Microbes and Infection 5 (2003) 715–721

www.elsevier.com/locate/micinf

Forum in immunology

Molecular and cellular mechanisms of action of the vacuolating cytotoxin

(VacA) and neutrophil-activating protein (HP-NAP) virulence factors
of Helicobacter pylori
Cesare Montecucco a,b,*, Marina de Bernard a,b
a
Dipartimento di Scienze Biomediche, Università di Padova, Via G. Colombo 3, 35121 Padua, Italy
b
Istituto Veneto di Medicina Molecolare, Via Orus 2, Padua, Italy

Abstract

Helicobacter pylori has elaborated a unique set of virulence factors that allow it to colonise the stomach wall. These factors include urease,
helicoidal shape, flagella and adhesion molecules. Here we discuss the molecular characteristics and mechanisms of action of the vacuolating
cytotoxin, VacA, and the neutrophil-activating protein, HP-NAP. Their activities are discussed in terms of tissue alterations, which promote the
release of nutrients necessary for the growth and survival of the bacterium in its nutrient-poor ecological niche.
© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: Helicobacter; Inflammation; Cell vacuolation; Epithelia; Neutrophils

1. Introduction
Helicobacter pylori infects the stomach of more than 50%
of the entire human population [1]. Since its isolation and
identification [2], this microaerophilic Gram-negative bacte-
rium has been associated with chronic superficial gastritis
(inflammation of the gastric mucosa), chronic active gastritis
(gastric mucosa inflammation with polymorphonuclear cell
infiltration), peptic ulcer disease (gastric and duodenal ul-
cers), and gastric cancer [3]. To date, a number of virulence
factors have been described and well characterised in H. py-
lori. The molecular mechanisms and involvement in the
H. pylori-associated disease processes of two of these viru-
lence factors will be dealt with in this chapter.
H. pylori occupies a particular ecological niche consisting
of the apical surface of stomach epithelial cells and within
the protective mucus layer. This environment has peculiar
features, which are worth recalling, to understand the activi-
ties of the virulence factors discussed below. To reach this
particular site, the bacterium must cross the oral cavity and
the oesophageal tract, which are characterised by the pres-
ence of hydrolytic enzymes and a neutral pH. Once through
the pylorus, the bacterium faces the acidity of the stomach
* Corresponding author. Tel.: +39-49-827-6058; fax: +39-49-827-6049.
E-mail address: cesare@civ.bio.unipd.it (C. Montecucco).
© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
DOI: 10.1016/S1286-4579(03)00124-2
lumen with values in the pH range 1–4. Such acid conditions
promote the denaturation of most proteins and their hydroly-
sis by gastric proteases. H. pylori can survive these harsh
conditions by buffering its internal and surrounding pH via
the enzymatic activity of urease with production of ammonia
and bicarbonate ions. It is believed that H. pylori rapidly
leaves the stomach lumen to enter the mucus layer which
covers and protects the mucosa against ulceration due to the
gastric fluids.
The mucus layer has unique barrier properties and is
relatively impermeable even to small molecules. In addition,
hydrogen ions can freely diffuse from the apical portion of
the mucosal epithelial cells into the gastric lumen, but not
vice versa. In contrast, bicarbonate anions (and probably iron
and nickel ions which are necessary for H. pylori growth) are
poorly permeable. Thus, a pH gradient exists inside the
mucus film characterised by slightly acidic values near the
apical cell membrane in normal conditions, or by more acid
pH if the thickness of the mucus layer is reduced in patho-
logical conditions.
H. pylori can enter the mucus because it releases mucus
hydrolysing enzymes, it has a helicoidal shape and it is
propelled by flagella. These bacteria adhere strongly to the
apical cell membrane via adhesins and by inducing a reor-
ganisation of the plasma membrane of the host gastric epi-
thelial cells [4]. In this particular region of the body, the
716 C. Montecucco, M. de Bernard / Microbes and Infection 5 (2003) 715–721

supply of nutrients, including ions essential for bacterial

growth, is very limited. Several H. pylori genes are, there-
fore, expected to encode for proteins that affect the physi-
ological state of the stomach cells and tissues in order to
favour H. pylori growth. Here, we will focus on two such
proteins, which are also major virulence factors: the vacu-
olating cytotoxin (VacA) and the neutrophil-activating pro-
tein (HP-NAP).

