THE JOURNAL OF BIOLOGICAL CHEMISTRY
2324 –2331, 2004
Interactions between p-33 and p-55 Domains of the Helicobacter
pylori Vacuolating Cytotoxin (VacA)*
Published, JBC Papers in Press, October 30, 2003, DOI 10.1074/jbc.M310159200
Victor J. Torres‡§, Mark S. McClain¶, and Timothy L. Cover‡¶储**
From the ‡Department of Microbiology and Immunology and the ¶Department of Medicine, Vanderbilt University
School of Medicine, Nashville, Tennessee 37232-2605 and the 储Department of Veterans Affairs Medical Center,
Nashville, Tennessee 37212
The VacA toxin secreted by Helicobacter pylori is con-
sidered to be an important virulence factor in the patho-
genesis of peptic ulcer disease and gastric cancer. VacA
monomers self-assemble into water-soluble oligomeric
structures and can form anion-selective membrane
channels. The goal of this study was to characterize
VacA-VacA interactions that may mediate assembly of
VacA monomers into higher order structures. We inves-
tigated potential interactions between two domains of
VacA (termed p-33 and p-55) by using a yeast two-hybrid
system. p-33/p-55 interactions were detected in this sys-
tem, whereas p-33/p-33 and p-55/p-55 interactions were
not detected. Several p-33 proteins containing internal
deletion mutations were unable to interact with wild-
type p-55 in the yeast two-hybrid system. Introduction of
these same deletion mutations into the H. pylori vacA
gene resulted in secretion of mutant VacA proteins that
failed to assemble into large oligomeric structures and
that lacked vacuolating toxic activity for HeLa cells. Ad-
ditional mapping studies in the yeast two-hybrid system
indicated that only the N-terminal portion of the p-55
domain is required for p-33/p-55 interactions. To charac-
terize further p-33/p-55 interactions, we engineered an
H. pylori strain that produced a VacA toxin containing an
enterokinase cleavage site located between the p-33 and
p-55 domains. Enterokinase treatment resulted in com-
plete proteolysis of VacA into p-33 and p-55 domains,
which remained physically associated within oligomeric
structures and retained vacuolating cytotoxin activity.
These results provide evidence that interactions be-
tween p-33 and p-55 domains play an important role in
VacA assembly into oligomeric structures.
Helicobacter pylori is a Gram-negative, spiral-shaped mi-
croaerophilic bacterium that colonizes the gastric mucosa of
more than 50% of the human population (1). Colonization of the
gastric mucosa by H. pylori results in mucosal inflammation
and is recognized as a major risk factor for the development of
peptic ulcer disease, distal gastric adenocarcinoma, and gastric
lymphoma (2– 4).
* This work was supported in part by National Institutes of Health
Grants AI39657 and DK53623 and by the Medical Research Depart-
ment of the Department of Veterans Affairs (to T. L. C.). The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a structural biology predoctoral fellowship from the
Vanderbilt University Center for Structural Biology.
** To whom correspondence should be addressed: Division of Infec-
tious Diseases, A3310 MCN, Vanderbilt University School of Medicine,
Nashville, TN 37232. Tel.: 615-322-2035; Fax: 615-343-6160; E-mail:
timothy.L.cover@vanderbilt.edu.
2324
Vol. 279, No. 3, Issue of January 16, pp.
Printed in U.S.A.
Received for publication, September 12, 2003
Most virulent H. pylori strains secrete a cytotoxin known as
VacA into the extracellular space (5– 8). Several lines of evi-
dence indicate that VacA contributes to the capacity of H. py-
lori to colonize the stomach and that this toxin is an important
virulence factor in the pathogenesis of peptic ulceration and
gastric cancer (6 –12). The most extensively characterized ac-
tivity of VacA is its capacity to induce vacuolation in mamma-
lian cells (5, 13). The membranes of these VacA-induced vacu-
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oles contain markers for late endosomal and lysosomal
compartments (14). The precise mechanism of VacA-induced
vacuole formation is not completely understood but is thought
to involve binding of the toxin to the plasma membrane, inter-
nalization of the toxin, and action of the toxin in an intracel-
lular site (6 – 8). One model proposes that VacA forms anion-
selective channels in the membrane of late endocytic
compartments (6 – 8, 15, 16). In addition to inducing the forma-
tion of intracellular vacuoles, VacA causes multiple other ef-
fects on target cells, including depolarization of the membrane
potential (16, 17), permeabilization of epithelial monolayers
(18), apoptosis (19 –22), detachment of epithelial cells from the
basement membrane (12), interference with the process of an-
tigen presentation (23), and inhibition of T lymphocyte activa-
tion (24).
The vacA gene is translated as a 139 –140-kDa protoxin and
shows no striking similarity to any other known bacterial toxin
(5, 11, 25, 26). Upon expression, this protoxin undergoes N- and
C-terminal processing to yield a mature 88-kDa secreted VacA
toxin (27). The mature secreted 88-kDa VacA toxin can undergo
spontaneous proteolytic degradation into fragments that are
about 33 and 55 kDa in mass (11, 27–29). This degradation has
been observed especially when preparations of the purified
toxin are stored for prolonged periods (11, 28). The site of
proteolytic cleavage is predicted to be located within a hydro-
philic surface-exposed loop (11, 27). It has been presumed that
the two fragments (termed p-33 and p-55) represent two do-
mains or subunits of VacA (11, 30, 31). It should be noted that
the nomenclature for describing these fragments has not been
uniform; several previous studies have described 37- and 58-
kDa fragments (11, 30, 32–36). In the current study, we desig-
nate the two fragments as p-33 and p-55, based on the results
of mass spectrometry analysis (27).
Previous studies have identified specific functions that are
attributable to either the p-33 or the p-55 domain. Several lines
of evidence indicate that amino acid sequences within the p-55
domain (located at the C terminus of the mature secreted toxin)
mediate binding of VacA to host cells (28, 31, 37, 38). VacA
binding to cells is inhibited by antiserum reactive with the p-55
fragment but not inhibited by antiserum to the p-33 fragment
(28). Also, a truncated form of VacA containing mainly the p-55
VacA fragment was reported to bind to target cells in a manner
similar to the full-length VacA (31). Amino acid sequences
This paper is available on line at http://www.jbc.org
 H. pylori VacA
located within a hydrophobic region near the N terminus
(within the p-33 domain) are required for membrane channel
formation by VacA (39, 40). When expressed in transiently
transfected cells, neither the p-33 nor the p-55 domain alone is
sufficient for vacuolating toxin activity (32, 34, 41). In tran-
siently transfected cells, expression of the entire p-33 domain
as well as about 111 amino acids from the N terminus of the
p-55 domain is required for vacuolating toxin activity (32).
