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• W2129983297 abstract "Zonula occludens toxin (Zot) is an enterotoxin elaborated by Vibrio cholerae that increases intestinal permeability by interacting with a mammalian cell receptor with subsequent activation of intracellular signaling leading to the disassembly of the intercellular tight junctions. Zot localizes in the bacterial outer membrane of V. cholerae with subsequent cleavage and secretion of a carboxyl-terminal fragment in the host intestinal milieu. To identify the Zot domain(s) directly involved in the protein permeating effect, several zot gene deletion mutants were constructed and tested for their biological activity in the Ussing chamber assay and their ability to bind to the target receptor on intestinal epithelial cell cultures. The Zot biologically active domain was localized toward the carboxyl terminus of the protein and coincided with the predicted cleavage product generated by V. cholerae. This domain shared a putative receptor-binding motif with zonulin, the Zot mammalian analogue involved in tight junction modulation. Amino acid comparison between the Zot active fragment and zonulin, combined with site-directed mutagenesis experiments, confirmed the presence of an octapeptide receptor-binding domain toward the amino terminus of the processed Zot. Zonula occludens toxin (Zot) is an enterotoxin elaborated by Vibrio cholerae that increases intestinal permeability by interacting with a mammalian cell receptor with subsequent activation of intracellular signaling leading to the disassembly of the intercellular tight junctions. Zot localizes in the bacterial outer membrane of V. cholerae with subsequent cleavage and secretion of a carboxyl-terminal fragment in the host intestinal milieu. To identify the Zot domain(s) directly involved in the protein permeating effect, several zot gene deletion mutants were constructed and tested for their biological activity in the Ussing chamber assay and their ability to bind to the target receptor on intestinal epithelial cell cultures. The Zot biologically active domain was localized toward the carboxyl terminus of the protein and coincided with the predicted cleavage product generated by V. cholerae. This domain shared a putative receptor-binding motif with zonulin, the Zot mammalian analogue involved in tight junction modulation. Amino acid comparison between the Zot active fragment and zonulin, combined with site-directed mutagenesis experiments, confirmed the presence of an octapeptide receptor-binding domain toward the amino terminus of the processed Zot. zonula occludens toxin tight junction(s) phosphate-buffered saline tissue resistance amino acid Vibrio cholerae produces a variety of extracellular products including zonula occludens toxin (Zot)1 (1Fasano A. Baudry B. Pumplin D.W. Wasserman S.S. Tall B.D. Ketley J. Kaper J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5242-5246Crossref PubMed Scopus (448) Google Scholar). The zot gene, along with other genes encoding virulence factors such as ctxA, ctxB (2Gill D.M. Biochemistry. 1996; 15: 1242-1248Crossref Scopus (209) Google Scholar, 3Gill D.M. Meren M. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3050-3054Crossref PubMed Scopus (478) Google Scholar), and ace(4Trucksis M. Galen J.E. Michalski J. Fasano A. Kaper J.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5267-5271Crossref PubMed Scopus (215) Google Scholar), is part of the chromosomally integrated genome of a filamentous phage designated CTXΦ (5Waldor M.K. Mekalanos J.J. Science. 1996; 272: 1910-1914Crossref PubMed Scopus (1351) Google Scholar, 6Pearson G.D. Woods A. Chang S.L. Mekalanos J.J. Gene (Amst.). 1993; 67: 31-40Google Scholar, 7Taylor R.K. Miller V.L. Furlong D.B. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2833-2837Crossref PubMed Scopus (768) Google Scholar, 8Karaolis D.K.R. Samara S. Maneval Jr., D.R. Johnson J.A. Kaper J.B. Nature. 1999; 399: 375-379Crossref PubMed Scopus (310) Google Scholar, 9Koonin E.V. FEBS Lett. 1992; 321: 3-6Crossref Scopus (35) Google Scholar, 10Horabin J.I. Webster R.E. J. Mol. Biol. 1986; 188: 403-413Crossref PubMed Scopus (29) Google Scholar). The zot product seems to be involved in the CTXΦ morphogenesis because Zot mutagenesis studies demonstrated the inability of CTX elements to be self-transmissible under appropriate conditions (5Waldor M.K. Mekalanos J.J. Science. 1996; 272: 1910-1914Crossref PubMed Scopus (1351) Google Scholar). The high concurrence among V. cholerae strains of the zot gene and the ctxgenes (11Johnson J.A. Morris J.G. Kaper J.B. J. Clin. Microbiol. 