Persistence of Helicobacter
pylori VacA toxin and vacuolating
potential in cultured gastric epithelial cells
Patrizia
Sommi1,
Vittorio
Ricci1,
Roberto
Fiocca2,
Vittorio
Necchi2,
Marco
Romano3,
John L.
Telford4,
Enrico
Solcia2, and
Ulderico
Ventura1
1 Institute of Human Physiology
and 2 Department of Human
Pathology, University of Pavia and Istituto Ricovero e Cura a
Carrattere Scientifico Policlinico San Matteo, 27100 Pavia;
3 Department of
Medicine-Gastroenterology II, University of Naples, 80131 Naples;
and 4 Immunological Research
Institute of Siena-Chiron Vaccine, 53100 Siena, Italy
 |
ABSTRACT |
The vacuolating
toxin A (VacA) is one of the most important virulence factors in
Helicobacter
pylori-induced damage to human gastric
epithelium. Using human gastric epithelial cells in
culture and broth culture filtrate from a VacA-producing
H.
pylori strain, we studied
1) the delivery of VacA to cells,
2) the localization and fate of
internalized toxin, and 3) the
persistence of toxin inside the cell. The investigative techniques used
were neutral red dye uptake, ultrastructural immunocytochemistry,
quantitative immunofluorescence, and immunoblotting. We found that VacA
1) is delivered to cells in both
free and membrane-bound form (i.e., as vesicles formed by the bacterial
outer membrane), 2) localizes inside
the endosomal-lysosomal compartment, in both free and membrane-bound form, 3) persists within the cell
for at least 72 h, without loss of vacuolating power, which, however,
becomes evident only when NH4Cl is
added, and 4) generally does not
degrade into fragments smaller than ~90 kDa. Our findings suggest
that, while accumulating inside the endosomal-lysosomal compartment, a
large amount of VacA avoids the main lysosomal degradative processes
and retains its apparent molecular integrity.
outer membrane vesicles; VacA internalization; VacA metabolism; VacA immunocytochemistry
 |
INTRODUCTION |
HELICOBACTER pylori is
a gram-negative curved or spiral bacterium that plays a major
role in the development of chronic gastritis, peptic ulcer, and gastric
cancer (22, 25, 34, 38, 39). H. pylori
is specifically suited to the colonization of the human stomach, in
which it causes an inflammatory reaction and epithelial damage with
cellular swelling and cytoplasmic vacuolation, both in vivo and in
vitro (4, 11, 13, 16, 24, 29, 30, 37). The two main bacterial factors
involved in this cellular damage are urease (14, 20) and vacuolating
toxin A (VacA) (2), as expressed by 100% and 50-60%,
respectively, of H. pylori clinical
isolates. Urease acts by producing ammonia via urea hydrolysis. The
mechanism through which ammonia (and other weak bases) exerts its
cytopathic effect is well known (9, 23, 26). Ammonia crosses cell
membranes in an uncharged state, is trapped by protonation within
acidic intracellular compartments, and thereafter induces osmotic
swelling of these compartments, which in turn causes cell vacuolation.
VacA seems to play a key role in epithelial damage induced by
H. pylori infection (2, 13, 37), but
the mechanism through which vacuolation occurs remains poorly
understood. Monomeric VacA, with a molecular mass of ~90 kDa (3), is
synthetized by H. pylori as a 139-kDa
protoxin (2, 37), which is rapidly processed to form the native toxin
released in the extracellular environment. In bacterial culture, and
probably in vivo too, monomers of ~90 kDa gather together to form
high molecular mass (1,000 kDa) oligomers (2, 3, 10). Moreover, it has
been suggested that the monomeric toxin of ~90 kDa is further
processed to produce a 37-kDa
NH2-terminal fragment and a 58-kDa
COOH-terminal fragment and that these fragments remain associated after
cleavage (36, 37). VacA is believed to exert its cytotoxic activity by
acting inside the cells (12), but the molecular species involved in vacuolation and the fate of the internalized toxin remain unclear. Furthermore, it is unknown how long the toxin and/or its
fractions persist inside the cell.
In the present study, through using human gastric epithelial cells in
culture, we attempt to clarify the uptake, localization, and fate of
the internalized toxin and the time of persistence of toxin inside the
cell.
 |
MATERIALS AND METHODS |
Bacterial strains and filtrate production.