Secreted VacA proteins assemble into large water-soluble
and flower-shaped structures composed predominantly of
12–14 VacA monomers (29, 30, 42). Assembly of VacA into
oligomeric structures is presumably required for membrane
channel formation. This hypothesis is supported by electron
microscopy studies in which VacA associated with membranes
appears as oligomeric structures (15, 42), as well as electro-
physiologic studies in which the kinetics of membrane channel
formation by VacA have been investigated (43). Thus far, the
process by which VacA assembles into higher order structures
has not been studied in any detail. Therefore, the goal of this
study was to characterize the interactions that may mediate
assembly of VacA into oligomeric structures. We describe here
several lines of evidence indicating that interactions occur be-
tween the p-33 and p-55 domains of VacA, and we identify
candidate regions within these two domains that are required
for these interactions. In addition, we propose that interactions
between p-33 and p-55 domains are essential for the assembly
of VacA into oligomeric structures and for VacA vacuolating
cytotoxin activity.
EXPERIMENTAL PROCEDURES
Bacterial and Yeast Strains, Media, and Growth Condition—H. py-
lori strain 60190 (ATCC 49503) was grown on trypticase soy agar plates
containing 5% sheep blood at 37 °C in ambient air containing 5% CO2.
Liquid cultures were grown in sulfite-free Brucella broth supplemented
with either 5% fetal bovine serum or 0.5% activated charcoal. Esche-
richia coli XL1-Blue (Stratagene) was used for plasmid propagation and
was grown on Luria-Bertani (LB) broth or LB agar at 37 °C. Yeast
two-hybrid experiments were performed with Saccharomyces cerevisiae
strain YRG-2 (MAT␣ ura3-52 his3-200 ade2-101 lys2-801 trp1-901
leu2-3,112 gal4-542 gal80-538 LYS2::UASGAL1-TATAGAL1-HIS3
URA3::UASGAL4 17mers(x3)-TATACYC1-lacZ) (Stratagene). Yeast strains
were grown in rich medium (yeast extract, peptone, dextrose (YPAD)) or
in synthetic defined (SD) minimal medium (supplemented with re-
quired amino acids and glucose) at 30 °C as described in the GAL4
Two-hybrid Phagemid manual (Stratagene).
Construction of VacA Yeast Two-hybrid Vectors Containing vacA
Fragments—vacA sequences encoding the wild-type p-33 and p-55 VacA
domains were PCR-amplified from genomic DNA of H. pylori strain
60190 and cloned into plasmids encoding the transcription activation
domain (pAD)1 and/or the DNA binding domain (pBD) of the GAL4
Two-hybrid Phagemid system (Stratagene). The sequences of the prim-
ers used for cloning the different vacA fragments into the yeast two-
hybrid vectors are described in Table I. Primers combinations, restric-
tion site used for cloning the PCR products into the pAD and pBD
vectors, and the amino acid numbers of the VacA fragments are de-
scribed in Table II.
To facilitate the introduction of in-frame deletion mutations into the
pAD33 plasmid, vacA sequences encoding p-33 mutant domains were
PCR-amplified from genomic DNA of previously described H. pylori
strains harboring the specific in-frame vacA deletions (39) and cloned
into the pAD vector (Table II). vacA sequences encoding p-55 fragments
were PCR-amplified from genomic DNA of H. pylori strain 60190 (Table
II) and were cloned into the pBD vector. The entire vacA fragment in
each p-33 and p-55 encoding plasmid was analyzed by nucleotide se-
quence analysis in order to verify that no unintended mutations had
been introduced.
Yeast Methods—Yeast strains were co-transformed with 400 ng of
individual plasmids using the lithium acetate method (44) and cultured
1
The abbreviations used are: pAD, plasmids encoding the GAL4
transcription-activation domain; pBD, plasmids encoding the GAL4
DNA binding domain; CHAPS, 3-[(3-cholamidopropyl)-dimethyl-
ammonio]-1-propanesulfonate.
2325
TABLE I
Oligonucleotides used in this study
Primer Nucleotide sequence
name
AND510a 5⬘-TATAGCCCGGGCCGCCTTTTTTACAACCGTGAT
AND511 5⬘-CCCCTCGACTTATTTAGCACCACTTTGAGAAG
AND512 5⬘-CCCCGGATCCGCCTTTTTTACAACCGTGAT
AND515a 5⬘-CCCCGTCGACTTAAGCGTAGCTAGCGAAACGCG
AND516 5⬘-CCCCGGATCCAACGACAAACAAGAGAGCAG
AND6098 5⬘-CCCCGAATTCAACGACAAACAAGAGAGCAG
OP4175 5⬘-CCCCGGATCCGCCTTTTTTACAACCCTTGG
OP4176 5⬘-CCCCGTCGACTTACGTATCAATACCTTTAAAAT
BAR1556 5⬘-CCCCGAATTCGGTAATGGTGGTTTCAACAC
BAR1557 5⬘-CCCCGAATTCACTAGGTCAATCTTTTCTGG
BAR1558 5⬘-CCCCGTCGACTTAGCCAGTTTCCAAACGCACG
BAR1559 5⬘-CCCCGTCGACTTAGATTTTCGCTTTCAATAAAACA
OP2891 5⬘-CCGACTACAAGGATGACGACGACAAAG
OP2892 5⬘-CTTTGTCGTCGTCATCCTTGTAGTCGG
on SD medium supplemented with the required amino acids and glu-
cose at 30 °C as described in the GAL4 Two-hybrid Phagemid manual
(Stratagene). For a positive protein-protein interaction control, we co-
transformed the yeast with pBD-WT and pAD-WT plasmids, which
encode fusion proteins consisting of amino acids 132–236 of wild-type
␭cI, fragment C, together with either the GAL4-BD or -AD, respectively.
