Helicobacter
pylori secretes a toxin, VacA, that can form anion-selective
membrane channels. Within a unique amino-terminal hydrophobic region of
VacA, there are three tandem GXXXG motifs (defined by
glycines at positions 14, 18, 22, and 26), which are characteristic of
transmembrane dimerization sequences. The goals of the current study
were to investigate whether these GXXXG motifs are required
for membrane channel formation and cytotoxicity and to clarify the role
of membrane channel formation in the biological activity of VacA. Six
different alanine substitution mutations (P9A, G13A, G14A, G18A, G22A,
and G26A) were introduced into the unique hydrophobic region located
near the amino terminus of VacA. The effects of these mutations were
first analyzed using the TOXCAT system, which permits the study of
transmembrane oligomerization of proteins in a natural membrane
environment. None of the mutations altered the capacity of
ToxR-VacA-maltose-binding protein fusion proteins to insert into a
membrane, but G14A and G18A mutations markedly diminished the capacity
of the fusion proteins to oligomerize. We then introduced the six
alanine substitution mutations into the vacA chromosomal
gene of H. pylori and analyzed the properties of purified
mutant VacA proteins. VacA-G13A, VacA-G22A, and VacA-G26A induced
vacuolation of HeLa cells, whereas VacA-P9A, VacA-G14A, and VacA-G18A
did not. Subsequent experiments examined the capacity of each mutant
toxin to form membrane channels. In a planar lipid bilayer assay, VacA
proteins containing G13A, G22A, and G26A mutations formed
anion-selective membrane channels, whereas VacA proteins containing
P9A, G14A, and G18A mutations did not. Similarly, VacA-G13A, VacA-G22A,
and VacA-G26A induced depolarization of HeLa cells, whereas VacA-P9A,
VacA-G14A, and VacA-G18A did not. These data indicate that an intact
proline residue and an intact
G14XXXG18 motif within the
amino-terminal hydrophobic region of VacA are essential for membrane
channel formation, and they also provide strong evidence that membrane
channel formation is essential for VacA cytotoxicity.
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INTRODUCTION |
Helicobacter pylori is a Gram-negative bacterium that
colonizes the gastric mucosa of humans (1). Colonization of the stomach by H. pylori is a strong risk factor for the development of
peptic ulcer disease and distal gastric adenocarcinoma (2-4). Within the gastric mucosa, H. pylori organisms are found
predominantly in the gastric mucus layer, but they also adhere to
gastric epithelial cells (5).
Adherence of H. pylori to gastric epithelial cells triggers
the initiation of processes that ultimately result in alteration of
gastric epithelial cell function or gastric epithelial cell death. One
of the important effector molecules used by H. pylori to
modulate eukaryotic cell function is a secreted toxin known as VacA
(6-8). The secreted VacA toxin is an 88-kDa protein, consisting of
about 821 amino acids (9). These 88-kDa VacA monomers spontaneously
self-assemble into large water-soluble flower-shaped oligomeric
complexes, consisting predominantly of 12 or 14 monomeric subunits
(10-12). When added to eukaryotic cells in vitro, the
oligomeric form of VacA is nearly devoid of cytotoxic activity.
However, exposure of oligomeric VacA to either acidic or alkaline pH
results in disassembly of the oligomeric complexes into monomers that
exhibit potent cytotoxic activity (10, 13-15).
The addition of VacA to eukaryotic cells causes multiple alterations in
cell structure and function. When VacA is added to cells in the
presence of weak bases, the most apparent effect is the development of
cytoplasmic vacuolation (16, 17). VacA also induces permeabilization of
epithelial monolayers (18), apoptosis (19-21), depolarization of the
resting membrane potential (22, 23), and it interferes with the process
of antigen presentation (24). After binding of VacA to the plasma
membrane, the toxin is internalized by cells (15, 23, 25, 26). Based on
experiments in which cells are transiently transfected with
VacA-encoding plasmids (27, 28), it is presumed that VacA cytotoxicity
results from toxin action at an intracellular site. Endosomal
compartments and mitochondria have both been suggested as possible
intracellular sites of VacA action (19, 29, 30).