2. Vacuolating cytotoxin (VacA)

2.1. Structure and binding to cells

The production of a cytotoxin by H. pylori was deduced

by the seminal finding that the supernatants of H. pylori
isolates induced striking vacuolar degeneration in cultured
eukaryotic cells [5]. This effect was subsequently shown to
be caused by a secreted protein toxin, thereafter termed VacA
[6].
The vacA gene encodes a pro-toxin approximately
140 kDa in mass; both an amino-terminal signal sequence
and a carboxy-terminal fragment are proteolitically cleaved
during the process of VacA secretion and an ~88-kDa mature
toxin is exported [6,7]. Mature toxin molecules are released
into the extracellular space, or may be retained on the surface
of the bacterium [8]. The single polypeptide forming the
mature VacA tends to undergo cleavage at the site of an
exposed, protease-sensitive loop into N-terminal 34-kDa
(p37) and C-terminal 54-kDa (p58) fragments [9]. These two
fragments remain non-covalently associated, and may corre-
spond to distinct structural and functional subunits. VacA is
purified from the culture supernatant as an oligomer with a
mass >900 kDa [6]. Imaging by deep-etch electron micros-
copy shows individual VacA oligomers as flower-shaped
complexes about 30 nm in diameter (Fig. 1 panel a). These
structures are thought to be composed of one or two rings,
each made up of six to seven VacA monomers [9,10]. Acidic
[11,12] or basic [13] pH solutions disassemble the oligomers
into membrane-inserting monomers [14] and are associated
with a marked enhancement of VacA cytotoxicity [11,13].
Transfection of HeLa cells with plasmids expressing the
amino-terminal 422 amino acids of VacA is sufficient to
induce vacuolisation of HeLa cells [15,16]. Small trunca-
tions, internal deletions, and several point mutations in the
amino-terminal portion of VacA abrogate toxin activity when
assessed in transiently transfected cells [17]. Similarly, puri-
fied toxin from a mutant Helicobacter strain, carrying a
deletion of amino acid residues 6–27, fails to induce cyto-
plasmic vacuolation, is defective in the capacity to form
membrane channels (see below) and inhibits the activity of
the wild-type toxin [18]. Taken together, these data suggest
that the amino-terminal region of VacA plays a very impor-
tant role in toxin activity and that the active membrane-
inserted toxin is an oligomer.
The first step of VacA entry into target cells consists of the
binding to the plasma membrane. Residues 480 and 700 have
Fig. 1. Structure and activity of VacA. (a) Low-resolution structure of
oligomeric VacA: alternate monomers are shaded differently to represent the
interaction of the P37 portion of one monomer with the P58 portion of the
adjacent monomer. The dotted line represents the loop connecting P58 and
P37. (b) Current model of the cellular activities of VacA. The toxin binds to
the apical portion of epithelial cells and inserts into the plasma membrane
via hydrophobic protein–lipid interactions. This insertion leads to the for-
mation of anion-selective channels of low conductance, which are capable of
releasing bicarbonate, chloride and urea from the cell cytosol. Bicarbonate
and urea can thus enter the carbon and nitrogen cycles of H. pylori.
Eventually, the VacA toxin channel is internalised and changes the anion
permeability of late endosomal compartments, with enhancement of the
vacuolar ATPase proton pumping activity. In the presence of weak bases, and
in particular of the ammonia generated by the H. pylori urease, osmotically
active acidotropic ions (NH4+) will accumulate in the endosomes. This leads
to water influx and vesicle swelling, an essential step in vacuole formation.
Somehow, the VacA toxin is also capable of altering cell–cell junctions and
of modifying the trans-epithelial electrical resistance, with concomitant
increased fluxes of iron and nickel ions and small metabolites from the
mucosa to the bacterium, thus providing it with essential nutrients. Adapted
from [42].
been suggested to be especially important for cell line-
specific recognition and binding [19].
Although saturable binding of VacA to sensitive cell lines
suggests the presence of specific receptor sites, at least three
separate proteins have been implicated in toxin recognition
[12,20–22]. In addition, cell sensitivity to VacA has been
associated with the presence of one or several GPI-anchored
 C. Montecucco, M. de Bernard / Microbes and Infection 5 (2003) 715–721
protein(s) [23,24]. Depletion of plasma membrane choles-
terol decreases the amount of VacA internalised into HeLa
cells by more than 70%, and cell-bound VacA was found to
be associated with rafts, suggesting that VacA may directly
associate with cholesterol-rich microdomains [23–25].
2.2. Cell vacuolation and endocytic pathway alteration
VacA induces the formation of membrane-delimited
‘vacuoles’ in intoxicated cells. Membranes of these vacuoles
contain both late endosomal and lysosomal markers [26].
Vacuolation depends not only on VacA but also on the pres-
ence in the extracellular medium of permeant weak base,
such as ammonia [6]. Several cellular enzymes required for
vacuole development and maintenance have been identified
[27–29]. Vacuoles observed in biopsies of stomach mucosa
from H. pylori-infected patients and in HeLa cells in culture
contain electron dense material, but are largely devoid of the
multivesicular bodies which are characteristic of late endo-
somes (LE) and lysosomes (LY) [30]. It appears that VacA
induces considerable rearrangement of the organisation of
LE and LY, with extensive membrane fusion and swelling.
These events do not require SNARE proteins, and it has been