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For a negative control (i.e. two proteins that do not interact), we used
the pAD-WT plasmid co-transformed with a pBD-pLamin C plasmid,
which expresses the BD of GAL4 fused to amino acids 60 –230 of human
lamin C. We also used plasmids pAD-Mut and pBD-Mut, which encode
a mutated (E233K) ␭cI, fragment C, fused to either the GAL4-AD or
-BD. The cI-E233K mutation interferes with the interaction between
the cI monomers, resulting in a protein-protein interaction that is
weaker than that of wild-type proteins. Plasmids expressing positive
and negative interaction control proteins were obtained from
Stratagene.
One colony of each co-transformant was grown in 2 ml of SD medium
containing 2% (w/v) glucose and lacking Trp and Leu (SD⫺Leu,⫺Trp)
at 30 °C with aeration for ⬃24 h. Cultures were then normalized to an
A600 of about 0.26 in a final volume of 100 ␮l of SD⫺Leu,⫺Trp,⫺His
broth, and 10-fold serially diluted into SD⫺Leu,⫺Trp,⫺His broth in a
microtiter plate. Diluted cultures (5 ␮l) were seeded on duplicate
SD⫺Leu,⫺Trp plates (selection for plasmids) and SD⫺Leu,⫺Trp,⫺His
plates (selection for protein-protein interactions) and incubated at
30 °C for 3–10 days.
To confirm the occurrence of protein-protein interactions, a ␤-galac-
tosidase liquid assay was performed using the Yeast ␤-Galactosidase
Assay Kit as described by the manufacturer (Pierce). Briefly, individual
co-transformants were grown in 2 ml of SD⫺Leu,⫺Trp medium con-
taining 2% (w/v) glucose at 30 °C with aeration for ⬃16 –24 h until
cultures reached mid-log phase (A600 0.4 – 0.5). 150-␮l aliquots of the
cultures were then mixed with 150 ␮l of the working solution (equal
volume of 2⫻ ␤-Galactosidase Assay Buffer and Y-PERTM Reagent) and
then incubated at 37 °C until solutions turned yellow (⬃1– 4 h), at
which time reactions were stopped by the addition of 175 ␮l of ␤-Ga-
lactosidase Assay Stop Solution. The time required for yellow color
development was recorded for each tube. The A410 values of clarified
reaction supernatants (200 ␮l) were measured in a 96-well plate, and
the ␤-galactosidase activity was calculated by using the Miller equation
as described in the Yeast ␤-Galactosidase Assay Protocol (Pierce). The
values presented are the average of at least three independent co-
transformants (means ⫾ S.D.). Statistical significance was analyzed
using Student’s t test.
Introduction of DNA Encoding a FLAG Tag Epitope and Enteroki-
nase Site into the vacA Gene of H. pylori 60190 —To modify the vacA
gene so that it encoded a VacA protein containing a FLAG tag (DYK-
DADDDK) and an enterokinase cleavage site (after the underlined K),
complementary primers OP2891 and OP2892 encoding the FLAG
epitope (Table I) were annealed and ligated into the unique StuI site of
plasmid pA178 (45). The resulting plasmid, pA178-FLAG, contained a
sequence encoding the FLAG epitope in the proper orientation inserted
between amino acids 317 and 318 of VacA. Plasmid pA178-FLAG was
then used to transform H. pylori strain VM022 (39), and transformants
were selected by growth on 5.5% sucrose plates, as described previously
(39, 46). Analysis of a single transformant (H. pylori VM088) by PCR
and by DNA sequence analysis confirmed that the sequence encoding
the FLAG epitope and the enterokinase cleavage site had been intro-
duced into the desired location.
2326 H. pylori VacA
FIG. 1. Proteolytic degradation of VacA into 33- and 55-kDa
fragments. Proteins precipitated from broth culture supernatant of
H. pylori 60190 by a 50% saturated solution of ammonium sulfate (lanes
A and B) or purified VacA (lanes C and D) from H. pylori 60190 were
electrophoresed on a 10% SDS-polyacrylamide gel, transferred to a
nitrocellulose membrane, and immunoblotted with polyclonal anti-
VacA serum. These preparations exhibit varying degrees of spontane-
ous VacA proteolysis into p-33 and p-55 fragments.
Purification of Oligomeric Forms of VacA—VacA was purified from
broth culture supernatants of H. pylori as described previously (29).
Briefly, broth culture supernatant proteins were concentrated by pre-
cipitation with a 50% saturated solution of ammonium sulfate and
resuspended in phosphate-buffered saline. Oligomeric VacA (⬃1,000
kDa) was purified by gel filtration chromatography using a Superose
6HR 16/50 column. To analyze the oligomeric state of the enterokinase-
treated FLAG-VacA, 40 ␮g of cleaved toxin was applied to a Superose
6HR 16/50 column, and fractions (2 ml each) were collected. Fractions
were analyzed by immunoblot analysis with an anti-VacA serum.
Proteolysis of FLAG-VacA with Enterokinase—Purified FLAG-VacA
was proteolytically cleaved with enterokinase as described by the Re-
combinant Enterokinase Kit manual (Novagen). Briefly, 10 ␮g of puri-
fied FLAG-VacA was incubated with 1 unit of enterokinase and 5 ␮l of
10⫻ enterokinase cleavage buffer in a final volume of 50 ␮l for ⱖ16 h at
room temperature. For mock treatment, FLAG-VacA was treated in the
same manner but without enterokinase. Proteolysis of the full-length
FLAG-VacA was assessed by immunoblot analysis using an anti-VacA
serum or the M2 monoclonal anti-FLAG antibody (Sigma).
Cell Culture and Vacuolating Assays—HeLa cells were grown in
minimal essential medium (modified Eagle’s medium containing
Earle’s salts) supplemented with 10% fetal bovine serum. For vacuolat-
ing assays, HeLa cells were seeded (1.5 ⫻ 104) into 96-well plates 24 h
prior to each experiment. Protein concentrations of the purified VacA
toxins were determined by using a Micro-BCA assay (Pierce). Serial
dilutions of purified VacA toxins (standardized by protein concentra-
tion) were incubated with cells in serum-free medium containing 10 mM
ammonium chloride as described previously (5). Purified VacA prepa-
rations (VacA, FLAG-VacA, and cleaved FLAG-VacA) were acid-acti-
vated by adjusting them to pH 3 by the addition of 250 mM hydrochloric
acid before they were added to cell culture wells (47, 48). After 24 h of
incubation of VacA toxins with the cell monolayers, cells were examined
by inverted light microscopy. Toxins that induced vacuoles in more than
50% of the cells were scored positive for vacuolating cytotoxic activity.