Water-soluble VacA molecules are able to insert into lipid bilayers and
form anion-selective membrane channels (22, 31-33). It has been
hypothesized that VacA cytotoxicity results primarily from the
formation of such channels. One model proposes that VacA forms
anion-selective channels in the membranes of endosomes, which leads to
osmotic swelling of these compartments (for review, see Refs. 8 and
34). However, it also has been hypothesized that VacA cytotoxicity may
result primarily from an unrecognized enzymatic activity and that the
formation of anion-selective membrane channels may be a secondary
feature (for review, see Ref. 8). Support for the latter hypothesis is
based on the recognition that numerous A-B type bacterial toxins
exhibit an enzymatic activity and also exhibit a capacity for membrane
channel formation (for review, see Refs. 35 and 36).
In support of the view that VacA cytotoxicity results primarily from
its capacity to form membrane channels, it has been reported that an
inhibitor of anion-selective channels,
NPPB,1 blocks VacA
cytotoxicity (22). In addition, we have reported that VacA-
(6-27),
a VacA mutant containing a 22-amino acid deletion, lacks the capacity
to form membrane channels and also lacks cytotoxic activity (37).
Unfortunately, neither of these experimental approaches provides
conclusive evidence indicating that membrane channel formation is
essential for VacA cytotoxicity. Interpretation of experiments using
NPPB is limited by the possibility that NPPB might exert multiple
effects on cells or on the toxin rather than specifically inhibiting
VacA channel activity. Also, conclusions from experiments with
VacA-
(6-27) are limited by the possibility that a 22-amino acid
deletion might alter multiple functional activities of the toxin rather
than specifically ablating channel forming activity.
VacA contains a unique hydrophobic region located near its amino
terminus, and it seems possible that such a region might be utilized
for insertion of VacA into membranes during the process of membrane
channel formation. In a previous study, we used a model system known as
TOXCAT to analyze the functional role of the VacA hydrophobic region
(38, 39). The results indicated that the VacA hydrophobic region (amino
acids 1-32) mediated insertion of ToxR-VacA-maltose-binding
protein (MBP) fusions into the inner membrane of
Escherichia coli and also mediated protein
dimerization. In contrast, a fusion protein containing a mutant VacA
hydrophobic region (in which glycine 14 was replaced by alanine)
inserted into the inner membrane but dimerized less efficiently than
the fusion protein containing the wild-type VacA sequence. These
experiments demonstrated that the VacA amino-terminal hydrophobic
region is capable of inserting into membranes and undergoing dimerization.
Within the VacA hydrophobic region, there are three tandem
GXXXG motifs, which are characteristic of transmembrane
dimerization sequences (Fig. 1). In the
current study, we sought to investigate the functional role of these
three GXXXG motifs in membrane channel formation and
cytotoxicity and to clarify the role of membrane channel formation in
the biological activity of VacA. We report here that alanine
substitution mutations at the Gly-14 and Gly-18 positions abolish the
capacity of VacA to form membrane channels and also abolish the
capacity of VacA to induce cytotoxic effects. We also report that the
G14XXXG18 motif is required for
dimerization of VacA in a membrane using the TOXCAT system. These
results provide further evidence that VacA cytotoxicity occurs via a
process that requires membrane channel formation and indicate that the
amino-terminal hydrophobic region of VacA plays an essential role in
both membrane channel formation and cytotoxicity.

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Fig. 1.
Hydrophobicity plot of the mature secreted
VacA toxin from H. pylori 60190 (79).
The predicted hydrophobicity of the mature VacA toxin from H. pylori 60190 was analyzed by the method of Kyte and Doolittle
(80). Capital letters indicate the amino-terminal sequence
of VacA as well as a hydrophilic region of VacA which is susceptible to
proteolytic cleavage. The underlined segments represent
three tandem GXXXG motifs, characteristic of transmembrane
oligomerization regions, located near the amino terminus of VacA.