suggested that the vacuolar limiting membrane results from
successive processes of fusion between the internal mem-
branes among themselves and with the limiting membrane of
late endosomal compartments [31].
A role for dynamin, a GTPase functioning in intracellular
vesicle formation, in VacA-induced vacuolisation was sug-
gested by the finding that a dominant negative mutant of
dynamin inhibits cell vacuolisation in transfected cells with-
out affecting VacA binding or internalisation [32].
VacA alters the functions of the late endocytic pathway
and LY even in the absence of detectable vacuolation. With
no added weak bases, treatment of HeLa cells with VacA
results in missorting of lysosomal hydrolases to the extracel-
lular medium, and impairs the degradation of extracellular
ligands, such as epidermal growth factor. Both effects may be
ascribed to the capacity of VacA to partially neutralise the pH
of acidic cellular compartments [33]. VacA-induced alter-
ations in the function of the endocytic pathway also probably
account for the capacity of the toxin to interfere with antigen
processing by B lymphocytes [34].
The possible impairment of the intracellular endocytic
pathway may have cytotoxic implications. In fact, protein
degradation is a cellular function essential for the removal of
non-functional cell membrane proteins and extracellular
ligands and for the consequent re-utilisation of their amino
acids. Moreover, the processing of protein antigens by im-
mune antigen-presenting cells takes place mainly inside the
antigen-processing compartment, a specialised form of
LE/LY compartment, which is capable of fusing with the
plasma membrane. The inhibition of antigen processing and
presentation by VacA could be part of a strategy of survival
for H. pylori, because the depression of antigen processing
within antigen-presenting cells of the mucosa could signifi-
cantly contribute to the long-lasting infection with H. pylori
717
in the human stomach. Indeed, evidence for a reduced im-
mune response at the level of the stomach mucosa was found
[35].
2.3. Effect on polarised epithelial monolayers
and intracellular survival of H. pylori
Addition of VacA to various polarised epithelial monolay-
ers in vitro results in a rapid increase in ion conductivity,
which can be measured as a drop in trans-epithelial resis-
tance (TER). This appears to be due to modulation of the
resistance of cell–cell junctions (the paracellular pathway)
[36]. This effect of VacA is not accompanied by either vacu-
olation or inhibition of epidermal growth factor degradation;
TER decrease is not inhibited by bafilomycin A1 and is not
influenced by the presence of weak bases, in contrast to the
effect of these compounds on vacuolation. Not only ions but
also small neutral molecules with a molecular weight
<450 kDa (for example, mannitol and sucrose) exhibit in-
creased trans-epithelial diffusion upon VacA treatment.
These results indicate that VacA increases the supply of
essential nutrients that are necessary for bacterial growth on
the mucosa. This action has been proposed to enhance H.
pylori growth in the stomach by allowing increased diffusion
of nutrients from gastric mucosa into the mucus layer [36].
Recently, VacA was shown to improve the intracellular
survival of H. pylori within AGS cells (human gastric cell
line derived from an antral adenocarcinoma) [37]. If con-
firmed, this would represent another major role of VacA, as it
emerges that intracellular vacuoles may constitute a reservoir
of live H. pylori, one that is difficult to be reached by
antibiotics and by inflammatory cells [38]. Moreover, H. py-
lori strains expressing VacA interrupt phagosome maturation
in macrophage cell line RAW 264.7 and in human
macrophage-like cell line THP-1: failure of macrophages to
kill bacteria could explain, at least in part, the persistence of
H. pylori [39].
2.4. Biophysical and biochemical activities
Studies with planar lipid bilayers showed that VacA forms
anion-selective and voltage-dependent channels only at low
pH or after low-pH pre-activation [40]. The VacA channel in
the membrane is hexameric and assembles with the same
functional properties on the plasma membrane of HeLa cells
[41]. After binding and pore formation, VacA is slowly inter-
nalised by endocytosis, and the toxin anion channel, due to
its acid resistance, is expected to permeabilise to anions the
endocytic membrane as well. However, the activity of such
intracellular anion-specific channels is only expected to be of
importance to organelles endowed with the vacuolar ATPase
(v-ATPase) proton pump. This pump is electrogenic, i.e. as it
acidifies the lumen, it generates a proton gradient that de-
presses its further activity. The presence of an anion channel
should strongly promote the v-ATPase activity, leading to an
enhanced accumulation of protons, which in turn would drive
the uptake of weak bases that can permeate through the
718 C. Montecucco, M. de Bernard / Microbes and Infection 5 (2003) 715–721
membrane. The relevance of the VacA channel activity to the
proton pumping of v-ATPase is supported by the finding that
chloride channel defective cells have fewer acidic intracellu-
lar organelles. An increased lumenal concentration of anions
and cations has been suggested to cause an osmotically
driven swelling with generation of vacuoles [42] (Fig. 1
panel b). Accordingly, anion-channel blockers inhibit vacu-
olation of HeLa cells exposed to VacA [41,43]. Moreover,
mutations within the amino-terminal hydrophobic region of
VacA, which impair membrane channel formation inhibit