1993; 31: 143-145Crossref Google Scholar, 12Karasawa T. Mihara T. Kurazono H. Nair G.B. Garg S. Ramamurthy J. Takeda Y. FEMS Microbiol. Lett. 1993; 106: 143-145Crossref PubMed Scopus (34) Google Scholar) also suggests a possible synergistic role of Zot in the causation of acute dehydrating diarrhea typical of cholera. The recently completed genomic sequence of V. choleare El Tor N16961 revealed that the CTXΦ filamentous phage is integrated in one of the two circular chromosomes of the bacterium (13Heidelberg J.F. Eisen J.A. Nelson W.C. Clayton R.A. Gwinn M.L. Nair G.B. Karasawa T. Mihara T. Takeda Y. Nature. 2000; 406: 477-484Crossref PubMed Scopus (1447) Google Scholar). Beside its role in phage morphogenesis, Zot also increases the permeability of the small intestine by affecting the structure of the intercellular tight junctions (tj) (1Fasano A. Baudry B. Pumplin D.W. Wasserman S.S. Tall B.D. Ketley J. Kaper J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5242-5246Crossref PubMed Scopus (448) Google Scholar). This effect was initially described on rabbit ileal tissues mounted in Ussing chambers by using filtered supernatants from V. cholerae O1 strains, suggesting that Zot is secreted (1Fasano A. Baudry B. Pumplin D.W. Wasserman S.S. Tall B.D. Ketley J. Kaper J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5242-5246Crossref PubMed Scopus (448) Google Scholar, 14Baudry B. Fasano A. Ketley J. Kaper J.B. Infect. Immun. 1992; 60: 428-434Crossref PubMed Google Scholar). Zot also possesses a cell specificity related to the toxin interaction with a specific receptor whose surface expression differs on various cells (15Fasano A. Fiorentini C. Donelli G. Uzzau S. Kaper J.B. Margaretten K. Ding X. Guandalini S. Comstock L. Goldblum S.E. J. Clin. Invest. 1995; 96: 710-720Crossref PubMed Scopus (300) Google Scholar, 16Fasano A. Uzzau S. Fiore C. Margaretten K. Gastroenterology. 1997; 112: 839-846Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17Uzzau S. Lu R. Wang W. Fiore C. Fasano A. FEMS Microbiol. Lett. 2001; 194: 1-5Crossref PubMed Google Scholar). Zot induces modifications of cytoskeletal organization that lead to the opening of tj secondary to the transmembrane phospholipase C and subsequent protein kinase Cα-dependent polymerization of actin filaments strategically localized to regulate the paracellular pathway (15Fasano A. Fiorentini C. Donelli G. Uzzau S. Kaper J.B. Margaretten K. Ding X. Guandalini S. Comstock L. Goldblum S.E. J. Clin. Invest. 1995; 96: 710-720Crossref PubMed Scopus (300) Google Scholar). Furthermore, in vivo experiments suggested that the effect of Zot on tj might lead to intestinal secretion after the permeation of the intercellular space (16Fasano A. Uzzau S. Fiore C. Margaretten K. Gastroenterology. 1997; 112: 839-846Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). This modulation is reversible, time- and dose-dependent, and confined to the small intestine because Zot does not affect colon permeability (1Fasano A. Baudry B. Pumplin D.W. Wasserman S.S. Tall B.D. Ketley J. Kaper J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5242-5246Crossref PubMed Scopus (448) Google Scholar, 16Fasano A. Uzzau S. Fiore C. Margaretten K. Gastroenterology. 1997; 112: 839-846Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Furthermore, the number of Zot receptors seems to decrease along the intestinal villous axis (16Fasano A. Uzzau S. Fiore C. Margaretten K. Gastroenterology. 1997; 112: 839-846Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). To clarify the Zot bifunctional activity, we analyzed the structure-function properties of the toxin by constructing a series of deletion mutants that were tested for their ability to both modulate tj and bind to the Zot/zonulin receptor (18Lu R. Wang W. Uzzau S. Vigorito R. Zielke H.R. Fasano A. J. Neurochem. 2000; 74: 320-326Crossref PubMed Scopus (55) Google Scholar). The results provide evidence that the active domain responsible for Zot enterotoxic activity resides toward the carboxyl-terminal region of the toxin. In-frame deletion derivatives of the zot gene were obtained by using polymerase chain reaction techniques (TableI). To develop deletions of the Zot carboxyl or amino terminus, several oligonucleotides were designed to amplify various portions of the 5′ and 3′ ends of the zotgene (Table II). The zot gene was cloned in plasmid vector pQE30 (Qiagen, Inc., Valencia, CA), which provides high-level expression in Escherichia coli of proteins containing a 6-histidine (6xHis) affinity tag at their amino terminus. The His tag allows a one-step method for protein purification using the Nickel-nitrilotriacetic acid resin capture column. To obtain Zot internal in-frame deletions (ΔE and ΔC clones), pSU113 (19Uzzau S. Cappuccinelli P. Fasano A. Microb. Pathog. 