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MATERIALS AND METHODS |
Analysis of VacA Using the TOXCAT System--
The TOXCAT system
(Fig. 2) was originally developed by Russ
and Engelman (39) to study transmembrane helix-helix associations in a
natural membrane environment. In this system, a putative transmembrane
(TM) sequence is cloned between a sequence encoding the transcription
activator domain of Vibrio cholerae ToxR and a sequence
encoding the periplasmic domain of the E. coli MBP. To
determine whether the putative transmembrane sequence mediates transmembrane oligomerization, the ToxR-TM-MBP fusion proteins are
expressed in E. coli. Membrane localization of the fusion protein is detected based on complementation of a nonpolar
malE mutant E. coli strain. Insertion of the
ToxR-TM-MBP fusion protein into the inner membrane, such that the MBP
domain localizes to the periplasmic space, is detected by determining
whether the bacteria are able to transport maltose and thus grow on
maltose-minimal medium. Dimerization of the fusion protein is
determined based on expression of the cat gene, which is
under the control of the dimerization-dependent
transcription activator ToxR. E. coli strains expressing
ToxR-TM-MBP fusion proteins that dimerize are resistant to
chloramphenicol, whereas strains expressing fusion proteins that lack a
dimerization sequence are sensitive to chloramphenicol. Plasmid
pccVacA-wt and pccVacA-G14A have been described previously (38).
Additional plasmids containing mutations within the vacA amino-terminal sequence were constructed using the method of Perrin and
Gilliland (40). As a control, the portion of vacA
corresponding to amino acids 295-326 was also cloned into the TOXCAT
plasmid, generating pccVacA-295/326. Chloramphenicol acetyltransferase (CAT) enzyme was measured by an antigen-capture enzyme-linked immunosorbent assay (Roche Molecular Biochemicals)
according to the manufacturer's instructions.

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Fig. 2.
Schematic illustration of the
principles on which the TOXCAT assay is based (39). VacA sequences
(depicted as coiled lines) were cloned between a sequence
encoding the transcription activator domain of V. cholerae ToxR (squares) and a sequence encoding
the periplasmic domain of the E. coli MBP
(circles) so that ToxR-VacA-MBP fusion proteins would be
expressed. If ToxR-VacA-MBP fusion proteins insert into the E. coli inner membrane such that the MBP domain localizes to the
periplasmic space, cells are able to transport maltose and thus can
grow in maltose-minimal medium (designated
Mal+). Dimerization of the fusion proteins
results in the activation of the dimerization-dependent transcription
activator ToxR, which leads to expression of the cat gene
from the ToxR-activated cholera toxin promoter (pctx). Cells
expressing cat are designated Cat+.
The figure depicts four possible phenotypes expressed in the TOXCAT
assay.
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Construction of H. pylori Strains Expressing Mutant VacA
Proteins--
Plasmid pAV202 (41), which contains 567 bp of
cysS, the cysS-vacA intergenic region, 883 bp of
the vacA coding sequence from H. pylori 60190, and a CAT gene inserted at the 3'-terminus of cysS, was used
as the template for all site-directed mutagenesis reactions. Mutations
encoding single amino acid substitutions were introduced into pAV202
using the method of Perrin and Gilliland (40) or inverse PCR (42).
These mutations then were introduced into the H. pylori
chromosomal vacA gene by natural transformation and allelic
exchange, as described previously (37, 41, 43). Mutants were selected
on Brucella agar plates containing 5 µg/ml chloramphenicol. To confirm that the desired mutations had been introduced successfully into the chromosomal vacA gene,
fragments of the 5'-end of vacA were PCR amplified and
analyzed by restriction endonuclease digestion and nucleotide sequence analysis.
Purification of H. pylori VacA--
H. pylori strains
were grown in sulfite-free Brucella broth containing
activated charcoal (44). VacA was purified in an oligomeric form from
culture supernatants of H. pylori strains, as described
previously (10). Purified VacA preparations were routinely acid
activated before the addition of VacA to planar lipid bilayer chambers
or cell culture wells, as described previously (13, 15).
Planar Lipid Bilayer Methodology--
Planar lipid bilayers,
composed of egg
phosphatidylcholine/dioleoylphosphatidylserine/cholesterol (55:15:30
mol%) dissolved in n-decane were prepared as described
previously (31, 33, 37, 41). Purified acid-activated VacA toxins were
added to the lipid bilayers in a buffer consisting of 5 mM
citric acid (pH 4.0) and 2 mM EDTA, with the salt
composition as described under
"Results." Membrane currents were
measured as described previously (37, 41). The potential is indicated
relative to the cis side, defined as the chamber to which the protein
was added. Permeability ratios were determined from the
Goldman-Hodgkin-Katz equation (45), after the membrane voltage for zero
current (reversal potential) in asymmetric salt concentrations was
measured. Statistical significance was analyzed using Student's
t test.