Vacuolating activity was also quantified by a neutral red uptake assay
as described previously (49). Neutral red uptake data are presented as
A540 values (means ⫾ S.D.). Statistical significance was analyzed by
using Student’s t test.
RESULTS
Detection of p-33/p-55 Interactions in a Yeast Two-hybrid
System—Previous studies have reported that 88-kDa VacA
monomers commonly undergo spontaneous degradation, yield-
ing 33- and 55-kDa fragments (Fig. 1) (11, 27, 28). These two
fragments have been considered to represent two domains or
subunits of VacA (11, 20, 30, 31). To investigate potential
interactions among these two VacA fragments, we investigated
whether they were able to interact in a GAL4 transcription
activator yeast two-hybrid system (GAL4 Two-hybrid Phage-
mid; Stratagene) (50). This particular yeast two-hybrid system
was selected based on the low expression of the bait and prey
fusion proteins which, in turn, is thought to reduce the number
of false positive interactions (51). In this system, the yeast
GAL4 transcription activator has been divided into the follow-
ing two separate functional domains (52): (i) the transcription
activation domain (AD) present on plasmid pAD-GAL4 –2.1
(pAD), which encodes for the LEU2 gene as a selectable mark-
er; and (ii) the DNA binding domain (BD) present on the
plasmid pBD-GAL4-Cam (pBD), which encodes for the TRP1
gene as a selectable marker. If two fusion proteins interact in
this system, they will bring in close proximity the GAL4 tran-
scription activation domain and the GAL4 DNA binding do-
main to the GAL4/GAL1 promoters, which in turn will initiate
the transcription of the HIS3 and lacZ reporter genes. Protein-
protein interactions are then detected by the ability of co-
transformed yeast cells to grow in selective medium lacking
Leu, Trp, and His (SD⫺Leu,⫺Trp,⫺His) and by production of
␤-galactosidase activity.
vacA sequences encoding p-33 and p-55 fragments were
cloned into plasmids encoding the transcription activation do-
main (pAD) and the DNA binding domain (pBD), generating
plasmids pAD33, pBD33, pAD55, and pBD55 (Table II). In
order to test whether interactions among VacA fragments could
be detected in this system, we co-transformed all four possible
combinations of the p-33 and p-55 yeast two-hybrid plasmids
into the yeast reporter strain (Fig. 2A). Serial dilutions of
co-transformed yeast were then tested for their ability to grow
on SD medium lacking Leu and Trp, as well as SD medium
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lacking Leu, Trp, and His. All of the co-transformed yeast grew
on the former media, which confirmed that the transformation
had been successful and provided an indication of the relative
number of co-transformed yeast cells present in each dilution.
Yeast co-transformed with plasmids encoding p-33 and p-55
fusion proteins grew in SD medium lacking Leu, Trp, and His,
indicating that there was an interaction between these fusion
proteins (Fig. 2B). In contrast, yeast co-transformed with plas-
mids encoding p-33/p-33 or p-55/p-55 fusion proteins did not
grow on SD medium lacking Leu, Trp, and His (Fig. 2B). The
p-33/p-55 interaction observed within co-transformed yeast
was further confirmed by the analysis of ␤-galactosidase activ-
ity, which reflects the activation of the lacZ reporter gene (Fig.
2C). The p-33/p-55 interaction was independent of the protein
fused (AD or BD) to the p-33 or p-55 fragment, because both
combinations of tested plasmids (pAD33/pBD55 and pAD55/
pBD33) were able to activate the HIS3 and lacZ reporters (Fig.
2, B and C). Furthermore, none of the fusion constructs were
able to activate the reporter genes when transformed alone into
the yeast reporter strain (data not shown), indicating that the
interaction detected in the yeast two-hybrid system is due to a
specific interaction between p-33 and p-55 VacA fragments.
p-33/p-55 Interactions of VacA Mutant Fragments—Previ-
ously, we have reported the construction of H. pylori strains
containing in-frame deletion mutations in the vacA gene (39).
Results from this previous study indicated that a mutant VacA
protein with a deletion of amino acids 6 –27, VacA-(⌬6 –27),
could assemble into water-soluble oligomeric structures and
suggested that VacA proteins containing ⌬28 –108, ⌬56 – 83,
⌬85–127, or ⌬112–196 deletion mutations were defective in the
capacity for oligomer assembly (39) (Table III). We hypothe-
sized that the latter mutations might disrupt interactions be-
tween p-33 and p-55 domains, and that such a defect might
account for failure of these VacA mutant proteins to form
oligomeric structures. In order to test directly this hypothesis,
we introduced these same deletion mutations (i.e. ⌬6 –27, ⌬28 –
108, ⌬56 – 83, ⌬85–127, and ⌬112–196) into the pAD33 plas-
mid, as described under “Experimental Procedures” (Table II).
The pAD33 plasmid was chosen for mutagenesis based on the
observation that higher reporter activity was detected when
wild-type p-33 was fused to the transcription activation domain
and wild-type p-55 fused to the DNA binding domain, com-
pared with the opposite orientation (Fig. 2C). Mutant pAD33
plasmids were co-transformed into yeast along with the wild-
type pBD55 plasmid, and the activation of the reporter genes
 H. pylori VacA 2327
TABLE II
Yeast two-hybrid vectors containing vacA fragments
Y2H plasmidsa Forward primersb Reverse primersb Restriction enzymesc VacA amino acidsd

pBD33 AND510a AND511 SrfI/SalI 1–312

pAD33 AND512 AND511 BamHI/SalI 1–312
pBD55 AND6098 AND515a EcoRI/SalI 313–821
pAD55 AND516 AND515a BamHI/SalI 313–821
pAD⌬6–27 OP4175 AND511 BamHI/SalI 1–312 (⌬6–27)
pAD⌬28–108 AND512 AND511 BamHI/SalI 1–312 (⌬28–108)
pAD⌬56–83 AND512 AND511 BamHI/SalI 1–312 (⌬56–83)
pAD⌬85–127 AND512 AND511 BamHI/SalI 1–312 (⌬85–127)
pAD⌬112–196 AND512 AND511 BamHI/SalI 1–312 (⌬112–196)
pBD313–700 AND6098 BAR1559 EcoRI/SalI 313–700
pBD313–550 AND6098 BAR1558 EcoRI/SalI 313–550
pBD313–478 AND6098 OP4176 EcoRI/SalI 313–478
pBD550–821 BAR1557 AND515a EcoRI/SalI 550–821
pBD479–821 BAR1556 AND515a EcoRI/SalI 479–821
a
Name of the yeast two-hybrid (Y2H) plasmids expressing VacA fragments.
b
Oligonucleotides used to PCR-amplify the different vacA sequences. Oligonucleotide sequences are described in Table I.
c
Restriction sites used to clone the PCR products into the pAD and pBD yeast two-hybrid plasmids.
d
The VacA amino acid numbering system used in this table is based on designating the first amino acid (alanine) of the mature secreted VacA
toxin of strain 60190 (25) as amino acid 1. ⌬ indicates amino acids that are deleted in the mutant VacA fragments.