1999; 27: 377-385Crossref PubMed Scopus (48) Google Scholar) was subjected to enzymatic digestion (StuI andHindIII). The right end of Zot was replaced with polymerase chain reaction products obtained by using F7/F2 and F11/F2 primers pairs, respectively, that were subsequently re-ligated. Oligonucleotides were designed to introduce restriction sites needed for cloning procedures (Table II). Amplification products were analyzed by agarose gel electrophoresis and purified from salts and free nucleotides (Qiaquick polymerase chain reaction purification kit; Qiagen, Inc.). The fidelity of polymerase chain reaction amplifications was confirmed by DNA sequencing of the plasmid inserts (ABI PRISM 373; Applied Biosystems, Foster City, CA).Table IBacterial strains and plasmid vectors (top) and schematic description of Zot deletion mutants (bottom) Open table in a new tab Table IIPrimer sequences* Sequences with restriction sites (underlined) of F2, F8 (HindIII), S2, F71, F28, F205, F141 (BamHI), F46, F92 (KpnI). Sequences corresponding to zot sequences are initalics. Open table in a new tab * Sequences with restriction sites (underlined) of F2, F8 (HindIII), S2, F71, F28, F205, F141 (BamHI), F46, F92 (KpnI). Sequences corresponding to zot sequences are initalics. The QuickChangeTMSite-directed Mutagenesis Kit (Stratagene, Kingsport, TN) was used to develop point mutations in pSU129 hosted in the DH5α/His-ΔG strain. These mutations resulted in the substitution of either the glycine (G) in position 291 with a valine (V) (DH5α/His-ΔG291) or the glycine in position 298 with a valine (DH5α/His-ΔG298). The oligonucleotides used to obtain the two site-directed derivatives are listed in Table II. Thezot gene, its seven deletion mutants, and its point mutated constructs were each inserted into the pQE30 vector to add a 6xHis tag on the amino terminus of each protein. E. coliDH5α was then transformed with the plasmids listed in Table I. The clones obtained were grown in Luria Bertani LB medium with 20 g/liter glucose, 25 mg/liter kanamycin, and 200 mg/liter ampicillin at 37 °C with vigorous mixing until A 600 reached 0.7–0.9. Cultures were then induced with 2 mmisopropyl-1-thio-β-d-galactopyranoside (Fisher), followed by an additional 2-h culture incubation at 37 °C with vigorous shaking. The cells were harvested by centrifugation at 4,000 × g for 20 min and resuspended in buffer A (6m guanidine-HCl, 0.1 m sodium phosphate, and 0.01 m Tris-HCl, pH 8.0; 5 ml/g wet weight). After stirring for 1 h at room temperature, the mixture was centrifuged at 10,000 × g for 30 min at 4 °C. A 50% slurry of Superflow (Qiagen, Inc.; 1 ml/g wet weight) was added to the supernatant and stirred for 1 h at room temperature. The mixture was loaded onto a 5 × 1.5-cm nickel-nitrilotriacetic acid resin column and washed sequentially with buffer A and buffer B (8m urea, 0.1 m sodium phosphate, and 0.01m Tris-HCl, pH 8). Each wash step was continued until theA 280 of the flow-through was less than 0.01. The proteins that bound to the column were eluted by the addition of 250 mm imidazole in buffer C (8 m urea, 0.1m sodium phosphate, and 0.01 m Tris-HCl, pH 6.3), stirred with a 50% slurry of Superflow (1 ml/g wet weight) for 2 h at room temperature, loaded onto another 5 × 1.5-cm nickel-nitrilotriacetic acid column, washed with phosphate-buffered saline (PBS), and eluted with 250 mm imidazole in PBS. Purity of the His-proteins was established by SDS-polyacrylamide gel electrophoresis analysis followed by Coomassie Blue staining and Western immunoblotting using polyclonal anti-Zot antibodies (19Uzzau S. Cappuccinelli P. Fasano A. Microb. Pathog. 1999; 27: 377-385Crossref PubMed Scopus (48) Google Scholar). Adult male New Zealand White rabbits (2–3 kg) were sacrificed by cervical dislocation. Segments of rabbit small intestine were removed, rinsed free of the intestinal content, opened along the mesenteric border, and stripped of muscular and serosal layers. Eight sheets of mucosa thus prepared were then mounted in Lucite Ussing Chambers (1.12 cm2 opening) connected to a voltage clamp apparatus (EVC 4000; World Precision Instruments, Sarasota, FL) and bathed with freshly prepared buffer containing 53 mm NaCl, 5 mm KCl, 30.5 mm Na2SO4, 30.5 mmmannitol, 1.69 mm Na2HPO4, 0.3 mm NaH2PO4, 1.25 mmCaCl2, 1.1 mm MgCl2, and 25 mm NaHCO3. The bathing solution was maintained at 37 °C with water jacketed reservoirs connected to a constant temperature circulating pump and gassed with 95% O2/5% CO2. Potential difference (PD) was measured, and short-circuit current (ISC) and tissue resistance (Rt) were calculated as described previously (1Fasano A. Baudry B. Pumplin D.W. Wasserman S.S. Tall B.D. Ketley J. Kaper J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5242-5246Crossref