Analysis of Membrane Potential of HeLa Cells--
Experiments to
analyze the membrane potential of HeLa cells were performed as
described previously (22, 23). Briefly, HeLa cells were detached with
trypsin/EDTA, washed, and then incubated with
bis-(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol (oxonol VI)
(Molecular Probes) in a final concentration of 2.5 µM for 15 min at 37 °C. A cell suspension (2 ml) was placed in a stirred quartz cuvette at 37 °C in a PerkinElmer Life Sciences LS50B
fluorometer. After stabilization of the fluorescence signal
(excitation, 585 nm; emission, 645 nm), acid-activated VacA toxins
(final concentration 5 µg/ml) were added to the cells, and changes in
the fluorescence were monitored. Depolarization causes a
potential-dependent change in the cytoplasmic/transmembrane
distribution of the fluorophore, which is accompanied by a change in
fluorescence (46).
Cell Culture--
HeLa cells were grown in minimal essential
medium (modified Eagle's medium containing Earle's salts) containing
10% fetal bovine serum. Acid-activated VacA preparations were
incubated with cultured cells in a microtiter format, as described
previously (13, 15). After incubation for 24 h, the cells were
examined by inverted light microscopy. Samples that induced vacuolation in >50% of cells were scored as positive for the vacuolating
cytotoxin phenotype. In some experiments, vacuolation was also
quantified by neutral red uptake assay (47).
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RESULTS |
Identification of VacA Amino Acids Required for Transmembrane
Protein Dimerization in a TOXCAT Model System--
The amino-terminal
hydrophobic segment of VacA contains three tandem GXXXG
motifs, which are characteristic of transmembrane dimerization
sequences (Fig. 1). To investigate the role of these GXXXG
motifs in greater detail, we used the TOXCAT model system (Fig. 2),
which has been used to study transmembrane oligomerization of proteins
in a natural membrane environment (38, 39, 48-50). We used a
previously described plasmid designated pccVacA-wt (38), which encodes
a fusion protein in which the amino-terminal 32 amino acids of VacA are
inserted between the transcription activation domain of ToxR and the
periplasmic domain of MBP, and which also contains the cat
gene under the control of the V. cholerae ctx promoter. To
examine the role of the GXXXG motifs, we used four related
plasmids that were identical to pccVacA-wt except for the presence of
mutations within the vacA sequence which resulted in
substitution of the Gly-14, Gly-18, Gly-22, and Gly-26 residues by
alanine. We also constructed two related plasmids containing mutations
within the vacA sequence which resulted in substitution of
the Pro-9 and Gly-13 residues by alanine. Pro-9 was selected for
analysis because this residue is reported to be important for VacA
cytotoxic activity (51). Gly-13 was selected for analysis as a control
because this glycine residue is not located within a GXXXG
motif. As an additional control, a 32-amino acid segment (amino acids
295-326) from a predicted hydrophilic region of VacA (Fig. 1) was also
cloned into the TOXCAT vector. When analyzed by immunoblot analysis
using anti-MBP antiserum, each of these plasmid-containing E. coli strains produced ToxR-TM-MBP fusion proteins (Fig.
3A).

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Fig. 3.
Analysis of mutations in the VacA hydrophobic
region using the TOXCAT system (39). A, expression of
ToxR-VacA-MBP fusion proteins. E. coli strain MM39
carrying plasmid pccVacA-wt (lane a), pccVacA-P9A
(lane b), pccVacA-G13A (lane c), pccVacA-G14A
(lane d), pccVacA-G18A (lane e), pccVacA-G22A
(lane f), pccVacA-G26A (lane g), pccVacA-295/326
(lane h), and no plasmid (lane i) were cultured
in Luria-Bertani medium to an A600 of about 0.3. Equal culture volumes were pelleted, lysed in SDS-sample buffer,
separated by SDS-PAGE, and immunoblotted using anti-MBP antiserum (New
England Biolabs). B, membrane insertion of ToxR-VacA-MBP
fusion proteins determined by growth on M9-maltose. E. coli
strain MM39 carrying plasmid pccVacA-wt, pccVacA-P9A, pccVacA-G13A,
pccVacA-G14A, pccVacA-G18A, pccVacA-G22A, pccVacA-G26A, and
pccVacA-295/326 were grown on M9-glucose plates. Bacteria were
resuspended in M9-maltose broth at an A600 of
~0.3. Strains were then spotted onto either M9-glucose or M9-maltose
plates. All strains, except for MM39 pccVacA-295/326, grew on
M9-maltose, indicating that the fusion proteins inserted into the
E. coli inner membrane. C, expression of CAT by
E. coli strains. E. coli strains were
cultured in Luria-Bertani medium to an A600 of
about 0.3. CAT protein levels from each strain was quantified using a
CAT enzyme-linked immunosorbent assay. Results represent the mean ± S.D. from triplicate cultures. MM39 pccVacA-G14A, pccVacA-G18A,
pccVacA-G26A, and pccVacA-295/326 produced significantly less CAT than
did MM39 pccVacA-wt (Student's t test; p < 5 × 10 7).