TABLE III
Characterization of mutant VacA toxins

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Y2H Oligomer Cytotoxic
p-33a p-55b interactionc formationd activitye

WT WT ⫹ ⫹ ⫹
⌬6–27 WT ⫹ ⫹ ⫺
⌬28–108 WT ⫺ ⫺ ⫺
⌬56–83 WT ⫺ ⫺ ⫺
⌬85–127 WT ⫺ ⫺ ⫺
⌬112–196 WT ⫺ ⫺ ⫺
a
The p-33 VacA domain contains amino acids 1–312.
b
The p-55 VacA domain contains wild type (WT) amino acids 313–
821.
c
Interaction between p-33 and p-55 VacA fragments was determined
using the yeast two-hybrid system (Y2H) as described in the text.
d
Oligomer formation of full-length VacA toxins secreted by H. pylori
was analyzed by determining whether these proteins eluted as large
oligomeric structures (⬇1,000 kDa) from a Superose 6 HR 16/50 FPLC
column (5, 39).
e
Cytotoxic vacuolating activity of VacA toxins for HeLa cells was
analyzed as described under “Experimental Procedures.”

activate the reporter genes when co-transformed with wild-

FIG. 2. Interaction of VacA fragments in the yeast two-hybrid
system. A, representation of the different p-33 and p-55 fusion proteins
and plasmid combinations used in the co-transformation experiments.
B, yeast cells were co-transformed with the plasmids depicted in A or
with controls plasmids described under “Experimental Procedures.”
Co-transformed yeast cells (normalized based on absorbance) were 10-
fold serially diluted, and identical inocula were plated on two types of
SD agar plates. All of the co-transformed yeast grew on SD⫺Leu,⫺Trp
plates. Protein-protein interactions were assessed by growth of the
co-transformed yeast cells on SD⫺Leu,⫺Trp,⫺His plates. C, ␤-galacto-
sidase liquid culture assay of the co-transformed yeast cells. Activation
of the protein-protein interaction reporters was only detected when
yeast were co-transformed with plasmids encoding wild-type p-33 to-
gether with plasmids encoding wild-type p-55. Results represent the
mean ⫾ S.D. from triplicate determinations, each representing a sepa-
rate colony. *, p ⬍0.05 when compared with the negative control; ⫹
cntrl, positive control; ⫺ cntrl, negative control.
was analyzed as described above. Wild-type pAD33 and
pAD⌬6 –27p-33 were each able to activate both the HIS3 and
lacZ reporter genes when co-transformed with wild-type
pBD55 (Fig. 3), whereas the other pAD33 mutants failed to
type pBD55. The p-55/⌬6 –27p-33 interaction was specific, be-
cause yeast cells co-transformed with pBD33 and pAD⌬6 –
27p-33 were not able to activate the reporter genes (data not
shown). Thus, mutations that disrupted oligomerization of
VacA secreted by H. pylori (39) also disrupted p-33/p-55 inter-
actions in the yeast two-hybrid system (Table III). Interest-
ingly, yeast co-transformed with plasmids expressing
⌬6 –27p-33 and p-55 consistently produced higher levels of
␤-galactosidase activity than did yeast expressing wild-type
p-33 and p-55 (Fig. 3B).
To further map putative amino acid sequences involved in
the p-33/p-55 interaction, we generated different p-55 frag-
ments (i.e. encoding amino acids 313–700, 313–550, 313– 478,
550 – 821, and 479 – 821) and cloned them into the pBD vector
as described under “Experimental Procedures” (Table II).
These new fusion proteins were tested for their ability to in-
teract with wild-type p-33 as described above (Fig. 4A). Yeast
co-transformed with the pAD33 plasmid and plasmids express-
ing p-55 fragments containing amino acids 313–700, 313–550,
or 313– 478 were able to grow on SD⫺Leu,⫺Trp,⫺His, whereas
yeast co-transformed with the pAD33 plasmid and plasmids
expressing p-55 fragments containing amino acids 479 – 821 or
550 – 821 were not able to grow (Fig. 4B). The former group of
co-transformed yeast grew slowly on SD⫺Leu,⫺Trp,⫺His
plates and expressed a relatively low level of ␤-galactosidase
activity (Fig. 4, B and C). This suggests that the interactions
2328 H. pylori VacA
FIG. 3. Interaction between wild-type p-55 and p-33 mutant
VacA fragments. A, co-transformed yeast cells (normalized based
on absorbance) were 10-fold serially diluted, and identical inocula
were plated on two types of SD plates. Protein-protein interactions
were assessed by growth of the co-transformed yeast cells on
SD⫺Leu,⫺Trp,⫺His plates. B, ␤-galactosidase liquid culture assay of
the co-transformed yeast cells. Activation of the protein-protein inter-
action reporters was only detected when yeast were co-transformed with
plasmids encoding wild-type p-33 or ⌬6 –27p-33 together with a plasmid
encoding wild-type p-55. Results represent the mean ⫾ S.D. from tripli-
cate colonies. *, p ⬍0.05 when compared with the negative control.
between the 313–700, 313–550, and 313– 478 p-55 fragments
and p-33 are weaker than the interaction between full-length
wild-type p-55 and p-33. When we co-transformed yeast with a
mutated version of the positive control plasmids (mutant ⫹
control described under “Experimental Procedures”) known to
encode proteins that interact with relatively low affinity (53), a
similar low level of ␤-galactosidase activity was detected (Fig.
4C). The data obtained with these mutant control plasmids
support our conclusion that the p-33 interacts weakly with
313–700, 313–550, and 313– 478 p-55 fragments.
