




FIG 7 Investigation of the direct effect of peptide AF-16 on Sat production by recombinant E. coli AAEC185psat and on the internalization of Sat into epithelial


of hDAF within raft fractions and of human Tfr within nonraft fractions. MBCD treatment (5 mM, 2 h) leads to the appearance of hDAF into nonraft fractions, whereas the treatment does not affect the localization of human TfR within nonraft fractions. Cells were lysed with buffer containing 0.5% Triton X-100 and separated into soluble and insoluble fractions, as described in Materials and Methods. Detergent-soluble and -insoluble fractions were resolved by SDS-PAGE and Western immunoblotted (IB) to detect hDAF or human TfR. (B) The number of fluid domes in recombinant E. coli AAEC185psat-infected cells which had or had not been treated with peptide AF-16 and had or had not been exposed to MBCD treatment. The number of fluid domes per monolayer was determined by counting under a phase-contrast light microscope. (C) Cells were indirectly immunolabeled with an antibody directed against the ZO-1 protein, and the samples were analyzed by CLSM. (Left) En face micrographs show the ZO-1 immunofluorescence labeling obtained by the maximum intensity projection (MIP) of optical sections. (Right) Quantification of cells displaying normal ZO-1 immunolabeling at the TJs. The blots in panel A and micrographs in panel C are representative of those from two or three independent experiments. (B and C) Each value shown is the mean ⫾ SD from three experiments (three successive passages of Caco-2/TC7 cells). *, P ⬍ 0.01 compared to the results for the control; **, P ⬍ 0.01 compared to the results for AAEC185psat; ***, P ⬍ 0.01 compared to the results for AAEC185psat treated with AF-16.

the cell monolayer and the underlying support. The biogenesis and properties of fluid domes have been investigated in Madin-Darby canine kidney cell monolayers (49, 56–62) and in LLC-PK1 pig kidney-derived cells (63, 64). For intestinal cells forming monolayers in culture (35), several studies have revealed that the fluid dome-forming cells are functionally equivalent to differentiated epithelial cells in the intestine involved in the transcellular transportation of solutes (45, 46, 48), since the functional properties of fluid domes are related to the differentiation-associated apical localization of functional sodium transporters, including sodium channels and Na⫹/H⫹ exchangers (NHEs) controlling the transcellular passage of ions/water (65–76). In several of these reports, the combined measures of the electrical and functional transport properties and the activities of brush border-associated transporters have clearly established the relationship between the transcellular passage of fluid and the formation of fluid domes. We found that the Sat-induced increase in the number of fluid domes in Caco-2/TC7 cell monolayers was concentration dependently in-


hibited in the presence of peptide AF-16, and this inhibition was accompanied by a decrease in the height and relative surface area of the fluid domes, indicating that peptide AF-16 had an inhibitory effect on the transcellular passage of fluid. We demonstrated that the effect of peptide AF-16 against the Sat-induced increase in the formation of fluid domes does not result from a blockade of cell entry of the toxin. It is reasonable to assume that the Satinduced secretory effect results from activation of the brush border membrane systems involved in the transcellular passage of fluid and electrolytes and expressed by these cells (35). Interestingly, we found here that peptide AF-16 is also able to antagonize the effect of CT on the formation of fluid domes in Caco-2/TC7 cell monolayers. Our finding is consistent with the previously observed in vivo and ex vivo antisecretory effect of protein AF and peptide AF-16 against the fluid secretion stimulated by CT (6, 14, 16, 17, 22–25). This secretory toxin is known to activate the transcellular transport of fluid and electrolytes by activating the cyclic AMP (cAMP)-adenylate cyclase that elevates the cellular level of


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FIG 8 Effect of MBCD-induced membrane lipid raft disorganization on the inhibitory effects of peptide AF-16. (A) The control shows the endogenous presence