vacuolation [44]. Recent evidence that cell vacuolation re-
quires extracellular chloride and is indeed associated with
intracellular accumulation of this anion, of NH4+ and of
water further support this model [45]. The importance of the
anion-channel activity of VacA is also confirmed by the fact
that the anion channel blocker NPPB prevents and reverses
the VacA-induced decrease of TER of polarised epithelia
[41]. Also the VacA-induced apical anion secretion found in
rat intestine [46] can be ascribed to its anion-channel activity.
Very recently, Debellis et al. [47] were able to monitor pH
changes in epithelial cells of the stomach mucosa of frogs
and have found that VacA forms an NPPB-sensitive perme-
ation pathway that allows the exit of bicarbonate anions from
the cell cytosol. Such anions could contribute to the buffering
of the apical domain of the tissue and could enter the carbon
cycle of H. pylori.
Given the importance of urea in H. pylori metabolism and
survival, the finding that urea permeates out of the cell
through the VacA channel may be a very relevant one [48], as
VacA could contribute to bacterial growth also by inducing
the release of urea on the apical domain. The urea permease
inhibitor phloretin inhibits urea permeation through the VacA
channel and also cell vacuolation and TER decrease in epi-
thelia [48], reinforcing the central role of the VacA channel in
its multiple biological effects.
3. H. pylori neutrophil-activating protein
The infiltration of neutrophils and mononuclear inflam-
matory cells within the H. pylori-infected stomach mucosa is
a common finding, and the degree of mucosal damage corre-
lates with neutrophil infiltration. This bacterium induces the
release of several pro-inflammatory cytokines from the tissue
[49]. In addition, one or more protein components present in
H. pylori extracts directly attract and activate neutrophils and
other inflammatory cells. Among these, a 150-kDa oligo-
meric protein isolated from H. pylori was found to promote
neutrophil adhesion to endothelial cells [50]. This protein
was designated HP-NAP (H. pylori neutrophil-activating
protein) because of its ability to induce neutrophils to pro-
duce reactive oxygen radicals [51]. HP-NAP is released in
the medium, most likely after cell lysis, and binds to the
bacterial surface, where it can act as an adhesin, mediating
binding to mucin [52] or to polymorphonuclear leukocyte
sphingomyelin [53]. Purified recombinant HP-NAP has been
produced in Bacillus subtilis to avoid contamination by
Escherichia coli LPS. This purified material is chemotactic
for human neutrophils and monocytes [51] and induces sur-
face expression of b2-integrins which are necessary for en-
dothelial trans-migration [50], suggesting that HP-NAP
plays a role in the accumulation of these cells at the site of
infection with H. pylori. HP-NAP is a powerful stimulant of
the production of reactive oxygen radicals. HP-NAP acts via
a cascade of intracellular activation events (Fig. 2), including
increase of cytosolic calcium ion concentration and phospho-
rylation of proteins, leading to the assembly of functional
NADPH oxidase on the neutrophil plasma membrane
through a PTX-sensitive pathway involving ERK and p38-
MAPK [54].
HP-NAP has also been shown to increase the synthesis of
tissue factor and the secretion of the inhibitor-2 of the plas-
minogen activator in mononuclear cells [55]. By inducing the
coordinate expression of cell pro-coagulant and antifibrin-
olytic activities, HP-NAP might favour fibrin deposition and
contribute to the inflammatory reaction of gastric mucosa
elicited by H. pylori [55]. HP-NAP is also capable of cross-
ing epithelial monolayers and of inducing the activation of
the underlying mast cells [56]. These data further support the
idea that HP-NAP has an important role in the in vivo trig-
gering and maintaining of the inflammatory events observed
during H. pylori infection. Once released from the bacte-
rium, HP-NAP would cross the stomach epithelial layer,
reaching the underlying tissue where mast cells reside. The
subsequent activation of mast cells by HP-NAP with release
of the content of granules and of the pro-inflammatory cytok-
ine IL-6 is known to recruit monocytes and neutrophils.
Thus, HP-NAP can act at different stages of the inflammatory
response by activating mast cells with release of pro-
inflammatory molecules able to activate neutrophils and
monocytes, and additionally by acting directly on neutro-
phils and monocytes by promoting their recruitment and
activation.
The napA gene is highly conserved within 12 different
isolates of H. pylori, at variance from vacA, which may
indicate a lack of immune selection for diversification of
HP-NAP. The atomic structure of HP-NAP shows a ball-
shaped docecamer formed by four-helix bundled subunits
with a hollow central part, similar to the E. coli DNA-binding
protein Dps [57]. Dps proteins are a diverse family of bacte-
rial stress proteins that are induced during periods of nutrient
limitation. However, unlike Dps, which binds DNA, HP-
NAP can bind up to 500 atoms of iron per dodecamer. Indeed,
HP-NAP was originally thought to be a bacterial ferritin,
based on the nucleotide sequence homologies [58]. The nap
gene has several A+T-rich regions of dyad symmetry imme-
diately upstream of its start codon, which could function as
Fur-regulated promoters for iron regulation. Similar A+T-
rich regions exist upstream of the H. pylori ferritin pfr gene.
All these data indicate that HP-NAP is important for iron
uptake by H. pylori. Iron is an essential nutrient for the
 C. Montecucco, M. de Bernard / Microbes and Infection 5 (2003) 715–721 719