PubMed Scopus (448) Google Scholar). Purified Zot (1 × 10−10m) and the seven purified Zot deletion mutant proteins (1 × 10−10m) were added to the mucosal side of each chamber. Intestinal tissues exposed to PBS were used as a negative control. Only tissues that, at the end of the experiment, showed an increase in ISC in response to the mucosal addition of glucose (confirming tissue viability) were included in the analysis. In selected experiments, tissues were exposed to the point mutation proteins (1 × 10−10m) derived from the ΔG clone (Table I), and the assay was conducted as described above. IEC6 cells derived from crypt cells of germ-free rat small intestine (20Quaroni A. May R.J. Meth. Cell Biol. 1980; 21: 403-427Crossref Scopus (140) Google Scholar) were grown in cell culture flasks (Corning Costar Co., Cambridge, MA) at 37 °C in an atmosphere of 95% air and 5% CO2. The complete medium consisted of Dulbecco's modified Eagle's medium with 4.5 g/liter glucose, containing 5% irradiated fetal bovine serum, 10 μg/ml insulin, 4 mml-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. The cells passage number varied between 15 and 20. IEC6 cells were grown in 8-chamber slides (1 × 105 cells/chamber). The monolayers were exposed to either 1 × 10−10m Zot or an equimolar amount of each of its deletion mutants and incubated for 30 min at 4 °C. IEC6 cells exposed to PBS were used as a negative control. After washing with PBS (pH 7.4), the cells were fixed in 4% formaldehyde for 10 min at room temperature and then permeabilized with 0.5% Triton X-100 (Sigma) in phosphate buffer (pH 7.4) for 10 min at room temperature. The cells were then washed with PBS and blocked with 0.1% bovine serum albumin for 45 min at room temperature. Primary rabbit polyclonal anti-Zot antibodies (1:500) (19Uzzau S. Cappuccinelli P. Fasano A. Microb. Pathog. 1999; 27: 377-385Crossref PubMed Scopus (48) Google Scholar) were then added, and the monolayers were incubated overnight at 4 °C. After washing with PBS, the cells were incubated for 30 min at room temperature with anti-rabbit IgG-fluorescein isothiocyanate-conjugated antibodies (Sigma) (1:100). Finally, the cells were washed with PBS and stained in red using Evans Blue Counterstain (Sigma) in phosphate buffer (1:1000) for 10 min at room temperature. The staining procedure was used to better visualize the binding particles (stained in green by the IgG-fluorescein isothiocyanate secondary antibodies) that appeared on a red background as fine yellow granules. The wells were washed with PBS, the coverslips were mounted with glycerol-PBS (1:1), pH 8, and then the cells were blindly analyzed by two independent observers with a fluorescence microscope (Optiplot; Nikon Inc., Melville, NY). In selected experiments, the IEC6 cells were incubated with the point mutation proteins (1 × 10−10m) derived from the ΔG clone listed in Table I, and the binding assay was conducted as described above. All values are the means ± S.E. The analysis of differences was performed by t test for either paired or unpaired varieties. p < 0.05 was considered statistically significant. To identify the Zot region(s) involved in both the toxin's permeating effect and its engagement to the target receptor, seven deletion mutants were generated. Each mutant showed the predicted M ras established by both SDS-polyacrylamide gel electrophoresis analysis (Fig. 1 A) and Western immunoblotting (Fig. 1 B). Gene sequencing of these constructs confirmed their correct design (data not shown). To identify the Zot domain(s) responsible for the enterotoxic activity, equimolar amounts of bacterial-expressed, purified His-Zot and its deletion mutants were each tested on rabbit small intestine mounted in Ussing chambers. The ability of the seven Zot mutants to affect the tj competency was analyzed by measuring the changes in Rt induced by a 90-min incubation with each deleted construct as compared with the effect obtained with wild-type His-Zot (positive control) and PBS (negative control). As shown in Fig.2 A, Rt reduction induced by His-ΔB and His-ΔC proteins was almost indistinguishable from that induced by His-Zot and was significantly different compared with the negative control. A significant Rt drop was also observed when tissues were exposed to His-ΔG, whereas no significant changes were detected after tissue incubation with His-ΔD, His-ΔE, His-ΔF, and His-ΔH (Fig. 2 A). These results suggest that the domain responsible for the Zot permeating effect on tj is located toward the protein's carboxyl-terminal section (Fig. 2 B). To identify the Zot domain(s) that specifically binds to the Zot/zonulin surface cell receptor, IEC6 cell monolayers were exposed to the same