cAMP, which in turn activates several brush border-associated ion channels and transporters (33). Membrane-associated lipid rafts are currently defined to be dynamic sterol-sphingolipid-enriched nanoscale assemblies of different sizes (77, 78). Quite large and highly stably organized super-lipid rafts are present at the membrane of the brush border of enterocytes (79–81). Brush border membrane-associated lipid rafts contain transporters involved in the transcellular passage of fluid and electrolytes, such as NHEs 1 to 3 (82, 83) and Cl⫺/HCO3⫺/OH⫺ exchangers (84). The antagonistic effect of peptide AF-16 against the AAEC1845psat-induced increase in fluid dome formation was abolished in cells pretreated with MBCD. However, considering that after cholesterol extraction with MBCD most of the hDAF was still associated with the detergent-insoluble membrane, suggesting that the majority of membrane lipid rafts were not entirely disrupted by MBCD, there is not a clear association between membrane raft integrity and the antagonistic effect of peptide AF-16 against the AAEC1845psatinduced increase in fluid dome formation. An increase of paracellular permeability at the paracellular level has been observed in Caco-2 cell monolayers as a result of rearrangements of TJ-associated structural and functional proteins induced by toxins or effectors of enterovirulent bacteria (85, 86). In polarized epithelial cells, TJs are a highly regulated domain (2) formed by the assembly of specialized proteins, including the cytoplasmic ZO-1, ZO-2, and ZO-3 proteins, which are connected to the F-actin cytoskeleton; the transmembrane protein occludin, which is connected to both the ZO proteins and the cytoskeleton; and junctional adhesion molecules and claudins, which are connected to the ZO proteins. TJs which form the closure of the intestinal epithelium through a gate activity also separate the apical from the basolateral membrane domains by means of fence activity (2). On the basis of functional studies (87), it has recently been demonstrated that this gate activity includes two functional paracellular pathways: one, which is known as the leaky pathway, includes occludin and controls the paracellular passage of larger molecules, and the other, which is known as the high-capacitypore pathway, involves the claudin family of proteins and acts as cation-selective and anion-selective protein-forming channels

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FIG 9 Summary of the concentration-dependent effects of peptide AF-16 on the recombinant E. coli AAEC185psat-induced increase in the number of transcellular and paracellular passages. The number, height, and relative surface area of fluid domes are related to transcellular passage in Caco-2/TC7 cell monolayers. The passage of D-[1-14C]mannitol and the rearrangements in the distribution of the ZO-1 and occludin proteins at the TJs are related to paracellular passage in Caco-2/TC7 cell monolayers.

and as protein-forming channels without any clearly established selectivity (88). It is noticeable that Sat affects the distribution of occludin at the TJs without modifying the claudin distribution (40). We observed here that the Sat-induced increase in the paracellular passage of D-[1-14C]mannitol and rearrangements at TJs and the loss of the ZO-1 and occludin at the protein level in Caco2/TC7 cell monolayers were concentration dependently inhibited by peptide AF-16. At the protein level, the loss of ZO-1 and occludin induced by Sat in enterocyte-like cells reported previously (40) and observed here suggests that these proteins can be substrates of Sat. The class 1 SPATEs Pet, EspC, SigA, and Sat have the common capacity to cleave a biological substrate, fodrin (␣-spectrin), a ubiquitous protein involved in actin cross-linking, in turn promoting severe cell cytoskeleton disassembly. Whether the TJassociated proteins ZO-1 to ZO-3 and occludin are substrates of class 1 SPATEs remains to be investigated. Glucosylating exotoxins A and B of C. difficile (34) are known to disrupt the intestinal barrier function by modifying structural and functional aspects of the organization of TJs, which in turn increases paracellular permeability (50, 51). We observed that peptide AF-16 also antagonized the CDT-induced disappearance of ZO-1 from TJs in Caco-2/TC7 cell monolayers. Our finding in vitro is consistent with previous observations showing the ability of peptide AF-16 to antagonize ex vivo the deleterious effects of CDT (14, 15, 21). In contrast to the lipid raft-dependent effect of peptide AF-16 on transcellular passage, we found that the disorganization of the membrane-associated lipid rafts by MBCD treatment did not abolish the inhibitory activity of peptide AF-16 against the Sat-induced disappearance of protein ZO-1 from TJs in Caco-2/TC7 cells. This result can be explained by the particular distribution of TJ-associated proteins within the membrane of this domain of polarized epithelial cells. TJs are highly regulated and formed by the assembly of specialized proteins, such as the cytoplasmic ZO-1, ZO-2, and ZO-3 proteins connected with the F-actin cytoskeleton, the transmembrane occludin connected with both ZO proteins and the cytoskeleton, and junctional adhesion molecules and claudins connected with ZO proteins (2). It has been proposed that membrane-associated raft microdomains play a pivotal role in the structural organization and functionality of TJs on the basis of the findings that the hyperphosphorylated form of occludin localized principally within lipid raft membrane domains (40, 52, 53, 89, 90) and that the localization of occludin at TJs was impaired after a metabolic lowering of membrane cholesterol levels, affecting the raft microdomain organization (89). However, it is noteworthy that the nonphosphorylated form of occludin and the ZO proteins localized within both raft and nonraft microdomains (40, 52, 53, 90). In conclusion, our results show that the peptide AF-16, which has a sequence that matches that of the active N terminus of AF, inhibits the increase of transcellular and/or paracellular passage of fluids promoted by the non-toxin A and B Sat and CT and CDT containing toxins A and B in cultured enterocyte-like Caco-2 cells and colon-like T84 cells. Bacterial toxins, such as CT, LT, ST, CDTs, and the class 1 SPATE toxin Sat, against which AF and peptide AF-16 exert inhibitory activities, use different mechanisms of action to develop their deleterious activities in intestinal epithelial cells. By its enzymatically active A1 chain, CT induces the ADP ribosylation of the heterotrimeric G protein Gs␣, constitutively activating adenylate cyclase and thereby increasing the cell


5. 6.

