Zonula Occludens Toxin Structure-Function Analysis
IDENTIFICATION OF THE FRAGMENT BIOLOGICALLY ACTIVE ON TIGHT
JUNCTIONS AND OF THE ZONULIN RECEPTOR BINDING DOMAIN*
Mariarosaria
Di Pierro
§,
Ruliang
Lu
,
Sergio
Uzzau¶,
Wenle
Wang
,
Klara
Margaretten
,
Carlo
Pazzani§,
Francesco
Maimone§
, and
Alessio
Fasano
**
From the
Division of Pediatric Gastroenterology and
Nutrition and Gastrointestinal Pathophysiology Section, Center for
Vaccine Development, ** Department of Physiology, University of Maryland
School of Medicine, Baltimore, Maryland 21201, ¶ Dipartimento di
Scienze Biomediche, Sezione di Microbiologia Sperimentale e Clinica,
Università di Sassari, Sassari, Italy 07100,
Centro
Interuniversitario di Ricerca sui Paesi in via di Sviluppo,
Università "La Sapienza," Rome, Italy 07100, and
§ Dipartimento di Anatomia Patologica e di Genetica,
Univerisità degli Studi, Bari, Italy 70126
Received for publication, October 23, 2000, and in revised form, January 29, 2001
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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.
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INTRODUCTION |
Vibrio cholerae produces a variety of extracellular
products including zonula occludens toxin
(Zot)1 (1). The
zot gene, along with other genes encoding virulence factors
such as ctxA, ctxB (2, 3), and ace
(4), is part of the chromosomally integrated genome of a filamentous
phage designated CTX
(5-10). The zot product seems to be
involved in the CTX
morphogenesis because Zot mutagenesis studies
demonstrated the inability of CTX elements to be self-transmissible
under appropriate conditions (5). The high concurrence among V. cholerae strains of the zot gene and the ctx
genes (11, 12) 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 CTX
filamentous phage is integrated in one
of the two circular chromosomes of the bacterium (13).
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) (1). 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 (1, 14). Zot also possesses a cell
specificity related to the toxin interaction with a specific receptor
whose surface expression differs on various cells (15-17). Zot induces
modifications of cytoskeletal organization that lead to the opening of
tj secondary to the transmembrane phospholipase C and subsequent
protein kinase C
-dependent polymerization of actin
filaments strategically localized to regulate the paracellular pathway
(15). Furthermore, in vivo experiments suggested that the
effect of Zot on tj might lead to intestinal secretion after the
permeation of the intercellular space (16). This modulation is
reversible, time- and dose-dependent, and confined to the
small intestine because Zot does not affect colon permeability (1, 16).
Furthermore, the number of Zot receptors seems to decrease along the
intestinal villous axis (16).
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 (18). The results provide evidence
that the active domain responsible for Zot enterotoxic activity resides
toward the carboxyl-terminal region of the toxin.
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EXPERIMENTAL PROCEDURES |
Construction of His-Zot In-frame Deletion Mutants--
In-frame
deletion derivatives of the zot gene were obtained by using
polymerase chain reaction techniques (Table
I). 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 zot
gene (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 (
E and
C clones), pSU113
(19) was subjected to enzymatic digestion (StuI and
HindIII). 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).
Site-directed Mutagenesis--
The QuickChangeTM
Site-directed Mutagenesis Kit (Stratagene, Kingsport, TN) was used to
develop point mutations in pSU129 hosted in the DH5
/His-
G strain.
These mutations resulted in the substitution of either the glycine (G)
in position 291 with a valine (V) (DH5
/His-
G291) or the glycine
in position 298 with a valine (DH5
/His-
G298). The
oligonucleotides used to obtain the two site-directed derivatives are
listed in Table II.
Purification of His-Zot and Its Deletion Mutants--
The
zot 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. coli
DH5
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 A600 reached
0.7-0.9. Cultures were then induced with 2 mM
isopropyl-1-thio-
-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 (6 M 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 (8 M urea, 0.1 M sodium phosphate, and 0.01 M Tris-HCl, pH 8). Each wash step was continued until the
A280 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, 0.1 M 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 (19).
In Vitro Ussing Chambers Experiments--
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 mM mannitol, 1.69 mM Na2HPO4, 0.3 mM NaH2PO4, 1.25 mM
CaCl2, 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 (1). Purified Zot (1 × 10
10 M) and the seven purified
Zot deletion mutant proteins (1 × 10
10
M) 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
10 M)
derived from the
G clone (Table I), and the assay was conducted as
described above.
Cell Cultures--
IEC6 cells derived from crypt cells of
germ-free rat small intestine (20) 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 mM L-glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin. The cells passage number varied between 15 and 20.
Binding Experiments--
IEC6 cells were grown in 8-chamber
slides (1 × 105 cells/chamber). The monolayers were
exposed to either 1 × 10
10
M 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) (19)
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
10 M)
derived from the
G clone listed in Table I, and the binding assay
was conducted as described above.
Statistical Analysis--
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.
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RESULTS |
Construction of Zot Deletion Mutants--
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 Mr
as established by both SDS-polyacrylamide gel electrophoresis analysis
(Fig. 1A) and Western
immunoblotting (Fig. 1B). Gene sequencing of these
constructs confirmed their correct design (data not shown).