purified proteins tested in Ussing chambers. The ability of each deleted Zot derivative to bind to IEC6 cells (Fig.3, B–H) was evaluated using fluorescence microscopy and compared with the binding observed with both wild-type His-Zot (Fig. 3 A) and the PBS negative control (Fig. 3 I). Cells incubated with His-ΔG (Fig.3 G) showed an amount of binding particles similar to that seen in cells incubated with His-Zot (Fig. 3 A). The number of fluorescent particles was only minimally decreased in cells incubated with His-ΔB (Fig. 3 B), His-ΔC (Fig.3 C), or His-ΔD (Fig. 3 D). In contrast, binding capability was completely lost by His-ΔE (Fig. 3 E), His-ΔF (Fig. 3 F), and His-ΔH (Fig. 3 H) mutants. Analysis of these results revealed that the Zot region corresponding to AA residues 265–301 was partially (His-ΔE and His-ΔH) or completely (His-ΔF) deleted in the binding-negative mutants, whereas it was spared in the binding-positive constructs (Fig.3 J), suggesting that this region may represent the putative Zot domain involved in receptor binding function. We have recently demonstrated that Zot localizes in the V. cholerae outer membrane, to which it is anchored through its single spanning domain (19Uzzau S. Cappuccinelli P. Fasano A. Microb. Pathog. 1999; 27: 377-385Crossref PubMed Scopus (48) Google Scholar). The molecule then undergoes to a cleavage that leads to the formation of a ∼33-kDa amino-terminal fragment that remains associated to the microorganism and a ≥12-kDa carboxyl-terminal peptide that supposedly is secreted in the host intestinal lumen milieu (19Uzzau S. Cappuccinelli P. Fasano A. Microb. Pathog. 1999; 27: 377-385Crossref PubMed Scopus (48) Google Scholar). Based on the Ussing chamber assay results and the binding experiments of the Zot deletion mutants reported above, it is conceivable to hypothesize that this secreted fragment engages to the zonulin receptor and, consequently, causes tj disassembly. Comparison of the amino termini of the secreted Zot fragment (AA residues 288–399) and its eukaryotic analogue zonulin (21Wang W. Uzzau S. Goldblum S.E. Fasano A. J. Cell Sci. 2000; 113: 4435-4440PubMed Google Scholar) revealed an 8- amino acid shared motif (Table III, shaded area) that encompasses the 265–301-AA region identified by the binding experiments described above as the putative binding domain.Table IIIComparison of the amino-terminal sequences of human intestinal zonulin and ZotThe shaded amino acids (Zot AA residues 291–298, zonulin AA residues 8–15) represent the putative Zot/zonulins receptor-binding site characterized by the following shared motif: non-polar (G), variable, non-polar, variable, non-polar (V), polar (Q), variable, non-polar (G). Open table in a new tab The shaded amino acids (Zot AA residues 291–298, zonulin AA residues 8–15) represent the putative Zot/zonulins receptor-binding site characterized by the following shared motif: non-polar (G), variable, non-polar, variable, non-polar (V), polar (Q), variable, non-polar (G). Both zonulin and Zot domains revealed a motif in which 4 of the 8 amino acid residues were identical (GXXXVQXG). To confirm that this motif is involved in target receptor engagement, a synthetic octapeptide (GGVLVQPG containing the shared motif) named FZI/0 (see Table III) was engineered and tested on ileal tissue mounted in Ussing chambers either alone or in combination with Zot or zonulin. No changes in Rt in tissues exposed to either FZI/0 or to a scrambled peptide were observed (Fig. 4). Treatment of the ileal tissue preparations with FZI/0 before and throughout the study period prevented Rt changes in response to both Zot and zonulin, whereas the permeating effect of the two proteins was unaffected by pretreatment with the scrambled peptide (Fig. 4). These data suggest that Zot and zonulin bind to the same receptor through a common binding motif (shaded in Table III) localized at the amino termini of both molecules. To establish the structure requirements to engage the target zonulin receptor, two His-ΔG site-directed mutants within the putative binding motif were engineered and tested for both their binding capability on IEC6 cells and their biological activity in Ussing chambers. IEC6 cells incubated with His-ΔG 291 (in which the G in position 291 was substituted with V) showed a reduced number of binding particles (Fig.5 A, 3) as compared with cells incubated with His-ΔG (Fig. 5 A, 2), whereas no binding was observed on cells incubated with His-ΔG 298 (G298V) mutant (Fig.5 A, 4). Biological assays in Ussing chambers showed that His-ΔG291 had a residual but not significant effect on tj disassembly (Fig. 5 B). The permeating effect was completely ablated when the intestinal tissues were incubated with His-ΔG 