The VacA-producing H. pylori strain
used was CCUG 17874 (from Culture Collection University of
Gotebörg, Gotebörg, Sweden). Bacteria were grown in
Brucella broth, supplemented with 5%
FCS (GIBCO, Grand Island, NY), for 24-36 h at 37°C in a
thermostatic shaker under microaerophilic conditions. As previously
described (35), to obtain the broth culture filtrate (BCF), we then
removed bacteria by centrifugation and sterilized the supernatants by passage through a 0.22-µm cellulose acetate filter (Nalge, Rochester, NY). Uninoculated broth filtrate served as a control. To remove ammonia, we dialyzed control and BCF against Hanks' balanced salt solution (HBSS) for 36 h in dialysis tubing with a 12-kDa molecular mass cutoff (Sigma Chemical, St. Louis, MO). The presence of VacA in the BCF was tested by means of SDS-PAGE, followed by
immunoblotting with anti-VacA serum (27).
Gastric epithelial cells and cell incubation.
For this study, we used the MKN 28 cell line. This cell
line, derived from a human gastric tubular adenocarcinoma, shows
moderate gastric-type differentiation (15, 31). MKN 28 cells were grown as monolayers in DMEM/Ham's nutrient mixture F-12 (Sigma Chemical) supplemented with 10% FCS (GIBCO) in 35-mm petri dishes (Corning Glass
Works, Corning, NY) at 37°C in a humidified atmosphere of 5%
CO2 in air.
Subconfluent cell monolayers were washed twice with HBSS before
incubation for 16 h (loading period;
step
1) with either uninoculated broth
filtrate (diluted 1:3 in HBSS), BCF (diluted 1:3 in HBSS, both with and
without 4 mM NH4Cl), or 4 mM
NH4Cl (dissolved in HBSS). After
the initial loading, cell monolayers were incubated for 5 h
(step
2) with either HBSS or
NH4Cl and were finally treated for
16 h (step
3) with either HBSS, 4 mM
NH4Cl (dissolved in HBSS), BCF
(diluted in HBSS as above), or BCF plus 4 mM
NH4Cl. We also performed tests
with BCF preincubated with anti-VacA serum
II (see below; diluted
1:20) for 1 h at 37°C. In all the experiments, the incubation
medium was completely removed after each step, and the cell monolayers
were extensively washed (10 times with cold HBSS) before subsequent
incubation steps.
For electron microscopy study, MKN 28 cells were loaded for 16 h with
either BCF, unfiltered supernatant of broth culture, or uninoculated
broth filtrate (all diluted 1:3 in HBSS), extensively washed, and then
incubated in HBSS for 21 or 72 h. For quantitative immunofluorescence
analysis, cells previously incubated with BCF or uninoculated broth
filtrate (diluted as above) were extensively washed and then maintained
in HBSS alone for 5, 21, 48, or 72 h. In some experiments, to study the
intracellular persistence of VacA activity, cells previously incubated
with BCF or uninoculated broth filtrate (diluted as above) were
extensively washed and maintained in HBSS alone for 5, 21, 48, or 72 h
and then in HBSS or 4 mM NH4Cl for
an additional 16 h.
Neutral red dye uptake.
At the end of each step, the degree of cell vacuolation was assayed by
means of neutral red dye uptake, in accordance with the method of Cover
et al. (5), and was expressed as micrograms of neutral red dye per
microgram of cell protein (29). The protein content of cell monolayers
was measured in accordance with the method of Lowry et al. (18).
Neutral red dye is an acidotropic, membrane-permeant amine that
accumulates in the vacuolar lumen (5, 23). Neutral red dye uptake is a
widely accepted in vitro assay for H. pylori-induced cell vacuolation (5, 24, 29, 30).
SDS-PAGE and immunoblotting.
Cells loaded for 16 h with BCF and then incubated in HBSS for 21 or 72 h were washed extensively with HBSS and finally lysed with lysis buffer
(1.5 M Tris · HCl, pH 6.8, 8% SDS, and
40% glycerol) supplemented with 20% 2-mercaptoethanol. Controls
consisted of 1) cells incubated for
16 h without BCF, maintained in HBSS for 21 h, and lysed as above, and
2) BCF from H. pylori strain CCUG 17874. Each sample (40 µl) was
subjected to SDS-PAGE in 7% polyacrylamide gel with a 3% stacking
gel. Proteins were then blotted onto nitrocellulose (Bio-Rad
Laboratories, Richmond, CA); the subsequent immunologic analysis used
polyclonal antisera. The following rabbit antisera raised against
native VacA or its fragments (as obtained by recombinant DNA
techniques) were used:
II, directed against native VacA;
B,
against region B (amino acid residues 262-428 of the toxin),
BK, against region BK (amino acids 34-751); and
C, against
region C (amino acids 751-1,000) (37). Serum
II has been shown
to block the vacuolating activity of purified VacA in in vitro tests (19).