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To determine whether the putative transmembrane sequences encoded by
these plasmids promoted membrane localization of the ToxR-TM-MBP fusion
proteins, E. coli strains containing these plasmids were
cultured on M9-maltose medium. Consistent with previous results (38),
E. coli strain MM39 containing pccVacA-wt was able to grow
on M9-maltose medium, whereas MM39 with no plasmid was unable to grow
on this medium (Fig. 3B, data not shown). As expected,
E. coli containing pccVacA-295/326 was unable to grow on
this medium. The failure of this strain to grow on M9-maltose medium is
consistent with the failure of this VacA hydrophilic segment to mediate
insertion of the TOXCAT fusion protein into the inner membrane of
E. coli. Each of the plasmids containing mutant
vacA sequences grew on M9-maltose medium, similar to
E. coli containing the parental wild-type vacA
plasmid (Fig. 3B). These results indicate that none of the
single amino acid substitutions in the VacA hydrophobic region altered
the capacity of this VacA segment to mediate insertion of the
ToxR-TM-MBP fusion proteins into a membrane.
We next analyzed whether the mutant VacA segments were able to mediate
dimerization of the TOXCAT fusion proteins. As reported previously
(38), E. coli MM39 containing pccVacA-wt produced high
levels of CAT, whereas E. coli MM39 containing no
plasmid produced no CAT enzyme (Fig. 3C and data not shown).
The control strain containing pccVacA-295/326 produced barely any
detectable CAT. Compared with the E. coli MM39 containing
pccVacA-wt, E. coli containing plasmids encoding
P9A, G13A, and G22A mutations expressed similar high levels of CAT
enzyme. In contrast, E. coli containing plasmids
encoding G14A, G18A, or G26A mutations expressed significantly lower
levels of CAT than did the strain containing pccVacA-wt. These results
indicate that several glycine residues within the VacA hydrophobic
region contribute to transmembrane protein dimerization and that the
glycines located within the G14XXXG18 motif are particularly
important for protein dimerization in this system.
Role of the VacA Amino-terminal Hydrophobic Region in Vacuolating
Cytotoxic Activity--
Although the experiments described above using
the TOXCAT system provide useful insights into the function of the VacA
amino-terminal segment, it seems possible that the conformation and
function of this segment might be considerably different in the context of the native VacA protein interacting with eukaryotic cells, rather
than the TOXCAT fusion proteins interacting with the inner membrane of
E. coli. Therefore, we next undertook studies designed to
analyze the function of the amino-terminal hydrophobic region in the
context of the intact VacA protein produced by H. pylori.
For these studies, we introduced mutations encoding P9A, G13A, G14A,
G18A, G22A, and G26A into the H. pylori chromosomal
vacA gene, as described under "Materials and Methods."
Each of the mutant strains expressed and secreted VacA proteins of the
expected size (Fig. 4A). The
level of VacA produced by each of these mutant strains was lower than
the level of VacA produced by a wild-type H. pylori strain
but similar to the level of VacA produced by a control strain without
any vacA mutations in which a chloramphenicol resistance
cassette was introduced upstream from the vacA promoter, within the cysS-vacA intergenic region (data not
shown). This indicates that the presence of the chloramphenicol
cassette in this region results in diminished levels of VacA
expression, presumably by altering levels of vacA
transcription (52). All of the mutant VacA proteins were detected as
large oligomeric structures when H. pylori culture
supernatants were fractionated by gel filtration chromatography. Thus,
although TOXCAT experiments indicated an apparent role for Gly-14 and
Gly-18 in dimerization within a membrane, these residues were not
essential for oligomerization of the intact VacA proteins in solution.