298 (Fig.5 B). These results paralleled the effects obtained with these two mutants in the binding assay and confirmed that the G in position 298 plays a crucial role in the capability of His-ΔG to bind to its target receptor, and, consequently, its substitution ameliorated the ligand biological effect on tj.Figure 5Effect of the two His-ΔG site-directed mutants (His-ΔG291 and His-ΔG298) on receptor binding and tj disassembly. A, binding assay. Experiments were performed as described in the Fig. 3 legend. IEC6 cell monolayers incubated with His-ΔG291 showed binding particles (3) similar to those detected in His-ΔG-exposed monolayers (2), whereas no binding was detected in monolayers exposed to His-ΔG298 (4). Negative PBS control (1) is shown for comparison. B, Ussing chamber assay. The tissue was treated with 1 × 10−10m of each protein, and Rt change was compared with that in PBS-exposed tissues. His-ΔG291 induced a partial but not a significant reduction in Rt when compared with the changes induced by His-ΔG. The permeating effect was completely ablated when the rabbit ileum was exposed to His-ΔG298. All values were expressed as the means ± S.E. ∗,p < 0.009 compared with His-ΔG298 andp < 0.0001 as compared with control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) V. cholerae, the human intestinal pathogen responsible for the diarrheal disease cholera, elaborates a large number of extracellular proteins, including several virulence factors. A number of epidemiological studies (22Colombo M.M. Mastrandrea S. Santona A. de Andrade A.P. Uzzau S. Rubino S. Cappuccinelli P. J. Infect. Dis. 1994; 170: 750-751Crossref PubMed Scopus (33) Google Scholar, 23Kurazono H. Pal A. Bag P.K. Dodson R.J. Haft D.H. Hickey E.K. Peterson J.D. Umayam L. Gill S.R. Nelson K.E. Read T.D. Tettelin H. Richardson D. Ermolaeva M.D. Vamathevan J. Bass S. Qin H. Draqoi I. Sellers P. McDonald L. Utterback T. Fleishmann R.D. Nierman U.C. White D. Microb. Pathog. 1995; 18: 231-235Crossref PubMed Scopus (49) Google Scholar) have shown a concurrent occurrence of the CT genes (ctx A and ctx B) and the genes for two other virulence factors elaborated by V. cholerae, Zot (1Fasano A. Baudry B. Pumplin D.W. Wasserman S.S. Tall B.D. Ketley J. Kaper J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5242-5246Crossref PubMed Scopus (448) Google Scholar) and accessory cholera toxin (Ace) (4Trucksis M. Galen J.E. Michalski J. Fasano A. Kaper J.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5267-5271Crossref PubMed Scopus (215) Google Scholar). This cluster of genes has been recently described as part of a filamentous phage (CTXΦ) that lives in symbiosis with its bacterial host. It has been reported previously that Zot is involved in both CTXΦ morphogenesis (5Waldor M.K. Mekalanos J.J. Science. 1996; 272: 1910-1914Crossref PubMed Scopus (1351) Google Scholar) and disassembly of the host intestinal intercellular tj (1Fasano A. Baudry B. Pumplin D.W. Wasserman S.S. Tall B.D. Ketley J. Kaper J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5242-5246Crossref PubMed Scopus (448) Google Scholar, 14Baudry B. Fasano A. Ketley J. Kaper J.B. Infect. Immun. 1992; 60: 428-434Crossref PubMed Google Scholar, 15Fasano A. Fiorentini C. Donelli G. Uzzau S. Kaper J.B. Margaretten K. Ding X. Guandalini S. Comstock L. Goldblum S.E. J. Clin. Invest. 1995; 96: 710-720Crossref PubMed Scopus (300) Google Scholar, 16Fasano A. Uzzau S. Fiore C. Margaretten K. Gastroenterology. 1997; 112: 839-846Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The study of the subcellular localization of Zot by affinity-purified anti-Zot antibodies (19Uzzau S. Cappuccinelli P. Fasano A. Microb. Pathog. 1999; 27: 377-385Crossref PubMed Scopus (48) Google Scholar) revealed that Zot localizes in the V. cholerae outer membrane with a molecular mass of ∼45 kDa, which is consistent with the predicted primary translation product from the first methionine of Zot (44.8 kDa) (19Uzzau S. Cappuccinelli P. Fasano A. Microb. Pathog. 1999; 27: 377-385Crossref PubMed Scopus (48) Google Scholar). A second immunoreactive molecule, corresponding to the 33-kDa amino-terminal region of Zot, was also detected at the outer membrane site (19Uzzau S. Cappuccinelli P. Fasano A. Microb. Pathog. 1999; 27: 377-385Crossref PubMed Scopus (48) Google Scholar). Both molecules are exposed at the bacterial cell surface (19Uzzau S. Cappuccinelli P. Fasano A. Microb. Pathog. 1999; 27: 377-385Crossref PubMed Scopus (48) Google Scholar). The 33-kDa Zot processing product is generated by a cleavage site at AA residue 287 (19Uzzau S. Cappuccinelli P. Fasano A. Microb. Pathog. 