Fig. 2. Scheme of the intracellular events involved in HP-NAP activation of human leukocytes. HP-NAP binds to a specific receptor, which is coupled to a
trimeric G protein sensitive to pertussis toxin. Binding triggers the entry of calcium via plasma membrane calcium channels and via the activation of channels
located on the endoplasmic reticulum, which are opened by IP3, produced by activated phospholipase C. The activated HP-NAP receptor also activates a PI3
kinase, which is inhibited by wortmannin. The rise in the cytosolic calcium concentration and the activity of PI3 kinase leads to phosphorylation of the cytosolic
subunits of the NADPH oxidase and to their migration to the plasma membrane, causing enzyme activation with production of superoxide anions. HP-NAP
rapidly activates ERK and p38, and this activation is essential for the HP-NAP-induced superoxide anion generation, adhesion and chemotaxis of human
neutrophils. Adapted from [42].

growth of most bacteria, including H. pylori. The unique References

niche inhabited by H. pylori is probably very poor in iron,
and H. pylori possesses several mechanisms for iron uptake.
These observations suggest that HP-NAP may have a role in
iron binding, but recent data from our laboratory reveal that
HP-NAP is constitutively expressed under iron-depleted
conditions, its expression is not regulated by the presence or
absence of iron and that it does not play a part in the metal
resistance of H. pylori [59]. An alternative possibility is that
HP-NAP has evolved unique properties of a pro-
inflammatory molecule to induce a very moderate state of
inflammation, which would promote H. pylori growth by the
release of nutrient factors from the inflammed tissue. The
comparison of the structure of HP-NAP with those of similar
molecules produced by other bacteria shows the unique pres-
ence of a cluster of positive charges, which could be involved
in the activation of inflammatory cells [57].
Acknowledgements
Work carried out in the authors’ laboratories is supported
by the CNR-MURST 5% Project and by MURST 40%
Project on Inflammation.
[1] M.J. Blaser, Helicobacter pylori: microbiology of a “slow” bacterial
infection, Trends Microbiol. 1 (1993) 255–259.
[2] J.R. Warren, B.J. Marshall, Unidentified curved bacilli on gastric
epithelium in active chronic gastritis, Lancet 1 (1983) 1273–1275.
[3] N.I.H. Consensus Development Panel on Helicobacter pylori in peptic
ulcer disease, Helicobacter pylori in peptic ulcer disease, J. Am. Med.
Assoc. 272 (1994) 65–69.
[4] A. Covacci, R. Rappuoli, Tyrosine-phosphorylated bacterial proteins:
Trojan horses for the host cell, J. Exp. Med. 191 (2000) 587–592.
[5] R.D. Leunk, P.T. Johnson, B.C. David, W.G. Kraft, D.R. Morgan,
Cytotoxin activity in broth-culture filtrates of Campylobacter pylori,
J. Med. Microbiol. 26 (1988) 93–99.
[6] T.L. Cover, M.J. Blaser, Purification and characterization of the vacu-
olating toxin from Helicobacter pylori, J. Biol. Chem. 267 (1992)
10570–10575.
[7] W. Schmitt, R. Haas, Genetic analysis of the Helicobacter pylori
vacuolating cytotoxin: structural similarities with the IgA protease
type of exported protein, Mol. Microbiol. 12 (1994) 307–319.
[8] J.L. Telford, P. Ghiara, M. Dell’Orco, M. Comanducci, D. Burroni,
M. Bugnoli, M.F. Tecce, S. Censini, A. Covacci, Z. Xiang, E. Papini,
C. Montecucco, L. Parente, R. Rappuoli, Gene structure of the Heli-
cobacter pylori cytotoxin and evidence of its key role in gastric
disease, J. Exp. Med. 179 (1994) 1653–1658.
[9] P. Lupetti, J.E. Heuser, R. Manetti, S. Lanzavecchia, P.L. Bellon,
R. Dallai, R. Rappuoli, J.L. Telford, Oligomeric and subunit structure
of the Helicobacter pylori vacuolating cytotoxin, J. Cell Biol. 133
(1996) 801–807.
720 C. Montecucco, M. de Bernard / Microbes and Infection 5 (2003) 715–721
[10] J.M. Reyrat, S. Lanzavecchia, P. Lupetti, M. de Bernard, C. Pagliac-
cia, V. Pelicic, M. Charrel, C. Ulivieri, N. Norais, X. Ji, V. Cabiaux,
E. Papini, R. Rappuoli, J.L. Telford, 3D imaging of the 58 kDa cell