Proteolytic Cleavage of H. pylori VacA into 33- and 55-kDa
Fragments—We sought to confirm the finding that p-33 inter-
acts with p-55 using VacA purified from H. pylori. Numerous
previous studies have demonstrated that VacA proteins pro-
duced by H. pylori can degrade into 33- and 55-kDa fragments
(11, 27, 28). However, it remains unclear whether proteolytic
cleavage of the 88-kDa VacA protein into 33- and 55-kDa frag-
ments alters VacA cytotoxic activity or alters its oligomeric
structure. One reason why this issue has not yet been ad-
dressed in any detail is that it has been difficult to experimen-
tally induce the desired cleavage. VacA preparations commonly
undergo partial proteolytic degradation during storage (Fig. 1),
but complete proteolysis of the 88-kDa VacA protein into
smaller fragments occurs uncommonly. Furthermore, sponta-
neous proteolytic degradation of VacA often involves cleavage
at multiple sites (27).
In order to test experimentally whether cleavage of VacA
into 33- and 55-kDa fragments alters its activity, we sought to
develop a system in which a protease could be used to induce
proteolytic cleavage at a specific site located between the 33-
and 55-kDa domains. To accomplish this, we constructed a
modified H. pylori strain in which an oligonucleotide encoding
the FLAG tag epitope and an enterokinase cleavage site was
inserted into the vacA allele of H. pylori 60190 as described
under “Experimental Procedures” (Fig. 5A). This enterokinase
cleavage site (Fig. 5B) provided a mechanism by which the
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FIG. 4. Interaction between wild-type p-33 and fragments of
p-55. A, representation of the different p-55 fragments used in the
co-transformation experiment. B, yeast cells co-transformed with the
pBD55 fragments depicted in A and wild-type pAD33 (normalized based
on absorbance) were 10-fold serially diluted, and identical inocula were
plated on two types of SD agar plates. C, ␤-galactosidase liquid culture
assay of the co-transformed yeast cells. Protein-protein interactions
were assessed by growth of the co-transformed yeast cells on
SD⫺Leu,⫺Trp,⫺His plates and by a ␤-galactosidase liquid culture as-
say. Sample numbers in C are identical to those in B. Wild-type p-33
was able to interact with p-55 fragments containing amino acids 313–
700, 313–550, and 313– 478 but not with amino acids 550 – 821 or
479 – 821. ␤-Galactosidase liquid culture assay results represent the
mean ⫾ S.D. from triplicate colonies. *, p ⬍0.05 when compared with
the negative control.
88-kDa VacA protein could be specifically cleaved into frag-
ments similar to the spontaneously arising 33- and 55-kDa
VacA fragments. Immunoblotting studies with an anti-VacA
serum indicated that an H. pylori strain (VM088) containing
this engineered vacA allele expressed and secreted VacA at
levels similar to wild-type H. pylori strain 60190 (data not
shown). The FLAG-VacA toxin was purified from H. pylori
VM088 culture supernatant as large oligomeric structures that
eluted from a gel filtration column in the same fractions as
wild-type VacA. Purified FLAG-VacA underwent spontaneous
partial degradation into two forms of the p-33 and p-55 VacA
fragments (Fig. 6B, 1st lane), which is consistent with previous
observations suggesting that proteolysis can occur at multiple
sites (27). As expected, enterokinase treatment resulted in the
complete cleavage of the full-length FLAG-VacA into p-55 and
FLAG-tagged p-33 (FLAGp-33) fragments as seen by immuno-
blot analysis using an anti-VacA serum or an anti-FLAG mono-
clonal antibody (Fig. 6). In contrast, enterokinase treatment of
the wild-type 60190 VacA toxin (lacking the FLAG tag epitope)
did not induce any detectable cleavage (data not shown).
Vacuolating Toxic Activity of Intact FLAG-VacA and Proteo-
lytically Cleaved FLAG-VacA—We next compared the cytotoxic
 H. pylori VacA
FIG. 5. Construction of H. pylori VM088 using a sacB-based
counter-selection approach. A, the pMM389 plasmid contains a
vacA fragment from H. pylori strain 60190 and a sacB/kan cassette (39,
46). Transformation of H. pylori 60190 with pMM389 yielded a kanamy-
cin-resistant transformant designated H. pylori VM022 (39). H. pylori
VM022 was transformed with pA178-FLAG plasmid; transformants
were selected on sucrose-containing medium, and a strain (H. pylori
VM088) with a sequence encoding a FLAG tag with an enterokinase site
inserted into the vacA gene was thereby obtained. B, VacA spontane-
ously degrades into two fragments, termed p-33 and p-55. Open arrows
indicate the most common site where wild-type VacA from H. pylori
60190 undergoes spontaneous cleavage and the resulting p-33 and p-55
fragments (27). Closed arrows indicate the enterokinase cleavage site
introduced with the FLAG tag epitope and the FLAGp-33 and p-55
fragments generated after enterokinase cleavage. The amino acid num-
bering system used in this figure is based on designating the first amino
acid (alanine) of the mature secreted VacA toxin of strain 60190 as
amino acid 1.
activities of intact FLAG-VacA and FLAG-VacA that had been
proteolytically cleaved into p-55 and FLAGp-33 fragments. Pu-
rified oligomeric FLAG-VacA was either treated with enteroki-
nase or mock-treated as described under “Experimental Proce-
dures.” Proteins were then acid-activated (a procedure used to
enhance vacuolating activity) and added to the neutral-pH
medium overlying HeLa cells (47, 48). The vacuolating activi-
ties of intact FLAG-VacA and proteolytically cleaved FLAG-
VacA were indistinguishable as determined by microscopic ex-
amination (data not shown) and analysis by a neutral red
uptake assay (Fig. 7). The vacuolating activities of full-length
FLAG-VacA and proteolytically cleaved FLAG-VacA were not
detectably different when tested in a range of toxin concentra-
tions from 2.5 to 10 ␮g/ml (Fig. 7 and data not shown).
VacA purified from wild-type H. pylori strain 60190 exhibits
very little vacuolating toxic activity unless it is acid- or alka-
line-activated prior to contact with cells (47, 48, 54). We hy-
pothesized that proteolytically cleaved VacA might exhibit vac-
uolating toxic activity even in the absence of acid activation.