It seems likely that oligomerization of water-soluble VacA monomers
involves interactions of multiple domains rather than only homotypic
interactions of the amino-terminal hydrophobic regions of different
monomers. In addition, there may be substantial differences between the
structure of VacA in solution compared with the structure of VacA in a
membrane.

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Fig. 4.
Expression and vacuolating activity of
wild-type and mutant VacA proteins secreted by H. pylori. A, expression of VacA
proteins. H. pylori mutant strains were grown in broth
culture, and broth culture supernatants were analyzed by immunoblot
assay using anti-VacA serum. Lane a, H. pylori
60190 expressing wild-type VacA; b, VacA-P9A; c,
VacA-G13A; d, VacA-G14A; e, VacA-G18A;
f, VacA-G22A; g, VacA-G26A. Each of the strains
expressed and secreted a VacA protein. B, vacuolating
activity. Equal amounts (20 µg/ml) of purified VacA proteins were
added to the medium overlying HeLa cells. Vacuolating activity was
measured by neutral red uptake assay (47). Results represent the
means ± S.D. from duplicate experiments, each performed in
triplicate, and are expressed as the percent of neutral red uptake
relative to control cells treated with wild-type VacA purified from
H. pylori strain 60190.
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Next, we sought to determine the effect of the various mutations
described above on the capacity of VacA to induce vacuolating cytotoxic
effects on eukaryotic cells. Purified preparations of each of the VacA
proteins were standardized by protein concentration, acid-activated,
and then added to HeLa cells. As a control, wild-type VacA was purified
from the wild-type H. pylori strain 60190. VacA proteins
containing G13A, G22A, or G26A mutations each induced vacuolation of
HeLa cells (Table I). However, quantification of VacA-induced
vacuolation by neutral red uptake assay (47) revealed that VacA-G13A
and VacA-G22A were consistently less active than wild-type toxin from
H. pylori strain 60190 (Fig. 4B). In contrast to
VacA-G13A, VacA-G22A, and VacA-G26A, VacA proteins containing P9A,
G14A, or G18A mutations did not induce any detectable vacuolation, even
at the highest protein concentration tested (Table I and Fig.
4B). These data indicate that the vacuolating activity of
VacA is detectably altered by several of the alanine substitution
mutations. However, an intact proline and an intact G14XXXG18 motif are absolutely
essential for vacuolating activity.
Role of the VacA Amino-terminal Hydrophobic Region in Formation of
Channels in Planar Lipid Bilayers--
To test the hypothesis that the
G14XXXG18 motif or perhaps one of
the other GXXXG motifs contributes to the capacity of VacA
to form anion-selective membrane channels, VacA proteins containing the
mutations described above were purified from H. pylori broth culture supernatant. These purified proteins then
were standardized according to protein concentration and were tested
for the capacity to form membrane channels in a planar lipid bilayer
system (37, 41). When tested at a VacA protein concentration of 30 nM, wild-type VacA formed anion-selective membrane channels
in the expected manner, as described previously (Table I). VacA
proteins containing G13A, G22A, or G26A mutations also formed membrane
channels, with an anion selectivity similar to that of channels formed
by wild-type VacA. The rate of membrane channel formation was similar
among these toxins, except for a slightly slower rate of membrane
channel formation by VacA-G22A. In contrast, formation of membrane
channels by VacA proteins containing P9A, G14A, or G18A mutations could not be detected under these conditions (Table I). These results indicate that Pro-9, Gly-14, and Gly-18 are essential for channel formation by VacA.
Role of the VacA Amino-terminal Hydrophobic Region in
Depolarization of HeLa Cells--
When added to HeLa cells, VacA forms
channels in the plasma membrane, which results in depolarization of the
resting membrane potential (22, 23). To analyze further the role of the
VacA amino-terminal hydrophobic region, we tested the capacity of each of the VacA preparations described above to induce depolarization of
HeLa cells. As expected, wild-type VacA induced membrane depolarization in a manner similar to that described previously (Fig.
5). VacA proteins containing G13A, G22A,
or G26A mutations also induced depolarization of the resting membrane
potential. In contrast, no depolarization was induced by VacA proteins
containing P9A, G14A, or G18A mutations. Thus, P9A, G14A, and G18A
mutations resulted in loss of the capacity to form membrane channels
when analyzed in the planar lipid bilayer system (Table I) as well as
loss of the capacity to depolarize HeLa cells (Fig. 5).