Electron microscopy.
At the end of incubation, cell monolayers were washed twice with
cacodylate buffer [0.2 M
(CH3)2AsO2Na · 3H2O,
pH 7.3 with HCl] and fixed with a freshly prepared mixture of one
part 2.5% glutaraldehyde and two parts 1% osmium tetroxide in
cacodylate buffer for 40 min at 4°C. Fixed monolayers were scraped
and collected in cacodylate buffer, centrifuged at 10,000 g for 10 min, and then embedded in
Epon-Araldite mixture. Uranyl lead-stained ultrathin sections were
viewed with a Zeiss EM 902 elecron microscope (Oberkochen, Germany).
For the ultrastructural immunolocalization of VacA, we used the
colloidal gold-labeling technique. Briefly, ultrathin
sections were collected on 300-mesh nickel grids, washed with
buffer
A (0.45 M NaCl, 1% Triton X-100, and
0.05 M Tris · HCl, pH 7.4), and incubated in
nonimmune goat serum at room temperature for 1 h to prevent nonspecific
binding of immunoglobulins. The sections were then
incubated at 4°C overnight with
II polyclonal rabbit antiserum
directed against native oligomeric VacA, diluted 1:600 in
buffer
B (0.45 M NaCl, 1% BSA, 0.5% sodium
azide, and 0.05 M Tris · HCl, pH 7.4). After further
washing in buffer
B, primary immunoglobulin binding was
revealed by gold-labeled goat anti-rabbit IgG (EM GAR 20, British
BioCell, Cardiff, United Kingdom) diluted 1:20 in
buffer
B. The sections were stained with
uranyl and lead before electron microscopy investigation (30).
Quantitative immunofluorescence analysis.
In accordance with Chavrier et al. (1), after incubation cell
monolayers were washed once with PBS and permeabilized by treatment for
15 min with 0.5% saponin in 80 mM PIPES (pH 6.8), 5 mM EGTA, and 1 mM
MgCl2. The cells were fixed for 15 min with 3% formaldehyde in PBS (pH 7.4). After fixation, the cells
were washed for 5 min with 0.5% saponin in PBS (saponin-PBS), and free aldehyde groups were quenched for 10 min with 50 mM
NH4Cl in PBS. Cell monolayers were
washed with saponin-PBS for 5 min and then incubated with
II serum
in saponin-PBS for 20 min. After triple rinsing of the cells and 20 min
incubation with goat anti-rabbit IgG conjugated with
tetramethylrhodamine isothiocyanate (TRITC) (Sigma Chemical) (1:400 in
saponin-PBS), primary antibody binding was visualized. After being
washed in saponin-PBS, the petri dishes were mounted on an upring
microscope (Axiolab, Zeiss) equipped with a 100-W mercury lamp, a
water-immersion objective (Achroplan, Zeiss), and a standard filter set
for TRITC (filter set 15, Zeiss). Image acquisition was by means of a
high-sensitivity camera (Extended-ISIS camera, Photonic Science,
Millam, United Kingdom) interfaced by a frame grabber (CX100,
ImageNation, Beaverton, OR) to a high-end personal computer. Locally
developed software was used for the recording and the simultaneous
analysis, in triplicate, of the fluorescence obtained from 50 cells for
each condition.
Statistics.
All data were expressed as means ± SE of four independent
experiments. The statistical significance of the differences was evaluated by Student's t-test and by
ANOVA followed by Newman-Keuls Q-test
(33). Data expressed as a percentage of control were analyzed before
being normalized vs. control.
 |
RESULTS |
Neutral red dye uptake.
To investigate VacA activity in the presence or absence of ammonia, we
studied MKN 28 cells loaded in the following four different conditions
(step
1):
1) uninoculated broth filtrate
(control), 2) BCF plus
NH4Cl,
3) BCF alone, or
4)
NH4Cl alone. At the end of each
treatment, cell monolayers were extensively washed and further
incubated for 5 h (step
2) and then for 16 h
(step
3) in HBSS or
NH4Cl as depicted in Fig.
