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INTRODUCTION |
Persistent Helicobacter pylori infections can progress
to peptic ulcer disease or stomach cancer in humans (1-8). Many
H. pylori strains secrete a cytotoxin that collective
evidence indicates is an important virulence factor in H. pylori-mediated disease (9, 10). The toxin was named vacuolating
cytotoxin (VacA) because it induces vacuolation of cultured mammalian
cells (11, 12). Toxigenic strains of H. pylori are
frequently cultured from biopsies of gastroduodenal ulcers (9, 13-15),
whereas H. pylori strains harboring an altered VacA toxin
gene are noncytotoxic (13-18). Oral administration of purified VacA to
mice induces gastric mucosa degeneration and inflammatory cell
recruitment, characteristic of H. pylori-mediated diseases
(20, 21).
VacA is synthesized as a 140-kDa protein (5, 13, 15, 22). The
carboxyl-terminal domain facilitates secretion of the 103-kDa mature
protein into the extracellular medium (5, 13, 15, 22). The secreted
peptide is often proteolytically nicked into amino- and
carboxyl-terminal fragments, which remain associated by noncovalent
interactions and retain vacuolating activity (5).
VacA exhibits cellular behavior similar to that of protein toxins with
intracellular targets (24-26). Most intracellularly acting toxins
possess an overall structure corresponding to the A-B family of
bacterial exotoxins, which includes diphtheria, cholera, and anthrax
toxins (27). In general, the B component of an A-B toxin binds to
specific mammalian cell-surface receptors and facilitates the membrane
translocation of an enzymic A moiety into the cytosol. The A moiety
covalently modifies a specific intracellular target molecule, resulting
in the disruption of important cellular functions. VacA has recently
been shown to bind to specific, high affinity cell surface receptors
(28-30). The toxin is internalized inside vesicular-type compartments
by an unknown mechanism (26) and subsequently mediates the formation of
large acidic vacuoles comprising hybrid endocytic vesicles originating
from late endosomal and lysosomal compartments (31-34). By
transfecting HeLa cells with plasmids expressing VacA and thereby eliminating the necessity for receptor binding and membrane
translocation, de Bernard and co-workers (24) demonstrated that VacA
functions from within the host cell cytosol. Significantly, vacuolation of host cells was observed before VacA was detectable in the cells, indicating that the potency of the toxin may be attributed to a
discrete enzymatic moiety, which amplifies the effects of the toxin
within the cell (27, 35). Although these data strongly suggest that
VacA is an A-B toxin, distinct A and B fragments have not been identified.
Because a catalytic function has not yet been identified for VacA, it
is not currently feasible to identify the putative A-fragment by
testing truncated toxin fragments for enzymatic activity. However, we
hypothesized that the A-fragment would induce vacuolation from the host
cell cytosol in the absence of the B fragment of the toxin. To localize
the putative VacA A-fragment, we genetically constructed amino and
carboxyl-terminal truncations of VacA. Plasmids encoding these
fragments were used to transfect HeLa cells, which were analyzed for
vacuolation. These investigations revealed that a discrete VacA
fragment can function from the host cell cytosol to induce vacuoles
with properties similar to those caused by full-length toxin added
externally to cells. In addition, we discovered that determinants from
both fragments of proteolytically nicked toxin can function in
trans within the host cell cytosol to induce degenerative
vacuolation. Finally, we discuss the implications of these findings
with respect to the molecular organization of VacA. Importantly, the
identification of a discrete A fragment may be a critical step
preceding discovery of the enzymatic activity of the toxin, because a
number of bacterial toxins, including diphtheria and cholera, are
secreted as proenzymes, which require activation to elaborate their
catalytic activities (36, 37).
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EXPERIMENTAL PROCEDURES |
Materials--
Cell culture medium, fetal bovine calf serum, and
neutral red were purchased from Life Technologies, Inc. Restriction
enzymes and T4 DNA ligase were purchased from New England Biolabs
(Beverly, MA). High fidelity Pfu DNA polymerase and
deoxyribonucleotides were obtained from Stratagene (La Jolla, CA). The
plasmid pET20b was obtained from Novagen (Madison, WI). Vaccinia virus
expressing T7 polymerase (ATCC 2153-VR) and H. pylori strain
60190 (ATCC 49503) were purchased from American Type Culture Collection
(Manassas, VA). Bafilomycin A1 and monensin were obtained from Sigma
and Calbiochem, respectively.