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Fig. 5.
Analysis of membrane depolarization induced
by mutant VacA proteins. VacA proteins were purified from H. pylori broth culture supernatants as described under "Materials
and Methods." HeLa cells were loaded with oxonol VI (a probe used to
monitor membrane potential). After the addition of 5 µg/ml
acid-activated VacA proteins, changes in fluorescence were
monitored. The arrow indicates the time at which toxin was
added to the cuvette. Wild-type VacA, VacA-G26A, VacA-G13A, and
VacA-G22A induced membrane depolarization, whereas VacA-P9A, VacA-G14A,
and VacA-G18A did not.
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DISCUSSION |
Several previous studies have shown that amino acid sequences near
the amino terminus of VacA play an important role in the process by
which VacA exerts cytotoxic effects. The experimental system used in
most of these previous studies has involved transiently transfecting
HeLa cells with plasmids encoding VacA (27, 28, 37, 51, 53-55). When
analyzed in this manner, intracellular expression of wild-type VacA
induces cell vacuolation, whereas intracellular expression of truncated
forms of VacA which lack as few as 10 amino acids at the amino terminus
fails to induce cell vacuolation (28, 53). To identify individual amino
acids that might be important for cytotoxic activity, alanine scanning mutagenesis of VacA residues 6-17 has been undertaken (51). When
transfected into HeLa cells, plasmids encoding VacA-P9A and VacA-G14A
fail to induce intracellular vacuolation, whereas plasmids encoding
other single alanine mutations induce cell vacuolation in a manner
indistinguishable from wild-type VacA (51). Additional studies
indicating the importance of amino acid sequences near the amino
terminus of VacA have used allelic exchange mutagenesis of the
chromosomally encoded vacA gene in H. pylori to
produce mutant toxins (37, 41, 43). When analyzed in this manner, VacA
toxins in which a portion of the amino-terminal region was deleted
(VacA-(
6-27)) or in which a 12-amino acid extension was added to
the amino terminus (s2/m1 VacA) exhibit defects in vacuolating activity
(37, 41).
In the current study, we investigated further the functional role of
the VacA amino terminus by analyzing a panel of mutant H. pylori toxins. In one or more assays of VacA function, each of the
mutant toxins examined exhibited at least a partial diminution in
activity compared with wild-type VacA. However, the most dramatic and
consistent defects were observed with VacA-P9A, VacA-G14A, and
VacA-G18A. Our data confirm results from transient transfection experiments which indicated that P9A and G14A mutations ablate cytotoxic activity (51), and they indicate that a G18A mutation also
ablates cytotoxic activity. The essential role played by Pro-9, Gly-14,
and Gly-18 in VacA cytotoxic activity thus helps to explain the defect
in cytotoxic activity observed in the previously characterized mutant
toxin VacA-(
6-27) (37).
It has been proposed that the cytotoxicity of VacA is dependent on the
capacity of this toxin to form anion-selective membrane channels (22,
31-33, 37, 41). In the present study, we found that the three nontoxic
mutant VacA proteins (VacA-P9A, VacA-G14A, and VacA-G18A) were each
defective in membrane channel formation, whereas the other mutant VacA
proteins retained the capacity for both cytoplasmic vacuolation and
membrane channel formation. The results obtained in a planar lipid
bilayer assay were similar to those obtained in a HeLa cell
depolarization assay. Thus, the current data provide further support
for the hypothesis that VacA channel forming activity is essential for
the formation of intracellular vacuoles. However, the current data do
not exclude the possibility that VacA may have other biological
activities in addition to the capacity for channel formation.