binding subunit of the Helicobacter pylori cytotoxin, J. Mol. Biol. 290
(1999) 459–470.
[11] M. de Bernard, E. Papini, V. de Filippis, E. Gottardi, J.L. Telford,
R. Manetti, A. Fontana, R. Rappuoli, C. Montecucco, Low pH acti-
vates the vacuolating toxin of Helicobacter pylori, which becomes
acid and pepsin resistant, J. Biol. Chem. 270 (1995) 23937–23940.
[12] T.L. Cover, P.I. Hanson, J.E. Heuser, Acid-induced dissociation of
VacA, the Helicobacter pylori vacuolating toxin, reveals its pattern of
assembly, J. Cell Biol. 138 (1997) 759–769.
[13] K. Yahiro, T. Niidome, M. Kimura, T. Hatakeyama, H. Aoyagi,
H. Kurazono, K. Imagawa, A. Wada, J. Moss, T. Hirayama, Activation
of Helicobacter pylori VacA toxin by alkaline or acid conditions
increases its binding to a 250-kDa receptor protein–tyrosine phos-
phatase beta, J. Biol. Chem. 274 (1999) 36693–36699.
[14] M. Molinari, C. Galli, M. de Bernard, N. Norais, J.M. Ruysschaert,
R. Rappuoli, C. Montecucco, The acid activation of Helicobacter
pylori toxin VacA: structural and membrane binding studies, Bio-
chem. Biophys. Res. Commun. 24 (1998) 334–340.
[15] M. de Bernard, D. Burroni, E. Papini, R. Rappuoli, J.L. Telford,
C. Montecucco, Identification of the Helicobacter pylori VacA toxin
domain active in the cell cytosol, Infect. Immun. 66 (1998)
6014–6016.
[16] D. Ye, D.C. Willhite, S.R. Blanke, Identification of the minimal
intracellular vacuolating domain of the Helicobacter pylori vacuolat-
ing toxin, J. Biol. Chem. 274 (1999) 9277–9282.
[17] D. Ye, S.R. Blanke, Mutational analysis of the Helicobacter pylori
vacuolating toxin amino terminus: identification of the amino acids
essential for cellular vacuolation, Infect. Immun. 68 (2000)
4354–4357.
[18] A.D. Vinion-Dubiel, M.S. McClain,M. Czajkowsky, H. Iwamoto,
D. Ye, P. Cao, W. Schraw, G. Szabo, S.R. Blanke, Z. Shao, T.L. Cover,
A dominant negative mutant of Helicobacter pylori vacuolating toxin
(VacA) inhibits VacA-induced cell vacuolation, J. Biol. Chem. 274
(1999) 37736–37742.
[19] H.J. Wang, W.C. Wang, Expression and binding analysis of GST-
VacA fusions reveals that the C-terminal approximately 100-residue
segment of exotoxin is crucial for binding in HeLa cells, Biochem.
Biophys. Res. Commun. 278 (2000) 449–454.
[20] P.I. Padilla, A. Wada, K. Yahiro, M. Kimura, T. Niidome, H. Aoyagi,
A. Kumatori, M. Anami, T. Hayashi, J. Fujisawa, H. Saito, J. Moss,
T. Hirayama, Morphologic differentiation of HL-60 cells is associated
with appearance of RPTPbeta and induction of Helicobacter pylori
VacA sensitivity, J. Biol. Chem. 275 (2000) 15200–15206.
[21] K. Seto, Y. Hayashi-Kuwabara, T. Yoneta, H. Suda, H. Tamaki, Vacu-
olation induced by cytotoxin from Helicobacter pylori is mediated by
the EGF receptor in HeLa cells, FEBS Lett. 431 (1998) 347–350.
[22] K. Yahiro, T. Niidome, T. Hatakeyama, H. Aoyagi, H. Kurazono,
P.I. Padilla, A. Wada, T. Hirayama, Helicobacter pylori vacuolating
cytotoxin binds to the 140-kDa protein in human gastric cancer cell
lines, AZ-521 and AGS, Biochem. Biophys. Res. Commun. 238
(1997) 629–632.
[23] V. Ricci, A. Galmiche, A. Doye, V. Necchi, E. Solcia, P. Boquet, High
cell sensitivity to Helicobacter pylori VacA toxin depends on a
GPI-anchored protein and is not blocked by inhibition of the clathrin-
mediated pathway of endocytosis, Mol. Biol. Cell 11 (2000)
3897–3909.
[24] W. Schraw, Y. Li, M.S. McClain, F.G. van der Goot, T.L. Cover,
Association of Helicobacter pylori vacuolating toxin (VacA) with
lipid rafts, J. Biol. Chem. 277 (2002) 34642–34650.
[25] H.K. Patel, R.M. Willhite, R.M. Patel, D. Ye, C.L. Williams,
E.M. Torres, K.B. Marty, R.A. MacDonald, S.R. Blanke, Plasma
membrane cholesterol modulates cellular vacuolation induced by the
Helicobacter pylori vacuolating cytotoxin, Infect. Immun. 70 (2002)
4112–4123.
[26] M. Molinari, C. Galli, N. Norais, J.L. Telford, R. Rappuoli, J.P. Luzio,
C. Montecucco, Vacuoles induced by Helicobacter pylori toxin con-
tain both late endosomal and lysosomal markers, J. Biol. Chem. 272
(1997) 25339–25344.
[27] E. Papini, E. Gottardi, B. Satin, M. de Bernard, J.L. Telford, P. Mas-