However, neither intact nor proteolytically cleaved FLAG-
VacA exhibited detectable vacuolating activity in the absence
of acid activation, as determined by microscopic examination
(data not shown) and analysis by a neutral red uptake assay
(Fig. 7).
2329
FIG. 6. Enterokinase treatment of full-length FLAG-VacA re-
sults in production of 33- and 55-kDa VacA fragments. Purified
VacA from H. pylori VM088 (10 ␮g) was either treated with enteroki-
nase (1 unit) or mock-treated as described under “Experimental Proce-
dures.” Toxins (100 ng) were then electrophoresed on a 10% SDS-
polyacrylamide gel, transferred to a nitrocellulose membrane, and
reacted with anti-FLAG M2 monoclonal antibody (Sigma) (A). The blot
was then stripped and reacted with polyclonal anti-VacA serum (B).
Enterokinase treatment induced complete cleavage of full-length
FLAG-VacA into ⬃33- and ⬃55-kDa fragments. In preparations of
FLAG-VacA not treated with enterokinase, the FLAG tag epitope is
detected predominantly in full-length 88-kDa VacA and in the p-55
fragment (A, 1st lane). Only the ⬃33-kDa fragment retained the FLAG
tag epitope after enterokinase treatment (A, 2nd lane).
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FIG. 7. Vacuolating cytotoxic activity of intact and proteolyti-
cally cleaved FLAG-VacA. Identical aliquots of purified FLAG-VacA
were either treated with enterokinase as described under “Experimen-
tal Procedures” or left untreated. Toxins were then either acid-activated
(pH 3) or left untreated and added to tissue culture medium overlying
HeLa cells. The final VacA concentration of all samples was 5 ␮g/ml.
Vacuolating activity was quantified using a neutral red uptake assay.
Enterokinase-treated FLAG-VacA and untreated FLAG-VacA did not
differ significantly in vacuolating cytotoxin activity. Furthermore, both
enterokinase-treated and untreated FLAG-VacA required acid activa-
tion in order to induce vacuolating activity. Results represent the
mean ⫾ S.D. from triplicate samples and are expressed as a percent of
neutral red uptake induced by full-length acid-activated FLAG-VacA.
Oligomeric State of Proteolytically Cleaved FLAG-VacA—
The secreted FLAG-VacA assembles into large water-soluble
oligomeric structures (Fig. 8A), in a manner similar to wild-
type VacA (5, 29). To determine whether p-33 and p-55 frag-
ments of VacA remained physically associated following pro-
teolytic cleavage of FLAG-VacA, enterokinase-treated FLAG-
VacA was fractionated by gel filtration chromatography, and
its elution pattern was monitored by immunoblot analysis with
an anti-VacA serum. Proteolytically cleaved FLAG-VacA and
intact FLAG-VacA were found to elute in the same high mo-
lecular mass fractions (Fig. 8) but not in any of the lower
molecular mass fractions (data not shown), indicating that
VacA fragments (i.e. FLAGp-33 and p-55) remain associated
within an oligomeric structure (Fig. 8).
DISCUSSION
Over the past decade, multiple lines of investigation have
provided evidence for the occurrence of homotypic interactions
among VacA proteins (5, 11, 31, 34, 35, 39, 45). The earliest
evidence suggesting oligomerization of VacA proteins was the
observation that purified VacA from H. pylori broth culture
supernatant assembles into structures of ⬃1,000 kDa in mass,
corresponding to a buoyant density of about 22 S (5, 29). Ul-
trastructural studies have shown that these VacA complexes
2330 H. pylori VacA
FIG. 8. Oligomeric state of full-length FLAG-VacA and cleaved
FLAG-VacA. Concentrated broth culture supernatant from H. pylori
strain VM088 (A) and 40 ␮g of purified FLAG-VacA treated with en-
terokinase as described under “Experimental Procedures” (B) were
applied to a Superose 6 HR FPLC column. Forty fractions of 2 ml each
were collected, with fraction 1 corresponding to the void volume. Sam-
ples (40 ␮l) were then electrophoresed on a 10% SDS-polyacrylamide
gel, transferred to a nitrocellulose membrane, and immunoblotted with
anti-VacA serum. Full-length VacA as well as enterokinase-cleaved
VacA eluted in the same fractions with an elution peak at fraction 13,
corresponding to a molecular mass of about 1,000 kDa (5, 29). VacA was
not detected in any of the lower molecular mass fractions (data not
shown). p-55 fragments are detected more prominently than p-33 frag-
ments in B due to the relatively weaker reactivity of the anti-VacA
serum against the p-33 fragment. When the membrane shown in B was
stripped and immunoblotted with polyclonal anti-FLAG antibody, the
p-33 band was detected in fractions 8 –16 (data not shown).
have a flower-like shape and that they contain anywhere from
6 to 14 subunits (29, 30, 42). These flower-shaped structures
disassemble into VacA monomers after exposure to acid or
alkaline pH and then reassemble into oligomeric structures
after neutralization (29, 54). Further evidence for the assembly
of VacA into oligomeric structures has resulted from experi-
ments in which two different VacA toxins have been shown to
form mixed oligomeric structures (34, 39, 45). VacA-VacA in-
teractions have also been detected by transiently transfecting
cells with different fluorescently tagged VacA-expressing plas-
mids and using fluorescent resonance energy transfer method-
ology (35).
Although many lines of evidence indicate that VacA can form
oligomeric structures, thus far there has been relatively little
investigation of the specific VacA-VacA interactions that are
required for assembly into higher order structures. In this
study we investigated the role of p-33 and p-55 interactions in
oligomer formation by VacA. These two VacA fragments have
been considered to represent two domains or subunits of VacA.
In the present work, we demonstrated that p-33/p-55 interac-
tions can be detected using the yeast two-hybrid system. In
contrast, p-33/p-33 and p-55/p-55 interactions were not de-
tected in this system. The lack of detectable p-33/p-33 and
p-55/p-55 interactions was not due to the lack of expression of
these fusion proteins in the yeast, because all four fusion pro-
teins were able to interact when tested for p-33/p-55 interac-
tions. Importantly, the failure to detect p-33/p-33 or p-55/p-55
interactions in the yeast two-hybrid system does not exclude
the possibility that such interactions might occur in other en-
vironments. A previous study analyzed the properties of a
modified p-55 fragment secreted by H. pylori, and reported that
this protein could form homodimers but not large oligomeric
structures (31). However, the VacA protein analyzed in that
study contained at least 27 residues of the p-33 domain in
addition to the p-55 fragment (31). Another previous study
provided evidence that an N-terminal hydrophobic portion of
the p-33 fragment could undergo transmembrane protein ho-
modimerization within E. coli membranes (40, 55). The inabil-
ity to detect p-33/p-33 interactions with the yeast two-hybrid
system is consistent with a model in which p-33/p-33 interac-
tions occur only within membranes (40, 55).