Preparation of Plasmids Encoding VacA and Fragments of
VacA--
Standard protocols were utilized for isolation of plasmid
DNA, restriction endonuclease digestions, the polymerase chain
reaction, subcloning, and transformation of Escherichia coli
(38). The gene for VacA was amplified directly from H. pylori strain 60190 genomic DNA using the polymerase chain
reaction and cloned into pTRE (CLONTECH; Palo
Alto, CA), which was the parent plasmid used as template for
all the VacA-GFP fusions and VacA fragments generated for these investigations.
The gene encoding enhanced green fluorescence protein (GFP) was
extracted from pEGFP-N3 (CLONTECH; Palo Alto, CA)
and subcloned into pET20b replacing the BamHI and
NotI fragment in the polyclonal region. All the VacA
fragments were cloned into the modified pET20b-GFP, or pET-20b alone,
which harbors a T7 promoter, replacing the
NdeI-BamHI fragment of the polyclonal region.
Polymerase chain reaction primers were designed for the two fragments
of nicked VacA based on previous reports describing the cleavage site
between the two fragments of the nicked protein (5, 39). These two
fragments comprise residues 1-311 and 312-953, and are referred to as
p33 and p70 respectively based on their calculated molecular weights of
approximately 33 and 70 kDa. Plasmids were recovered from transformed
E. coli XL1-blue, purified, and used for HeLa cell
transfections as described below. Full-length VacA and all VacA
fragments were sequenced across the length of the entire open reading
frame using the Thermo Sequenase dye terminator cycle sequencing
pre-mix kit from Amersham Pharmacia Biotech.
Cell Culture--
HeLa cells were cultured as monolayers in
25-ml plastic flasks (Corning; Cambridge, MA) in Dulbecco's modified
minimal essential medium
(DMEM)1 containing 10% fetal
bovine calf serum under 5% CO2 at 37 °C. Twenty-four h
before experiments, cells were seeded in 96-well titration plates in
DMEM, 10% fetal bovine calf serum at a density of 1 × 105 cells/ml.
Transfection of Cells--
HeLa cells were plated (200 µl) at
a density of 1.0 × 105 cells/ml in 96-well tissue
culture plates (Corning; Cambridge, MA) in DMEM supplemented with 2.5%
fetal bovine calf serum, 100 units penicillin/ml, and 100 mg of
streptomycin/ml. HeLa cells were first infected with recombinant
vaccinia virus (vT7) bearing the T7 RNA polymerase gene (39). Vaccinia
virus stock was trypsinized at 37 °C for 30 min and added to HeLa
cells (24). After infection for 30 min, virus stock was removed, and
the HeLa cells were transfected using the calcium phosphate method
(38). After the transfection procedure, the cells were incubated in
DMEM + 5 mM NH4Cl at 37 °C for 20 h
before analysis.
Analysis of Transfected HeLa Cells--
We quantified relative
vacuolation based on the HeLa cell uptake of the dye neutral red as
described previously (40). The experiments were performed in 96-well
plates, and neutral red uptake was determined using a Dynatech MR5000
microtiter plate reader to measure the absorbances at 530 nm (minus the
absorbance at 410 nm).
To confirm green fluorescence protein (GFP) fluorescence in transfected
HeLa cells, the cells were visualized using a Nikon Diaphot inverted
microscope outfitted with a Xenon lamp and fluorescence filter
combinations for fluorescence microscopy.
Pharmacological Reagent Effects on Vacuolating Activity--
Two
h after transfection of HeLa cells, transfection reagents were replaced
with DMEM and 5 mM NH4Cl supplemented with the indicated concentrations of bafilomycin A1 or monensin and incubated for 20 h under 5% CO2 at 37 °C.