1. We found that
1)
NH4Cl alone induced a slight but
significant neutral red dye uptake, whereas BCF alone did not;
2) simultaneous treatment with BCF and NH4Cl greatly enhanced neutral
red uptake compared with NH4Cl alone; 3) treatment with BCF alone
in step
1 significantly enhanced neutral red
dye uptake induced by subsequent treatment with
NH4Cl, whereas neutral red dye
uptake induced by treatment with
NH4Cl in
step
1 was not increased by subsequent
treatments with NH4Cl. The
enhancing effect of BCF on neutral red dye uptake was suppressed by
previous incubation with
II anti-native VacA serum (data not shown).

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Fig. 1.
MKN 28 cells were loaded for 16 h with broth culture filtrate (BCF)
alone, BCF + 4 mM NH4Cl, 4 mM
NH4Cl alone, and uninoculated
broth filtrate (control) (step 1).
At the end of step
1, cells were washed extensively and
incubated for 5 h (step
2) and then for 16 h
(step
3) in Hanks' balanced salt solution
(HBSS) or NH4Cl. At the end of
each step, cell vacuolation was quantitated by neutral red dye uptake
assay. Each SE was <10% of the respective mean.
* P < 0.05, ** P < 0.001 vs. control at
the same step.
|
|
To investigate whether preloading of cells with a weak base was
important for VacA activity and/or potentiation, we incubated MKN 28 cells with NH4Cl before BCF
(alone or with addition of NH4Cl)
treatment. Figure 2 shows that
1) incubation with BCF
(step 3) was ineffective if cells were
loaded with NH4Cl
(step
1) and then incubated with HBSS for
5 h (step
2) and
2) BCF plus
NH4Cl caused neutral red dye
uptake to a similar extent, irrespective of pretreatment with
NH4Cl. Finally, when cells were
loaded with BCF plus NH4Cl and
washed in HBSS, subsequent incubation with BCF alone was ineffective.
Altogether, these results suggest that the vacuolating activity of VacA
was evident only when the weak base and VacA were added simultaneously
to the cells or when NH4Cl was
added to cells previously loaded with VacA.

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Fig. 2.
MKN 28 cells were loaded for 16 h
(step
1) with either BCF alone, 4 mM
NH4Cl alone, or BCF + 4 mM
NH4Cl. After extensive washing,
MKN 28 cells were restored with HBSS alone for 5 h
(step
2); subsequently, cells were
incubated for an additional 16 h with BCF (with and without
NH4Cl) or
NH4Cl alone
(step
3). At the end of each step, cell
vacuolation was quantitated as described in Fig. 1 legend. Each SE was
<10% of the respective mean.
* P < 0.05, ** P < 0.001 vs. BCF alone at
the same step.
|
|
In addition, we studied the persistence of the vacuolating activity of
VacA in cells that had been incubated for 16 h with BCF, maintained in
HBSS for differing time spans, and finally incubated in HBSS or
NH4Cl for an additional 16 h.
Figure 3 shows that incubation with
NH4Cl invariably increased neutral
red dye uptake in MKN 28 cells pretreated with either uninoculated
broth filtrate or BCF and subsequently maintained in HBSS for differing time spans. However, the increase in neutral red dye uptake, as evaluated for each time span considered, was 100% for cells previously loaded with control (Fig. 3A) and
400% for cells previously incubated with BCF (Fig.
3B), representing a statistically
significant difference (P < 0.001).

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Fig. 3.
Persistence of vacuolating toxin A (VacA) vacuolating power. MKN 28 cells were incubated for 16 h with uninoculated broth filtrate
(A) or BCF
(B). After being washed extensively
with HBSS, MKN 28 cells were maintained in HBSS alone for 5, 21, 48, or
72 h and then in HBSS (open bars) or
NH4Cl (hatched bars) for an
additional 16 h. Each SE was <10% of the respective mean.
* P < 0.05, ** P < 0.001 vs. paired
condition.
|
|
Electron microscopy.