sari, R. Rappuoli, S.B. Sato, C. Montecucco, The vacuolar ATPase
proton pump is present on intracellular vacuoles induced by Helico-
bacter pylori, J. Med. Microbiol. 44 (1996) 1–6.
[28] E. Papini, B. Satin, C. Bucci, M. de Bernard, J.L. Telford, R. Manetti,
R. Rappuoli, M. Zerial, C. Montecucco, The small GTP binding
protein rab7 is essential for cellular vacuolation induced by Helico-
bacter pylori cytotoxin, EMBO J. 16 (1997) 15–24.
[29] N.A. Hotchin, T.L. Cover, N. Akhtar, Cell vacuolation induced by the
VacA cytotoxin of Helicobacter pylori is regulated by the Rac1
GTPase, J. Biol. Chem. 275 (2000) 14009–14012.
[30] T.L. Cover, A.H. Susan, M.J. Blaser, Characterization of HeLa cell
vacuoles induced by Helicobacter pylori broth culture supernatant,
Hum. Pathol. 23 (1992) 1004–1010.
[31] M. de Bernard, M. Moschioni, A. Habermann, G. Griffiths, C. Mon-
tecucco, Cell vacuolization induced by Helicobacter pylori VacA
cytotoxin does not depend on late endosomal SNAREs, Cell. Micro-
biol. 4 (2002) 11–18.
[32] J. Suzuki, H. Ohnsihi, H. Shibatam, A. Wada, T. Hirayama, T. Iiri,
N. Ueda, C. Kanamaru, T. Tsuchida, H. Mashima, H. Yasuda,
T. Fujita, Dynamin is involved in human epithelial cell vacuolation
caused by the Helicobacter pylori-produced cytotoxin VacA, J. Clin.
Invest. 107 (2001) 363–370.
[33] B. Satin, N. Norais, J.L. Telford, R. Rappuoli, M. Murgia, C. Mon-
tecucco, E. Papini, Vacuolating toxin of Helicobacter pylori inhibits
maturation of procathepsin D and degradation of epidermal growth
factor in HeLa cells through a partial neutralization of acidic intracel-
lular compartments, J. Biol. Chem. 272 (1997) 25022–25028.
[34] M. Molinari, M. Salio, C. Galli, N. Norais, R. Rappuoli, A. Lanzavec-
chia, C. Montecucco, Selective inhibition of Li-dependent antigen
presentation by Helicobacter pylori toxin VacA, J. Exp. Med. 187
(1998) 135–140.
[35] M. Shirai, T. Arichi, T. Nakazawa, J.A. Berzofsky, Persistent infection
by Helicobacter pylori down-modulates virus-specific CD8+ cyto-
toxic T cell response and prolongs viral infection, J. Infect. Dis. 177
(1998) 72–80.
[36] E. Papini, B. Satin, N. Norais, M. de Bernard, J.L. Telford, R. Rap-
puoli, C. Montecucco, Selective increase of the permeability of polar-
ized epithelial cell monolayers by Helicobacter pylori vacuolating
toxin, J. Clin. Invest. 102 (1998) 813–820.
[37] A.M. Petersen, K. Sorensen, J. Blom, K.A. Krogfelt, Reduced intrac-
ellular survival of Helicobacter pylori vacA mutants in comparison
with their wild-types indicates the role of VacA in pathogenesis,
FEMS Immunol. Med. Microbiol. 30 (2001) 103–108.
[38] M.R. Amieva, N.R. Salama, L.S. Tompkins, S. Falkow, Helicobacter
pylori enter and survive within multivesicular vacuoles of epithelial
cells, Cell. Microbiol. 4 (2002) 677–690.
[39] P.Y. Zheng, N.L. Jones, Helicobacter pylori strains expressing the
vacuolating cytotoxin interrupt phagosome maturation in macroph-
ages by recruiting and retaining TACO (coronin 1) protein, Cell.
Microbiol. 5 (2003) 25–40.
[40] F. Tombola, C. Carlesso, I. Szabò, M. de Bernard, J.M. Reyrat,
J.L. Telford, R. Rappuoli, C. Montecucco, E. Papini, M. Zoratti,
Helicobacter pylori vacuolating toxin forms anion-selective channels
in planar lipid bilayers: possible implications for the mechanism of
cellular vacuolation, Biophys. J. 96 (1999) 1401–1409.
[41] I. Szabò, S. Brutsche, F. Tombola, M. Moschioni, B. Satin, J.L. Tel-
ford, R. Rappuoli, C. Montecucco, E. Papini, M. Zoratti, Formation of
anion-selective channels in the cell plasma membrane by the toxin
VacA of Helicobacter pylori is required for its biological activity,
EMBO J. 18 (1999) 5517–5527.
 C. Montecucco, M. de Bernard / Microbes and Infection 5 (2003) 715–721
[42] C. Montecucco, R. Rappuoli, Living dangerously: how Helicobacter
pylori survives in the human stomach, Nat. Rev. Mol. Cell. Biol. 2
(2001) 457–466.
[43] F. Tombola, F. Oregna, S. Brutsche, I. Szabò, G. Del Giudice, R. Rap-
puoli, C. Montecucco, E. Papini, M. Zoratti, Inhibition of the vacu-