In an effort to map putative interacting regions within p-33
and p-55, we used the yeast two-hybrid system to analyze a
series of mutant proteins containing internal deletions, as well
as truncated VacA proteins. The yeast two-hybrid data suggest
that amino acids 28 –196 within the p-33 domain and 313– 478
within the p-55 domain contribute to the p-33/p-55 interaction.
Only one of the p-33 deletion mutants (⌬6 –27p-33) was able to
interact with wild-type p-55. VacA amino acids 6 –27 comprise
a hydrophobic region of VacA that inserts into lipid membranes
and is required for VacA cytotoxic and membrane anion-chan-
nel activity (39, 40, 55). Interestingly, when co-expressed with
p-55, the ⌬6 –27p-33 mutant fragment induced higher reporter
activity in the yeast two-hybrid assay than did wild-type p-33.
A possible explanation for this result could be that the ⌬6 –
27p-33 mutant fragment is able to translocate to the yeast
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nuclei more efficiently than can the wild-type p-33, since pre-
viously it has been reported (56) that hydrophobic sequences
decrease nuclear protein translocation. Another possibility
could be that the ⌬6 –27p-33 mutant fragment interacts more
strongly with the wild-p-55 fragment than does wild-type p-33.
By constructing a FLAG-VacA toxin, which can be efficiently
cleaved into p-33 and p-55 domains, we were able to study the
p-33/p-55 interactions that occur within the VacA oligomer
produced by H. pylori. Our results (Fig. 8) clearly indicate that
p-33 and p-55 fragments remain physically associated after
proteolytic cleavage of VacA. In agreement with this interpre-
tation, when we incubated proteolytically cleaved FLAG-VacA
with an Anti-FLAG M2 Affinity Gel (Sigma), both the p-55 and
FLAGp-33 fragments were captured (data not shown). Neither
FLAGp-33 nor p-55 fragments could be eluted from the affinity
gel under pH conditions ranging from 7.5 to 4.5, nor with 1%
CHAPS, 10% Triton X-100, or 2 M urea, suggesting that there is
a stable physical interaction between the p-33 and p-55 VacA
domains (data not shown). Furthermore, our results demon-
strate that the vacuolating activity of intact FLAG-VacA and
fully proteolytically cleaved FLAG-VacA is indistinguishable
(Fig. 7). A likely explanation for intact activity of proteolyti-
cally cleaved VacA is that the fragments remain associated and
do not undergo any extensive adverse conformational changes
(unfolding) following proteolytic cleavage.
Two lines of evidence presented in the current study suggest
that the p-33/p-55 interactions play an important role in VacA
oligomer assembly. First, the intact oligomeric structure of
proteolytically cleaved VacA indicates that p-33/p-55 interac-
tions occur within VacA oligomers. Second, the properties of
mutant p-33 fragments in the yeast two-hybrid system corre-
late perfectly with the capacity of secreted H. pylori mutant
proteins to form oligomeric structures. This suggests that mu-
tations disrupting the p-33/p-55 interaction also disrupt the
formation of large VacA oligomeric structures. It is possible
that there may be two different types of interactions: (i) in-
tramolecular interactions between the p-33 and p-55 domains
of an individual VacA monomer that might be important for
proper folding of the VacA monomers within the oligomeric
structures, and (ii) intermolecular interaction between p-33
and p-55 domains of different 88-kDa molecules to form the
larger oligomeric structures. Currently, we are not able to
differentiate between these two interactions.
VacA toxins that fail to oligomerize consistently lack vacuo-
lating cytotoxic activity, suggesting that oligomerization is im-
 H. pylori VacA
portant for VacA activity (39). This hypothesis is supported by
the observation that neither p-33 nor p-55 is able to exhibit
vacuolating cytotoxic activity in a transient transfection assay
when expressed individually within cells (32, 34, 41). In con-
trast, when both domains are co-expressed within cells, they
interact and exhibit vacuolating cytotoxic activity (32, 34).
Thus, the data presented in the current study, as well as data
obtained using other systems, indicate that p-33/p-55 interac-
tions are essential for VacA assembly into oligomeric struc-
tures and also for vacuolating cytotoxic activity.
Oligomerization is an important feature of many proteins, in
particular pore-forming proteins (57, 58). Examples of these
proteins include perforin, Bcl-2 family members, as well as
several bacterial toxins. Several bacterial pore-forming toxins,
including protective antigen of Bacillus anthracis, ␣-hemolysin
of Staphylococcus aureus, streptolysin O of Streptococcus pyo-
genes, ␣-toxin of Clostridium septicum, El Tor cytolysin of
Vibrio cholera, and aerolysin of Aeromonas hydrophila, assem-
ble in target cell membranes forming transmembrane channels
(59 – 65). These proteins are interesting because, like VacA,
they are synthesized as water-soluble molecules but then insert
into membranes. Further studies of the processes by which
these toxins assemble into oligomeric structures may ulti-
mately lead to the development of therapeutic inhibitors that
block the oligomerization and the activity of these toxins
in vivo.
Acknowledgments—We thank Ping Cao, Beverly Hosse, Arlene D.
Vinion-Dubiel, Leslie Morrison, and Mark Hansberger for technical
assistance and helpful discussions. DNA oligonucleotides were synthe-
sized by the Vanderbilt University DNA Chemistry Core Facility, and
DNA sequence analysis was performed by the Vanderbilt University
DNA Sequencing Laboratory. The Vanderbilt University DNA Sequenc-
ing Laboratory is supported by the Vanderbilt-Ingram Cancer Center.
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Interactions between p-33 and p-55 Domains of the Helicobacter pylori Vacuolating
Cytotoxin (VacA)
Victor J. Torres, Mark S. McClain and Timothy L. Cover
J. Biol. Chem. 2004, 279:2324-2331.
doi: 10.1074/jbc.M310159200 originally published online October 30, 2003

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