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Fig. 1.
SDS-polyacrylamide gel electrophoresis
(A) and Western immunoblotting (B) of
Zot and its in-frame deletion mutants. Lane 0, molecular mass standards; lane 1, His-Zot;
lane 2, His- B (1-98); lane 3, His- C
(118); lane 4, His- D (301); lane 5, His- E (118); lane 6, His- F (222); lane
7, His- G (1); lane 8, His- H (1)
(deleted amino acid residues are shown in parentheses).
Conditions were as described under "Experimental Procedures."
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Biological Activity of the Zot Mutants in the ex Vivo Ussing
Chamber Assay--
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.
2A, Rt reduction induced by
His-
B and His-
C 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-
G, whereas no significant changes were detected
after tissue incubation with His-
D, His-
E, His-
F, and His-
H
(Fig. 2A). These results suggest that the domain responsible
for the Zot permeating effect on tj is located toward the protein's
carboxyl-terminal section (Fig. 2B).

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Fig. 2.
A, effect of Zot and its deletion mutant
derivatives on rabbit ileal Rt in Ussing chambers. Tissues were exposed
to either 1 × 10 10 M of
each protein or to a negative control (PBS), and variation in Rt
between the baseline ( ) and 90 min postincubation ( ) was
monitored. All values were expressed as the means ± S.E. *,
p between 0.03 and 0.0003 compared with either control,
D, E, F, or H. **, p < 0.01 compared
with control. ***, p < 0.000006 compared with
control. ****, p < 0.0001 compared with
control. B, schematic description of Zot deletion mutants.
The deletion for each protein is shown as a broken filled
line. The putative Zot functional domain responsible for tj
disassembly (delimited by the two vertical lines) maps in
the toxin's carboxyl-terminal region (AA 295-399). Deleted amino acid
residues are shown in parentheses.
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Binding Activity of Zot Deletion Mutants on IEC6 Cells--
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. 3A) and the PBS negative
control (Fig. 3I). Cells incubated with His-
G (Fig.
3G) showed an amount of binding particles similar to that
seen in cells incubated with His-Zot (Fig. 3A). The number of fluorescent particles was only minimally decreased in cells incubated with His-
B (Fig. 3B), His-
C (Fig.
3C), or His-
D (Fig. 3D). In contrast, binding
capability was completely lost by His-
E (Fig. 3E),
His-
F (Fig. 3F), and His-
H (Fig. 3H)
mutants. Analysis of these results revealed that the Zot region
corresponding to AA residues 265-301 was partially (His-
E and
His-
H) or completely (His-
F) deleted in the binding-negative
mutants, whereas it was spared in the binding-positive constructs (Fig.
3J), suggesting that this region may represent the putative
Zot domain involved in receptor binding function.

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Fig. 3.
Fluorescence microscopy of IEC6 cells.
Cells visualized in red by Evans blue counterstain were
exposed to either His-Zot (1 × 10 10
M; A) or an equimolar amount of Zot deletion
mutants (B-H) and incubated for 30 min at 4 °C. PBS was
used as a negative control (I). Green fluorescent binding
particles appear as yellow clusters on a red
background. Binding particles were present in cells exposed to
His-Zot (A) and G (G) and, to a lesser
extent, in cells exposed to B (B), C (C),
and D (D) (see arrows). No binding activity
was detected in cells incubated with E (E), F
(F), or H (H). J, identification of
the putative binding domain among the seven Zot deletion mutants. The
deletion for each protein is shown as a broken filled line.
The binding of each mutant was compared with His-Zot binding
(A) and to the PBS negative control (I).
Comparative binding analysis of the seven deletion mutants tested led
to the identification of a region corresponding to Zot amino acid
residues 265-301 (delimited by the two vertical lines)
necessary to display fluorescent signals. *, deleted amino acid
residues are shown in parentheses.
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Characterization of the Zot Binding Domain and the Structure
Requirements to Engage to the Zonulin Receptor--
We have recently
demonstrated that Zot localizes in the V. cholerae outer
membrane, to which it is anchored through its single spanning domain
(19). 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
(19). 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 (21) 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.
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Table III
Comparison of the amino-terminal sequences of human intestinal zonulin
and Zot
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).
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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-
G 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-
G 291 (in which the G
in position 291 was substituted with V) showed a reduced number
of binding particles (Fig.
5A, 3) as compared with cells
incubated with His-
G (Fig. 5A, 2), whereas no binding was
observed on cells incubated with His-
G 298 (G298V) mutant (Fig.
5A, 4). Biological assays in Ussing chambers showed that
His-
G291 had a residual but not significant effect on tj disassembly
(Fig. 5B). The permeating effect was completely ablated when
the intestinal tissues were incubated with His-
G 298 (Fig.
5B). 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-
G to bind
to its target receptor, and, consequently, its substitution ameliorated
the ligand biological effect on tj.