The ultrastructure of the MKN 28 cell line and its vacuolar changes
after incubation for 16 h with BCF from
VacA+ H. pylori strains have been reported in detail previously
(29, 30). In the presence of ammonia, H. pylori toxin gave rise to large vacuoles by expansion
and fusion of endosomes. Ultrastructural immunocytochemistry confirmed
the presence of internalized VacA within endosomal tubulovesicles and
related cytoplasmic vacuoles (Fig. 4). In
addition, VacA-immunoreactive bacterial outer membrane vesicles (OMV),
50-300 nm in size, were detected in MKN 28 cell cultures incubated
with H. pylori BCF or with unfiltered
supernatant of H. pylori broth culture
but not in cell cultures incubated with uninoculated broth filtrate.
The OMV were found to interact closely with the luminal-type surface of
MKN 28 cells (Fig. 5), to enter
invaginations of cell membrane and small endocytic vesicles immediately
beneath the cell surface (Fig. 6), and to
accumulate into dilated endosomes and related vacuoles, together with
VacA not bound to OMV (Fig. 4). At the end of 21 h and, especially, 72 h of HBSS treatment, both free and membrane-bound VacA
immunoreactivity levels were substantially reduced in endosomes and
vacuoles, while they were concentrated in discrete vacuolar structures
storing membranous or amorphous material and resembling lysosomes.
VacA-immunoreactive OMV and fragments were prominent in such structures
(Fig. 7).

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Fig. 4.
MKN 28 cell incubated for 16 h with BCF from the
VacA+ CCUG 17874 strain and then
maintained in HBSS for 21 h. VacA immunoreactivity [both free and
bound to outer membrane vesicles (OMV)] is concentrated within
cytoplasmic vacuoles (v). Note the dilated endosomes (e), from which
the vacuoles are believed to originate. Aldehyde-osmium fixation, VacA
immunogold labeling with II serum, and uranyl-lead counterstaining
were used. Original magnification: ×40,000; reduced by 14%.
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Fig. 5.
MKN 28 cell incubated for 16 h in BCF from
VacA+ CCUG 17874. VacA-immunoreactive OMV (arrows; note their evidently trilayered
membrane of bacterial origin) interact closely with microvillar
protrusions of luminal-type cell membrane. Note also the free (i.e.,
not bound to bacterial OMV) VacA reactivity and, inside a
multivesicular body (MV), a fragment of VacA-positive bacterial OMV.
Aldehyde-osmium fixation, VacA immunogold labeling with II serum,
and uranyl-lead counterstaining were used. Original magnification:
×67,000.
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Fig. 6.
A and
B: free and OMV-bound
VacA enter invaginations of luminal-type surface and
underlying endocytic vesicles (e) of BCF-incubated MKN 28 cells.
B: note prominently trilayered
membrane resembling bacterial outer membrane, while differing from
adjacent cell membrane. Aldehyde-osmium fixation, VacA immunogold
labeling with II serum, and uranyl-lead counterstaining were used.
Original magnification: A,
×72,000; B, ×120,000;
reduced by 30%.
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Fig. 7.
MKN 28 cell incubated for 16 h with BCF from the
VacA+ CCUG 17874 strain and then
maintained in HBSS for 72 h. VacA immunoreactivity is concentrated in
vesicular-membranous (lysosomal?) structures that store bacterial OMV
(arrows) and fragments. Note lack of reactivity of most of the vacuoles
(v) and of the dilated endosomal tubulovesicles (e). Aldehyde-osmium
fixation, VacA immunogold labeling with II serum, and uranyl-lead
counterstaining were used. Original magnification: ×67,000.
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|
Immunofluorescence.
The presence and persistence of internalized VacA inside the cell were
also assessed by quantitative immunofluorescence analysis. MKN 28 cells
incubated for 16 h with BCF and then maintained in HBSS for 5, 21, 48 or 72 h exhibited a specific fluorescence (ranging from 270% to 320%
of paired control; P < 0.05 vs.
paired control) that was stable (no statistically significant
differences between differing time points) throughout the entire time
course considered (not shown).
Immunoblotting.