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RESULTS |
Expression of VacA Fragments in HeLa Cells under Control of the T7
Promoter--
The minimal VacA vacuolating domain was mapped by
transfecting HeLa cells with pET-20b harboring genes encoding either
the mature VacA peptide (residues 1-953) cloned from the 60190 toxigenic strain of H. pylori or truncated fragments of VacA
fused to GFP. To improve protein expression, HeLa cells were first
infected with recombinant vaccinia virus (vT7) bearing the gene for
phage T7 RNA polymerase followed by transfection with pET-20b encoding VacA fragments fused to GFP (24, 39). Using this system, 50-70% of
the cells clearly demonstrated GFP fluorescence (data not shown), which
appeared essentially identical to previously published data (24). In
HeLa cells transfected with VacA-GFP, vacuolation was observed only in
those cells demonstrating GFP fluorescence. VacA-GFP fusions could be
detected by GFP fluorescence 5-8 h after transfection, whereas
vacuoles were already clearly visible after 4 h, also confirming
previously reported results that vacuolation was detected before GFP
fluorescence could be visualized in the cell cytosol (24).
Carboxyl-terminal Truncations of VacA--
Plasmids were
constructed to express multiple VacA fragments lacking portions of the
carboxyl terminus. These deletions yielded VacA amino-terminal
fragments of 349, 373, 394, 422, 478, 538, 670, and 741 amino acids
with or without GFP fusions (Fig.
1B). Purified plasmids
expressing either full-length toxin or truncation mutants were used to
transfect HeLa cells, which had been previously infected with vT7.
Twenty h after transfection, HeLa cells were analyzed for both GFP
fluorescence and uptake of neutral red.

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Fig. 1.
Analysis of VacA carboxyl-terminal truncation
mutants for intracellular vacuolating activity. HeLa cells were
transfected with pET20b plasmids expressing carboxyl-terminal
truncations of VacA, as described under "Experimental Procedures."
After 20 h, the cells were assayed for uptake of neutral red.
A, neutral red uptake of HeLa cells transfected with
plasmids expressing VacA fragments comprising residues 1-422 and
1-394 (without fusions) and GFP alone. Data is expressed as a
percentage of neutral red uptake by HeLa cells transfected with plasmid
expressing full-length VacA-GFP. The data were averaged from three
separate experiments performed at least in triplicate. B,
summary of vacuolating activity of carboxyl-terminal VacA truncation
fragments. VacA residues are indicated and shown in open
bars, whereas GFP is represented with shaded
bars.
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Analysis of the truncation mutants revealed that when VacA is directly
expressed in the host cell cytosol, the carboxyl terminus is not
essential for inducing vacuole formation (Fig. 1). Residues 1-478 (475 carboxyl-terminal deletion of VacA) fused to GFP induced vacuolation of
HeLa cells, whereas a fragment comprising VacA residues 1-422 fused to
GFP (531 carboxyl-terminal deletion of VacA) was unable to induce
detectable vacuolation (Fig. 1A). To test if the
carboxyl-terminal GFP fusion interfered with the vacuolating activity
of the 422-residue VacA fragment, this fragment was also expressed in
the HeLa cell cytosol without the GFP fusion. Although the 422-residue
VacA fragment with a free carboxyl terminus was sufficient to mediate
vacuolation, neither a shorter VacA fragment comprising residues 1-394
nor any smaller fragment induced vacuolation (Fig. 1B).
Amino-terminal Truncations of VacA--
To determine the
importance of the VacA amino terminus in the intracellular activity of
the toxin, a series of amino-terminal truncation mutants fused to GFP
were generated comprising residues 18-741, 57-741, and 198-741 (Fig.
2B). The plasmids encoding
these fragments were used to transfect HeLa cells after infection of the cells with vT7. The extent of vacuolation was determined by quantifying neutral red uptake. The expression of VacA fragments fused
to GFP was confirmed using fluorescence microscopy. None of the
amino-terminal truncations were able to induce intracellular vacuolation (Fig. 2). Deletion of only 17 residues resulted in the loss
of VacA-mediated vacuolating activity (Fig. 2A), indicating that in contrast to the carboxyl terminus, the VacA amino terminus appears to be essential for inducing intracellular vacuolation.