Studies of the mutant toxin VacA-
(6-27) indicated that the VacA
amino-terminal hydrophobic region does not play a role in toxin binding
to HeLa cells or toxin internalization by cells (37), but they
suggested that the amino-terminal hydrophobic region plays a role in
membrane channel formation. The analysis of mutant toxins containing
single alanine substitutions in the current study now provides strong
evidence that the amino-terminal hydrophobic region plays an essential
role in membrane channel formation. It may be presumed that the VacA
amino-terminal hydrophobic region either forms part of the channel or
plays an indirect role by stabilizing the structure of the channel. In
support of the former possibility, analysis of the predicted
hydrophobicity of VacA suggests that the amino-terminal segment is the
only contiguous stretch of hydrophobic amino acids long enough to span
a membrane, and results obtained using the TOXCAT system indicate that
this amino-terminal hydrophobic region is indeed capable of insertion into a membrane (Ref. 38 and the present study). It is not uncommon for
the pores of ion channels to be lined with hydrophobic amino acids
(56-58). However, it is possible that, in addition to the amino-terminal hydrophobic region, other VacA sequences may also insert
into the membrane to form a functional channel. In support of this
hypothesis, results of several studies have suggested that multiple
regions of the VacA protein are capable of inserting into lipid
membranes (59-61).
The appearance of VacA oligomers associated with membranes, as well as
the kinetics of membrane channel formation by VacA, suggest that VacA
channels are hexameric structures (10, 12, 31, 33). It seems likely
that multiple VacA-VacA interactions contribute to the formation of
VacA membrane channels. Results obtained using the TOXCAT system
indicate that the VacA amino-terminal hydrophobic region is capable of
mediating dimerization within a membrane environment (Ref. 38 and the
present work). In addition, previous studies have provided evidence
indicating the occurrence of interactions between other domains of the
toxin (54, 55, 62). The existence of multiple VacA-VacA interaction
domains may explain why deletion of the amino-terminal hydrophobic
region does not disrupt the formation of VacA oligomeric structures in solution (37).
VacA sequences containing G14A and G18A mutations exhibited defects in
the TOXCAT assay of protein dimerization, whereas the P9A mutation did
not alter the activity of the VacA sequence in this assay. This
suggests that the P9A mutation alters VacA function in a manner
different from the effects conferred by the G14A and G18A mutations.
Proline residues are known to be associated with turns or bends in
secondary structure, and therefore we speculate that the P9A mutation
alters the secondary structure of VacA within this region.
GXXXG motifs (such as that formed by Gly-14 and Gly-18) are
known to be important in the ability of transmembrane
-helices to
form homo-oligomers within a membrane environment (38, 39, 48-50). The
significance of the GXXXG motif has been most widely studied
using glycophorin A as a model transmembrane protein (39, 63-66). In
addition, the importance of GXXXG motifs has been reported in a variety of other transmembrane proteins (49, 67-69). Recently, the GXXXG motif has been identified as a motif involved in
helix-helix interactions in soluble proteins as well (70). Thus, the
inactivity of VacA sequences containing G14A and G18A mutations in the
TOXCAT system is consistent with the established role of
GXXXG motifs in protein-protein interactions.
The structure of the membrane insertion domain is known for only a few
bacterial toxins. In the case of toxins such as colicins and the
-endotoxins of Bacillus thuringiensis, the membrane
insertion domain consists of a bundle of hydrophobic and
amphipathic
-helices (71, 72), and in the case of toxins such as
Staphylococcus aureus
-toxin and Clostridium
perfringens perfringolysin O, the membrane insertion domain
consists of a
-barrel (73, 74). Like VacA, the
-toxin from
S. aureus also contains three tandem copies of the motif
GXXXG (75). However, unlike the VacA amino-terminal hydrophobic region, the glycine-rich region of
-toxin is
amphipathic. Structural analyses have indicated that this glycine-rich
or "hinge" region of
-toxin inserts into the membrane of
sensitive cells to form a transmembrane
-barrel (76-78). Thus,
although GXXXG motifs are frequently associated with
transmembrane
-helices (48), these motifs occasionally may be found
within transmembrane
-barrels (76-78). In future investigations, it
will be important to correlate the functional studies of the VacA
amino-terminal hydrophobic region described in this study with high
resolution structural analysis of VacA membrane channels.
We thank Beverly Hosse and Wayne Schraw for
technical assistance and William Russ and Donald Engelman (who
developed TOXCAT with support from the National Institutes of Health)
for providing reagents. DNA oligonucleotides were synthesized by the
Vanderbilt University DNA Chemistry Core Facility, and DNA sequence
analysis was performed by the Vanderbilt University DNA Sequencing Laboratory.
Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M212595200
The abbreviations used are:
NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid;
CAT, chloramphenicol
acetyltransferase;
MBP, maltose-binding protein;
TM, transmembrane;
wt, wild-type.
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