Cell uptake of VacA was further confirmed by immunoblotting analysis of
MKN 28 cell lysates at the end of 16 h of incubation with BCF plus an
additional 21 or 72 h of HBSS treatment (Fig. 8). Cells not loaded with BCF were negative
controls. As shown in Fig. 8, all anti-VacA sera tested recognized an
immunoreactive ~90-kDa protein in cell lysates, with the exception of
cells not loaded with BCF. In each Western blot, BCF-treated cells
(Fig. 8, lanes
1 and
2) clearly differed from control
cells (Fig. 8, lane
3, MKN 28 cells not loaded with BCF),
because BCF-treated cells possessed this ~90-kDa band (Fig. 8). The
persistence of VacA as a ~90-kDa peptide indicates that at least a
large amount of internalized VacA was not degraded into fragments of
smaller molecular mass.

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Fig. 8.
Immunoblotting analysis of MKN 28 cell lysates. Cells were loaded with
BCF and then, after extensive washing, incubated in HBSS for 21 h
(lane
1) or 72 h
(lane
2).
Lane
3, MKN 28 cells not loaded with toxin
(negative control). Lane
4, BCF from VacA-producing
Helicobacter pylori strain CCUG 17874. The following antisera raised against native VacA or its fragments were
used: II (directed against native VacA), B (against region B of
the toxin), BK (against region BK), and C (against region C).
Molecular size markers are indicated at
left. A specific VacA-immunoreactive
~90-kDa band is found in all BCF-incubated cell lysates and in
corresponding BCF; some cross-reacting nonspecific bands are also
present in lysates of cells not incubated with BCF.
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|
 |
DISCUSSION |
Using human gastric epithelial cells in culture and BCF from a
well-characterized VacA+
H. pylori strain, we attempted to
clarify the mechanisms of VacA internalization, action, and fate in the
present study. Our main findings were that
1) only in the presence of
NH4Cl does VacA induce significant
neutral red dye uptake, 2) both free
and membrane-bound (attached to bacterial OMV) VacA is present in BCF
and both forms interact with the cell membrane and are internalized by
the cell, 3) VacA accumulates inside
cells, and does so specifically in endosomal vesicles and related
endolysosomal vacuoles, 4) a large part of internalized VacA retains apparent molecular integrity, and
5) a vacuolating potential persists
in VacA-storing cells.
The neutral red dye uptake study showed that VacA does not induce large
vacuoles in the absence of ammonia, while enhancing NH4Cl vacuolating power. In
agreement with previous findings (30), the specific role of VacA in
enhancing NH4Cl vacuolating power is supported by our tests using BCF preincubated with neutralizing anti-VacA serum. We also confirmed previous observations (28, 30) that
VacA enters cells independently of ammonia but that its vacuolating
action is fully expressed only when
NH4Cl is added.
Internalized VacA persists inside the cell (for up to 72 h)
and seems to preserve a latent vacuolating power that can be activated by the addition of a weak base. An alternative hypothesis is that VacA
induces an unknown permanent cell change that allows the weak base to
cause cellular vacuolation. It should be outlined that
other weak bases not investigated here, such as nicotine or
trimethylamine, have been shown to give the same VacA potentiating effect as NH4Cl (7).
Immunoblotting analysis showed that the bulk of internalized VacA, as
localized into the endosomal compartments, does not undergo cleavage.
The endosomal acidic environment possibly induces some modifications in
the toxin itself. An acid-induced increase in the stability of VacA has
recently been reported by de Bernard et al. (8). It is possible that
protonation of VacA [predicted isoelectric point of 9.1 and 12%
arginine content (6)] takes place inside the acidic endosomal
compartments. This could prevent toxin cleavage (17). In addition, we
should consider the possibility that internalized toxin resists the low
concentration of hydrolitic enzymes present in the endosomal
compartment and never reaches the hydrolase-rich lysosome. This is
supported by the observation that H. pylori toxin interferes with processes controlling the late stages of the endocytic pathway (24, 36). Our findings fit with
recent observations (21, 32) that VacA induces the accumulation of a
postendosomal hybrid compartment, resembling both late endosomes and
lysosomes, but with a reduced proteolytic activity compared with normal
late endosomes and lysosomes.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge F. Tanzi (Pavia, Italy) for help with the
quantitative immunofluorescence analysis.
 |
FOOTNOTES |
This research was supported in part by grants from the Italian Ministry
of Health to Instituto Recovero e Cura a Carattere Scientifico
Policlinico San Matteo Hospital, the Italian Ministry of University and
Research, and the University of Pavia.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: E. Solcia, Dept. of Human Pathology, Via
Forlanini 16, 27100 Pavia, Italy.
Received 20 March 1998; accepted in final form 5 June 1998.
 |
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