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Fig. 2.
Analysis of VacA amino-terminal truncation
mutants for intracellular vacuolating activity. HeLa cells were
transfected with pET20b plasmids expressing amino-terminal truncations
of VacA, as described under "Experimental Procedures." After
20 h, the cells were assayed for uptake of neutral red.
A, neutral red uptake of HeLa cells transfected with
plasmids expressing VacA fragments comprising residues 1-741 fused to
GFP, 18-741 fused to GFP, and GFP alone. Data is expressed as the
percentage of neutral red uptake by HeLa cells transfected with plasmid
expressing full-length VacA-GFP. The data were averaged from three
separate experiments performed at least in triplicate. B,
summary of vacuolating activity of amino-terminal VacA truncation
fragments. VacA residues are indicated and shown in open
bars, whereas GFP is represented with shaded
bars.
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Co-transfection of HeLa Cells with Multiple VacA
Fragments--
VacA is often isolated as a proteolytically nicked
protein of two fragments comprising residues 1-311 and 312-953 of the
mature protein (p33 and p70, respectively) (5, 41). As previously reported, HeLa cells transfected with plasmids encoding either of these
fragments separately were not vacuolated (24). To determine whether p33
and p70 can induce vacuolation when introduced together into cells, we
co-transfected HeLa cells with separate plasmids encoding these two
fragments. These co-transfection experiments revealed that the two
fragments were able to functionally complement each other to induce
fully vacuolated cells (Fig. 3). Notably, GFP fused to the carboxyl terminus of p33 did not interfere with p70
complementation. If VacA is an A-B toxin, this would be the first
example in which two distinct fragments functionally complement each
other from within the host cell cytosol to mediate toxin activity.

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Fig. 3.
Analysis of HeLa cells co-transfection
experiments. HeLa cells were transfected with single pET20b
plasmids expressing VacA fragments or co-transfected with separate
plasmids, as described under "Experimental Procedures." After
20 h, the cells were assayed for uptake of neutral red.
A, neutral red uptake of HeLa cells transfected with
plasmids expressing VacA fragments comprising residues 1-311 fused to
GFP, 312-953 fused to GFP, and GFP alone. In addition, data is shown
for HeLa cells co-transfected with the plasmids encoding the VacA
fragment comprising residues 1-311 fused to GFP along with separate
plasmids encoding VacA residues 312-953 fused to GFP, VacA residues
312-478 (without GFP fusion), or VacA residues 312-422 (without GFP
fusion). Data is expressed as the percentage of neutral red uptake by
HeLa cells transfected with plasmid expressing full-length VacA-GFP.
The data were averaged from three separate experiments performed at
least in triplicate. B, summary of vacuolating activity of
co-transfection experiments with VacA fragments. VacA residues are
indicated and shown in open bars, whereas GFP is represented
with shaded bars.
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Having demonstrated that more than 500 amino acids at the carboxyl
terminus of nonnicked VacA are not required for intracellular vacuolation (Fig. 1), we next tested whether the entire p70 carboxyl terminus was required when co-expressed with p33. Plasmids encoding smaller fragments of p70 were designed based on the carboxyl-terminal truncations described above. Co-transfection experiments revealed that
a 167-residue VacA fragment comprising amino acids 312-478 with or
without GFP fusions functionally complemented inactive p33 to induce
intracellular vacuolation (Fig. 3A). However, the smaller
111-residue p70 fragment comprising VacA residues 312-422 did not
complement p33, regardless of whether or not the fragment was expressed
as a fusion protein with GFP.
Vacuoles Induced by Cytosolic VacA Fragments Respond to the Same
Reagents as Vacuoles Induced by Externally Added Toxin--
Ammonium
ions potentiate VacA-mediated cellular vacuolation regardless of
whether toxin is added externally or introduced directly into the
cytosol of HeLa cells (42). The potentiation mechanism is believed to
involve osmotic swelling of vacuolar vesicles, which have taken up
NH4Cl subsequent to a VacA-induced perturbation of cellular
endocytic trafficking (42). As shown in Fig.