7.

8.

9. 10. 11.

12.

13.

14.

15.

16. 17. 18.

ACKNOWLEDGMENTS This study was funded by Lantmännen AS-Faktor AB (Stockholm, Sweden) through a research contract with the University Paris-Sud (contract no. 10258A10). The funding provider had no role in the study design, collection or analysis of the data, or preparation of the manuscript.

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levels of cAMP; these increased levels of cAMP activate protein kinase A and, in turn, stimulate the action of a chloride ion efflux channel for the massive movement of water (33). The closely related LT from E. coli has the same mechanism of action as LT toxin inducing the ADP ribosylation of Gs␣, which activates adenylyl cyclase (91). ST from E. coli activates the intracellular catalytic domain of guanylyl cyclase, promoting the intracellular accumulation of cyclic GMP (cGMP), in turn activating a cGMP-dependent protein kinase II, which leads to the phosphorylation of the cystic fibrosis transmembrane regulator (91). The deleterious effects of CDTs result from the biologically active glucosyltransferase A domain of the toxins, which inactivates by glucosylation the rho GTPases essential for epithelial barrier functions (34). For the class 1 SPATE toxins, including Sat, a majority of the effects reported to be deleterious to cells resulted from their proteolytic activities (37, 38). It is therefore unlikely that the peptide AF-16 exerts its inhibitory activity by action at the level of interactions between the enzymatically active domains of these toxins and their cell molecular targets. The peptide AF-16 may be considered to have inhibitory activity at the level of intracellular traffic of these toxins. CT traffics to early and recycling endosomes and through the trans-Golgi network for delivery to the endoplasmic reticulum (ER), after which the CT enzymatic A1 chain is retrotranslocated to the cell cytosol by hijacking components of the ER-associated degradation pathway for misfolded proteins (33). The retrograde traffic of ST is unknown, and that of LT (92) resembles the CT retrograde traffic (33). The precise retrograde pathway of CDTs has not been elucidated but includes the translocation of the catalytic domains of the toxins from an early acidic endosomal compartment into the cell cytosol (34). Here and previously (42), results showed that Sat resides intracellularly in vesicles in intoxicated epithelial cells. The fate of these vesicles containing Sat is currently unknown. It is interesting to note that Pet of enteroaggregative E. coli, which displays several similarities with Sat (37, 38), moves after endocytosis from the cell surface to endosomes, the Golgi apparatus, and ER and is ultimately delivered back to the cell cytosol (93–95). Recent studies have identified potential therapeutic compounds and antibodies targeting the retrograde route in order to prevent the pathological manifestations that are triggered by a variety of plant (96–101) and bacterial (98–100, 102) toxins that use retrograde cell trafficking to reach their intracellular targets. Whether the peptide AF-16 exerts its inhibitory activity against bacterial toxins by the blockage of their cell retrograde transports remains to be elucidated in the future.


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Uptake of the antisecretory factor peptide AF-16 in rat blood and cerebrospinal fluid and effects on elevated intracranial pressure.

Transcellular sodium transport in cultured cystic fibrosis human nasal epithelium.

Neutrophil migration across a cultured epithelial monolayer elicits a biphasic resistance response representing sequential effects on transcellular and paracellular pathways.

Simultaneous quantification of intracellular and secreted active and inactive glucagon-like peptide-1 from cultured cells.

Lysine transport across rat jejunum: distribution between the transcellular and the paracellular routes.

Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke.

Characterization of insulin-like growth factor binding proteins secreted by cultured bovine theca and granulosa cells.

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The role of paracellular pathways in isotonic fluid transport.

Somatostatin inhibits the activity of adenylate cyclase in cultured human meningioma cells and stimulates their growth.

Atrial natriuretic peptide inhibits fluid intake in hyperosmolar subjects.

Synthesis of factor VIII antigen by cultured human endothelial cells.

Human amniotic epithelial cells cultured in substitute serum medium maintain their stem cell characteristics for up to four passages.

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Transforming growth factor beta 1 inhibits gonadotropin action in cultured porcine Sertoli cells.

Fibroblast growth factor inhibits 5 alpha-reductase activity in cultured immature Leydig cells.

Stem cells from human amniotic fluid exert immunoregulatory function via secreted indoleamine 2,3-dioxygenase1.

Inducible endothelin mRNA expression and peptide secretion in cultured human vascular smooth muscle cells.