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Fig. 4.
Structural requirements for receptor binding
and permeabilizing activity. To establish whether the Zot/zonulin
domain shaded in Table III represents the receptor binding motif
involved in the permeabilizing activity of both proteins, the ability
of either the synthetic peptide FZI/0 (GGVLVQPG) mimicking the putative
binding domain or of a scrambled peptide (VGVLGRPV) to block both Zot
and zonulin-induced reduction of ileal Rt was tested. Left
bars represent baseline Rt values, whereas right bars
are Rt values after a 90-min incubation. 0.1 µg/ml either Zot or
zonulin ( ) each induced a significant Rt decrement compared with the
negative control, whereas 1 µg/ml either FZI/0 or the
scrambled peptide did not. Pretreatment with 1 µg/ml FZI/0 for 20 min
before and throughout the exposition to Zot and zonulin ( ) prevented
the Rt reduction, whereas pretreatment with the scrambled
peptide ( ) did not. n = 3-5; *, p < 0.008 compared with Zot alone. **, p < 0.005 compared with zonulin alone.
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Fig. 5.
Effect of the two
His- G site-directed mutants
(His- G291 and
His- G298) 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- G291 showed binding particles (3) similar to those
detected in His- G-exposed monolayers (2), whereas no
binding was detected in monolayers exposed to His- G298
(4). Negative PBS control (1) is shown for
comparison. B, Ussing chamber assay. The tissue was treated
with 1 × 10 10 M of each
protein, and Rt change was compared with that in PBS-exposed tissues.
His- G291 induced a partial but not a significant reduction in Rt
when compared with the changes induced by His- G. The permeating
effect was completely ablated when the rabbit ileum was exposed to
His- G298. All values were expressed as the means ± S.E. *,
p < 0.009 compared with His- G298 and
p < 0.0001 as compared with control.
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DISCUSSION |
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 (22, 23) 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 (1) and accessory cholera toxin (Ace) (4). This cluster of genes
has been recently described as part of a filamentous phage (CTX
)
that lives in symbiosis with its bacterial host. It has been reported
previously that Zot is involved in both CTX
morphogenesis (5) and
disassembly of the host intestinal intercellular tj (1, 14-16).
The study of the subcellular localization of Zot by affinity-purified
anti-Zot antibodies (19) 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) (19). A second immunoreactive
molecule, corresponding to the 33-kDa amino-terminal region of Zot, was
also detected at the outer membrane site (19). Both molecules are
exposed at the bacterial cell surface (19). The 33-kDa Zot processing
product is generated by a cleavage site at AA residue 287 (19). 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 (1). Indeed, despite the
predicted toxin molecular mass (44.8 kDa) (14), its biological effect
on tj was found confined to the <30-kDa V. cholerae culture
supernatant fraction (1). 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 (14).
Taken together, these data suggest that Zot has a dual function:
whereas its ~33-kDa amino-terminal portion is possibly involved in
CTX
phage assembly (5, 9, 10), 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 and
Bacteroides fragilis) (24). A more physiological mechanism
of regulation of tj permeability has been proposed for Zot (1). Zot
activates a complex intracellular cascade of events that involve a
dose- and time-dependent protein kinase C
-related
polymerization of actin filaments strategically localized to regulate
the paracellular pathway (15). These changes are a prerequisite to the
opening of tj and are evident at a toxin concentration as low as
1.1 × 10
13 M (16). 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 (1, 16). The regional distribution of Zot receptor(s)
coincides with the different permeating effect of the toxin on the
various tracts of intestine tested (16). 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 (15, 16). Based on
the complexity of the intracellular signaling activated by Zot leading
to the tj disassembly (15), 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 (21). 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 (21).
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 (25). A century ago, the tj was conceptualized as
secreted extracellular cement forming an absolute and inert barrier
within the paracellular space (26). 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 (27). Furthermore, tj readily adapt to a variety of
developmental, physiological, and pathological circumstances (28, 29).
Data exist that support the linkage between the actin cytoskeleton and
the tj complex (30-32) and implicate signaling events that regulate
paracellular permeability (27). 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 (18).
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.
 |
FOOTNOTES |
*
Partially supported by National Institutes of Health Grant
DK-48373 (A. F.) and the European Commission Contract IC18-CT 97-0231 (F. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.

To whom correspondence should be addressed: Division of
Pediatric Gastroenterology and Nutrition, University of Maryland School of Medicine, 685 W. Baltimore St., HSF Bldg., Rm. 465, Baltimore, MD
21201. Tel.: 410-328-0812; Fax: 410-328-1072; E-mail:
afasano@umaryland.edu.
Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M009674200
 |
ABBREVIATIONS |
The abbreviations used are:
Zot, zonula
occludens toxin;
tj, tight junction(s);
PBS, phosphate-buffered saline;
Rt, tissue resistance;
AA, amino acid.
 |
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