1999; 27: 377-385Crossref PubMed Scopus (48) Google Scholar). This 33-kDa fragment remains associated to the bacterial membrane, whereas the carboxyl-terminal fragment of 12 kDa is excreted in the intestinal host milieu and is probably responsible for the biological effect of the toxin on intestinal tj. This toxin processing would explain the apparently contrasting results obtained when Zot was originally described (1Fasano A. Baudry B. Pumplin D.W. Wasserman S.S. Tall B.D. Ketley J. Kaper J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5242-5246Crossref PubMed Scopus (448) Google Scholar). Indeed, despite the predicted toxin molecular mass (44.8 kDa) (14Baudry B. Fasano A. Ketley J. Kaper J.B. Infect. Immun. 1992; 60: 428-434Crossref PubMed Google Scholar), its biological effect on tj was found confined to the <30-kDa V. cholerae culture supernatant fraction (1Fasano A. Baudry B. Pumplin D.W. Wasserman S.S. Tall B.D. Ketley J. Kaper J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5242-5246Crossref PubMed Scopus (448) Google Scholar). The importance of the carboxyl-terminal fragment of Zot for its action on tj is further supported by the observation that TnphoA insertions located at the Zot carboxyl-terminal region abolished the enterotoxic activity (14Baudry B. Fasano A. Ketley J. Kaper J.B. Infect. Immun. 1992; 60: 428-434Crossref PubMed Google Scholar). Taken together, these data suggest that Zot has a dual function: whereas its ∼33-kDa amino-terminal portion is possibly involved in CTXΦ phage assembly (5Waldor M.K. Mekalanos J.J. Science. 1996; 272: 1910-1914Crossref PubMed Scopus (1351) Google Scholar, 9Koonin E.V. FEBS Lett. 1992; 321: 3-6Crossref Scopus (35) Google Scholar, 10Horabin J.I. Webster R.E. J. Mol. Biol. 1986; 188: 403-413Crossref PubMed Scopus (29) Google Scholar), the ∼12-kDa carboxyl-terminal fragment of the toxin seems to be responsible for the permeating action on intestinal tj. Several microorganisms have been shown to exert a cytophatic, pathological effect on epithelial cells that involves the cytoskeletal structure and the tj function in an irreversible manner. These bacteria alter the intestinal permeability either directly (i.e.enteropathogenic E. coli) or through the elaboration of toxins (i.e. Clostridium difficile andBacteroides fragilis) (24Sears C.L. Guerrant R.L. Kaper J.B. Blaser M.J. Smith P.D. Ravdin J.I. Greenberg H.B. Guerrant R.L. Infection of the Gastrointestinal Tract. Raven Press, New York1995: 617-634Google Scholar). A more physiological mechanism of regulation of tj permeability has been proposed for Zot (1Fasano A. Baudry B. Pumplin D.W. Wasserman S.S. Tall B.D. Ketley J. Kaper J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5242-5246Crossref PubMed Scopus (448) Google Scholar). Zot activates a complex intracellular cascade of events that involve a dose- and time-dependent protein kinase Cα-related polymerization of actin filaments strategically localized to regulate the paracellular pathway (15Fasano A. Fiorentini C. Donelli G. Uzzau S. Kaper J.B. Margaretten K. Ding X. Guandalini S. Comstock L. Goldblum S.E. J. Clin. Invest. 1995; 96: 710-720Crossref PubMed Scopus (300) Google Scholar). These changes are a prerequisite to the opening of tj and are evident at a toxin concentration as low as 1.1 × 10−13m (16Fasano A. Uzzau S. Fiore C. Margaretten K. Gastroenterology. 1997; 112: 839-846Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The toxin exerts its effect by interacting with a specific surface receptor that is present on mature cells of small intestinal villi, but not in the colon (1Fasano A. Baudry B. Pumplin D.W. Wasserman S.S. Tall B.D. Ketley J. Kaper J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5242-5246Crossref PubMed Scopus (448) Google Scholar, 16Fasano A. Uzzau S. Fiore C. Margaretten K. Gastroenterology. 1997; 112: 839-846Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The regional distribution of Zot receptor(s) coincides with the different permeating effect of the toxin on the various tracts of intestine tested (16Fasano A. Uzzau S. Fiore C. Margaretten K. Gastroenterology. 1997; 112: 839-846Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Our previous results showed that Zot regulates tj in a rapid, reversible, and reproducible fashion and probably activates intracellular signals operative during the physiologic modulation of the paracellular pathway (15Fasano A. Fiorentini C. Donelli G. Uzzau S. Kaper J.B. Margaretten K. Ding X. Guandalini S. Comstock L. Goldblum S.E. J. Clin. Invest. 1995; 96: 710-720Crossref PubMed Scopus (300) Google Scholar, 16Fasano A. Uzzau S. Fiore C. Margaretten K. Gastroenterology. 