4, when HeLa cells were transfected with
the plasmid encoding the minimal functional VacA fragment (residues
1-422), vacuole formation was potentiated by the presence of 5 mM NH4Cl. Likewise, in co-transfection
experiments in which VacA fragments were expressed from separate
plasmids, vacuolation of HeLa cells was also potentiated by 5 mM NH4Cl (Fig. 4). In the absence of
NH4Cl, VacA fragments expressed in the cytosol of host cell
were attenuated in vacuolation activity, as demonstrated by the
inhibited uptake of neutral red by transfected HeLa cells.

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Fig. 4.
Potentiation of intracellular activity of
VacA fragments with NH4Cl. HeLa cells were transfected
with plasmids encoding the indicated VacA fragments and incubated in
the presence or absence of 5 mM NH4Cl. The data
were averaged from three separate experiments performed at least in
triplicate and is illustrated as the percentage of neutral red uptake
by HeLa cells transfected with plasmid expressing full-length VacA-GFP
in the presence of 5 mM NH4Cl.
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Cell vacuolation induced by VacA is strictly dependent on the function
of vacuolar-type ATPase proton pumps (34). To determine whether
vacuoles formed by the fragments of VacA expressed in the cytosol are
similar to those produced by full-length VacA, we transfected HeLa
cells with plasmids expressing full-length VacA fused to GFP or the
active VacA fragment comprising residues 1-422. In addition, we
co-transfected HeLa cells with plasmids expressing p33-GFP and p70-GFP.
We introduced bafilomycin A1 (2-25 nM), which targets
vacuolar-type ATPase proton pumps and has been shown to inhibit
VacA-induced cellular vacuolation (43). As shown in Fig.
5A, 5-10 nM
bafilomycin A1 inhibited neutral red uptake by approximately 50%.
Increasing, the drug to 25 nM nearly abolished vacuole
formation, indicating that the vacuoles formed intracellularly by
minimal VacA fragments also require active vacuolar-type ATPase proton
pumps.

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Fig. 5.
Inhibition of intracellular vacuolating
activity with bafilomycin A1 and monensin. Assays were performed
as described under "Experimental Procedures." The data were
averaged from three separate experiments performed at least in
triplicate and are illustrated as a percentage of neutral red uptake of
HeLa cells transfected with plasmids in the absence of bafilomycin A1
or monensin. HeLa cells were transfected with plasmids expressing
VacA-GFP ( ), VacA (residues 1-422) ( ), and co-transfected with
separate plasmids expressing p33-GFP and p70-GFP (×).
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Monensin was also reported in earlier studies to inhibit vacuole
formation when the toxin was added externally to the cells (42). To
compare whether monensin inhibits the intracellular activity of VacA as
well as the activity of externally added toxin, we tested the effects
of monensin (0.2-5.0 µg/ml) on these same VacA fragments expressed
from the cytosol of HeLa cells. Monensin addition yielded a
concentration-dependent blockage of VacA-induced vacuolation (Fig. 5B); 0.75-2.0 µg/ml monensin inhibited
neutral red uptake by approximately 50%, and 5 µg/ml almost
completely inhibited neutral red uptake. Collectively, these results
indicate that vacuoles induced by VacA fragments functioning directly
from within the host cell cytosol are similar to those induced by
full-length toxin.
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DISCUSSION |
In the absence of a defined biochemical activity for VacA, it is
not currently possible to localize an isolated catalytic domain using
enzyme activity assays to analyze smaller toxin fragments. However, we
expressed VacA fragments in the cytosol of HeLa cells to determine the
minimal domain, which induces vacuolation similar to whole toxin added
externally to cells. By eliminating the necessity for receptor binding
and translocation, we hypothesized that the B fragment of the toxin
could be entirely removed without affecting the ability of the toxin to
act intracellularly. Other toxin A fragments have been shown to be
entirely functional when translocated into host cells by alternative
means in the absence of the B fragment of the toxin. For example,
catalytically active A fragments of diphtheria toxin,
Pseudomonas exotoxin A, and shiga toxin have all been
successfully introduced into mammalian cells using the B fragment of
anthrax toxin (44-46).