olating and anion channel activities of the VacA toxin of Helicobacter
pylori, FEBS Lett. 460 (1999) 221–225.
[44] M.S. McClain, H. Iwamoto, P. Cao, A.D. Vinion-Dubiel, Y. Ly,
G. Szabo, Z. Shao, T.L. Cover, Essential role of a GXXXG motif for
membrane channel formation by Helicobacter pylori vacuolating
toxin, J. Biol. Chem. 278 (2003) 12101–12108.
[45] L. Morbiato, F. Tombola, S. Campello, G. Del Giudice, R. Rappuoli,
M. Zoratti, E. Papini, Vacuolation induced by VacA toxin of Helico-
bacter pylori requires the intracellular accumulation of membrane
permeant bases, Cl(–) and water, FEBS Lett. 508 (2001) 479–483.
[46] A. Guarino, M. Bisceglia, R.B. Canani, M.C. Boccia, G. Mallardo,
E. Bruzzese, P. Massari, R. Rappuoli, J.L. Telford, Enterotoxic effect
of the vacuolating toxin produced by Helicobacter pylori in Caco-2
cells, J. Infect. Dis. 178 (1998) 1373–1378.
[47] L. Debellis, E. Papini, R. Caroppo, C. Montecucco, S. Curci, Helico-
bacter pylori cytotoxin VacA increases alkaline secretion in gastric
epithelial cells, Am. J. Physiol. Gastrointest. Liver Physiol. 281
(2001) G1440–G1448.
[48] F. Tombola, L. Morbiato, G. Del Giudice, R. Rappuoli, M. Zoratti,
E. Papini, The Helicobacter pylori VacA toxin is a urea permease that
promotes urea diffusion across epithelia, J. Clin. Invest. 108 (2001)
929–937.
[49] J.E. Crabtree, Cytokine response in Helicobacter pylori infection, in:
M. Achtman, S. Suerbaum (Eds.), Helicobacter pylori: Molecular and
Cellular Biology, Norfolk Orizon Scientific Press, 2001, pp. 63–83.
[50] D.J. Evans Jr., D.J. Evans, T. Takemura, H. Nakano, H.C. Lampert,
D.Y. Graham, D.N. Dranger, P.R. Kvietys, Characterization of a
Helicobacter pylori neutrophil-activating protein, Infect. Immun. 63
(1995) 2213–2220.
[51] B. Satin, G. Del Giudice, V. Della Bianca, S. Dusi, C. Laudanna,
F. Tonello, D. Kelleher, R. Rappuoli, C. Montecucco, F. Rossi, The
neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a
protective antigen and major virulence factor, J. Exp. Med. 191 (2000)
1467–1476.
721
[52] F. Namavar, M. Sparrius, E.C. Veerman, B.J. Appelmelk, C.M. Van-
denbroucke-Grauls, Neutrophil-activating protein mediates adhesion
of Helicobacter pylori to sulfated carbohydrates on high-molecular-
weight salivary mucin, Infect. Immun. 66 (1998) 444–447.
[53] S. Teneberg, H. Miller-Podraza, H.C. Lampert, D.J. Evans Jr.,
D.G. Evans, D. Danielsson, K.A. Karlsson, Carbohydrate binding
specificity of the neutrophil-activating protein of Helicobacter pylori,
J. Biol. Chem. 272 (1997) 19067–19071.
[54] H. Nishioka, I. Baesso, G. Semenzato, L. Trentin, R. Rappuoli, G. Del
Giudice, C. Montecucco, The neutrophil activating protein of Helico-
bacter pylori (HP-NAP) activates the MAPK pathway in human
neutrophils, Eur. J. Immunol. 33 (2003) 840–849.
[55] P. Montemurro, G. Barbuti, W.G. Dundon, G. Del Giudice, R. Rap-
puoli, M. Colucci, P. De Rinaldis, C. Montecucco, N. Semeraro,
E. Papini, Helicobacter pylori neutrophil-activating protein stimu-
lates tissue factor and plasminogen activator inhibitor-2 production by
human blood mononuclear cells, J. Infect. Dis. 183 (2001)
1055–1062.
[56] P. Montemurro, H. Nishioka, W.G. Dundon, M. de Bernard, G. Del
Giudice, R. Rappuoli, C. Montecucco, The neutrophil-activating pro-
tein of Helicobacter pylori is a potent stimulant of mast cells, Eur. J.
Immunol. 32 (2002) 671–676.
[57] G. Zanotti, E. Papinutto, W.G. Dundon, R. Battistuta, M. Seveso,
G. Del Giudice, R. Rappuoli, C. Montecucco, Structure of the
Neutrophil-activating protein from Helicobacter pylori, J. Mol. Biol.
323 (2002) 125–130.
[58] D.J. Evans Jr., D.J. Evans, H.C. Lampert, H. Nakano, Identification of
four new prokaryotic bacterioferritins, from Helicobacter pylori,
Anabaena variabilis, Bacillus subtilis and Treponema pallidum, by
analysis of gene sequences, Gene 153 (1995) 123–127.
[59] W.G. Dundon, H. Nishioka, A. Polenghi, E. Papinutto, G. Zanotti,
P. Montemurro, G. Del Giudice, R. Rappuoli, C. Montecucco, The
neutrophil-activating protein of Helicobacter pylori, Int. J. Med.
Microbiol. 291 (2002) 545–550.