1997; 112: 839-846Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Based on the complexity of the intracellular signaling activated by Zot leading to the tj disassembly (15Fasano A. Fiorentini C. Donelli G. Uzzau S. Kaper J.B. Margaretten K. Ding X. Guandalini S. Comstock L. Goldblum S.E. J. Clin. Invest. 1995; 96: 710-720Crossref PubMed Scopus (300) Google Scholar), we postulated that Zot may mimic the effect of a functionally and immunologically related endogenous modulator of epithelial tj. The combination of affinity-purified anti-Zot antibodies and the Ussing chamber assay allowed us to identify an intestinal Zot analogue that we named zonulin (21Wang W. Uzzau S. Goldblum S.E. Fasano A. J. Cell Sci. 2000; 113: 4435-4440PubMed Google Scholar). When zonulin was studied in a nonhuman primate model, it reversibly opened intestinal tj after engagement to the same receptor activated by Zot and therefore acts with the same effector mechanism described for the toxin (21Wang W. Uzzau S. Goldblum S.E. Fasano A. J. Cell Sci. 2000; 113: 4435-4440PubMed Google Scholar). Structure analysis of the zonulin amino terminus and the Zot active fragment identified in this study revealed a shared motif between these two molecules (Table III). Our binding experiments with Zot deletion mutants demonstrated that this motif is crucial for the engagement of the toxin to its target receptor. The importance of this region was confirmed by the ablation of both Zot and zonulin binding and the biological effects on tj competency when intestinal tissues were pretreated with the synthetic octapeptide GGVLVQPG corresponding to the shared motif (Fig. 4). Site-directed mutagenesis revealed the key role of the glycine residue in position 298 (as referred to the Zot entire molecule) for Zot engagement to the target zonulin receptor and thus for the activation of intracellular signaling leading to the opening of intercellular tj. The paracellular route is the dominant pathway through which passive solutes flux across both the endothelial and epithelial barriers, and its functional status is regulated, in part, at the level of intercellular tj (25Pappo J. Ermak T.H. Clin. Exp. Immunol. 1996; 189: 487-490Google Scholar). A century ago, the tj was conceptualized as secreted extracellular cement forming an absolute and inert barrier within the paracellular space (26Coreijido M. Coreijado G. Tight Junctions. CRC Press, Boca Raton, FL1992: 1-13Google Scholar). It is now understood that tj are complex and dynamic structures whose physiological regulation appears to be tightly orchestrated through mechanisms that remain largely undefined (27Anderson J.M. Bolda M.S. Fanning A.S. J. Cell Biol. 1993; 5: 772-778Google Scholar). Furthermore, tj readily adapt to a variety of developmental, physiological, and pathological circumstances (28Milks L.C. Conyers G.P. Cramen E.B. J. Cell Biol. 1986; 103: 2729-2738Crossref PubMed Scopus (55) Google Scholar, 29Shasby D.M. Winter M. Shasby S.S. Am. J. Physiol. 1988; 255: C781-C788Crossref PubMed Google Scholar). Data exist that support the linkage between the actin cytoskeleton and the tj complex (30Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2129) Google Scholar, 31Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar, 32Wittchen E.S. Haskins J. Stevenson B.R. J. Biol. Chem. 1999; 274: 35179-35185Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar) and implicate signaling events that regulate paracellular permeability (27Anderson J.M. Bolda M.S. Fanning A.S. J. Cell Biol. 1993; 5: 772-778Google Scholar). The discovery of Zot shed some light on the intricate mechanisms that govern the permeability of tj and led to the discovery of zonulin, the natural ligand of the Zot target receptor. The partial characterization of this zonulin receptor revealed that it is a 45-kDa glycoprotein containing multiple sialic acid residues with structural similarities to myeloid-related protein, a member of the calcium-binding protein family possibly linked to cytoskeletal rearrangements (18Lu R. Wang W. Uzzau S. Vigorito R. Zielke H.R. Fasano A. J. Neurochem. 2000; 74: 320-326Crossref PubMed Scopus (55) Google Scholar). With the studies presented here, we have untangled at the molecular level the interplay between the V. cholerae Zot toxin and the eukaryotic zonulin pathway used by the microorganism to induce tj disassembly. Our findings on the structural requirements to engage to the zonulin receptor binding pocket and, consequently, to activate the zonulin pathway open new research opportunities to gain more insight on a system possibly involved in developmental, physiological, and pathological processes, including tissue morphogenesis, movement of fluid, macromolecules, and leukocytes between body compartments, and malignant transformation and metastasis." @default.
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