Our investigations demonstrated that an amino-terminal fragment of
approximately 422 residues comprises a minimal VacA domain that can
mediate vacuole formation from the host cell cytosol. More than 50% of
the 953 amino acids can be deleted from the VacA carboxyl terminus
without affecting intracellular vacuolating activity, supporting the
hypothesis that VacA contains a discrete A domain responsible for
catalytic activity. Furthermore, truncations of greater than 17 residues from the amino-terminal end of VacA abolished vacuolating
activity, emphasizing the importance of the amino terminus to
vacuolating activity. Although HeLa cells transfected with plasmids
encoding truncated VacA-GFP fusion proteins exhibited fluorescence, it
cannot be ruled out at this time that the lack of detectable cellular
vacuolation is because of amino-terminal loss or degradation by way of
selective proteolysis. Based on our data, we propose that the putative
A-fragment of VacA is located in the amino-terminal half of the toxin,
whereas the B-fragment receptor binding and translocation determinants
comprise the carboxyl-terminal half of the toxin.
We isolate VacA from H. pylori primarily as two distinct
fragments (p33 and p70) that remain noncovalently associated and retain
vacuolating activity subsequent to proteolytic nicking at amino acid
311 (5, 47). Although it was initially speculated that p33 and p70 may
constitute A and B fragments of the toxin, neither p33 nor p70 alone
were able to induce vacuolation when HeLa cells were transfected with
plasmids separately encoding these genes. This suggests that the A
fragment of the toxin may include domains from both fragments (25). Our
results support this proposal, indicating that both p33 and the amino
terminus of p70 are required to intracellularly induce vacuolation. In single transfection experiments, 422 residues of VacA were essential for vacuolating activity. However, in co-transfection experiments, a
larger fragment of p70 (residues 312-478) was required to complement inactive p33. It is not known whether residues 423-478 are required for direct interaction with p33 or alternatively, if the relatively short 111-amino acid polypeptide (residues 312-422) is unable to fold
into a functional domain.
Our results suggest an interesting and perhaps unique molecular
structure for VacA among the A-B toxins. Complementation of inactive
p33 with a 167-residue fragment of p70 indicates that both fragments
are essential. However, it is unclear whether both fragments comprise
essential domains of the putative catalytic core of the toxin. We also
show that GFP fused to the p33 carboxyl terminus does not interfere
with functional complementation by p70, suggesting that the essential
determinants of toxin activity are not contiguous across the p33-p70
cleavage site at residue 311. In addition, these results are consistent
with a recently published report that 46 residues at the interface
between the two VacA fragments could be eliminated without affecting
toxin activity (48). Thus, if VacA possesses a discrete catalytic domain, it is unlikely that it spans the p33-p70 interface. It is
possible that a putative VacA catalytic domain may be composed of
noncontiguous domains requiring assembly before elaborating the
catalytic activity of the enzyme. Although some enzymes can be
dissected and the fragments functionally reassembled (23, 19, 49), this
has not previously been demonstrated for any toxin A-fragment.
Alternatively, p33 and p70 could conceivably possess independent
functions, both of which are required for mediating an undefined
mechanism to induce formation of vacuoles within the host cell cytosol.
In summary, we have provided the first direct evidence for localization
of the putative VacA A-fragment to the amino-terminal half of the
toxin. Furthermore, determinants from both fragments of proteolytically
nicked VacA are required for the elaboration of toxin activity from
within the host cell cytosol. Investigations are ongoing to further
localize a discrete catalytic core. Mapping this functional domain as
well as the receptor binding and translocation domains, will not only
be important to begin understanding the mechanism of VacA cytotoxicity
and toxin structure-function relationships but will also be critical to
identify stable, nontoxic subdomains as immunogens for incorporation
into protective vaccines against H. pylori.