Infection and Immunity, October 1999, p. 5247-5252, Vol. 67, No. 10
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Kinetics and Mechanisms of Extracellular
Protein Release by Helicobacter pylori
Wayne
Schraw,1
Mark S.
McClain,1 and
Timothy L.
Cover1,2,3,*
Division of Infectious Diseases, Department
of Medicine,1 and Department of
Microbiology and Immunology,2 Vanderbilt
University School of Medicine, Nashville, Tennessee 37232-2605, and
Department of Veterans Affairs Medical Center, Nashville,
Tennessee 37232-26373
Received 9 April 1999/Returned for modification 24 May
1999/Accepted 16 July 1999
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ABSTRACT |
To investigate the kinetics and mechanisms of extracellular protein
release by Helicobacter pylori, we analyzed the entry of
metabolically radiolabeled bacterial proteins into broth culture supernatant. At early time points, vacuolating cytotoxin (VacA) constituted a major extracellular protein. Subsequently, culture supernatants accumulated many proteins that were components of intact
bacterial cells. This nonselective release of proteins was associated
with a decreasing turbidity of cultures and loss of bacterial
viability, indicative of an autolytic process. The rates of VacA
secretion and autolysis were each influenced by medium composition, and
therefore these may be regulated phenomena. Extracellular release of
proteins by H. pylori may be an important adaptation that
facilitates the persistence of H. pylori in the human
gastric mucus layer. Moreover, entry of proinflammatory proteins into
the gastric mucosa may contribute to the induction of a mucosal
inflammatory response.
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INTRODUCTION |
Helicobacter pylori are
noninvasive gram-negative bacteria that colonize the gastric mucosae of
more than half of the world's human population. Gastric colonization
by H. pylori is consistently associated with a mucosal
inflammatory response and is a risk factor for peptic ulcer disease and
gastric malignancies (6, 10, 18, 22). The mechanisms by
which H. pylori colonization leads to gastric mucosal
inflammation are not yet well understood, but entry of proinflammatory
bacterial components into the mucosa may be contributory. One such
bacterial component, urease, has been directly localized within the
lamina propria by immunohistochemical staining (15, 20, 21).
H. pylori proteins that are secreted or released into the
extracellular space may play important roles in enabling H. pylori to persistently colonize the gastric mucosa and may be
relevant to the pathogenesis of H. pylori-associated
diseases. Several previous studies have analyzed the release of
H. pylori proteins into broth culture supernatants during
bacterial growth in vitro (3, 5, 7, 29, 35). One of the
proteins found in broth culture supernatants is a vacuolating cytotoxin
(VacA) that alters the morphology and function of gastric epithelial
cells (4, 23). H. pylori broth culture
supernatants also contain an assortment of additional proteins,
including urease and heat shock proteins (HspA and HspB), which in most
bacterial species are localized exclusively in the cytoplasm (3,
5, 7, 29, 35). The pathways by which these proteins enter the
extracellular space are not yet completely understood. Therefore, in
this study we sought to investigate the kinetics and mechanisms of
extracellular protein release by H. pylori.
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MATERIALS AND METHODS |
Bacterial culture conditions.
H. pylori 60190 (ATCC
49503) is a cag+ strain containing a type s1a/m1
vacA allele (8). This strain was routinely grown at 37°C on blood agar plates (Trypticase soy agar containing 5% sheep blood) in room air supplemented with 8% CO2. Broth
cultures were inoculated by scraping bacteria from plates into sterile 0.9% saline solution and immediately adding this suspension to liquid
media. The initial optical density at 600 nm (OD600) of all
cultures was about 0.13. Broth cultures were agitated with a rotary
shaker and incubated at 37°C in room air containing 8% CO2. Bacterial growth was monitored by measuring the
OD600 of cultures at serial time points, using uninoculated
culture media as blanks. Concentrations of viable bacteria in broth
cultures were quantified by plating 10-µl aliquots of appropriate
10-fold dilutions on blood agar plates. Plates were incubated at 37°C for 3 days, colonies were counted, and results were expressed as
CFU/ml.
Metabolic labeling of bacterial proteins.
H. pylori
were serially passaged at 24-h intervals on blood agar plates and then
inoculated into sulfite-free brucella broth (SFBB) (16)
supplemented with 20 to 50 µCi of [35S]cysteine and
[35S]methionine per ml (Express protein labeling mix; NEN
Dupont), 0.025% (wt/vol) finely granulated activated charcoal, and 10 µg of vancomycin per ml. Activated charcoal was placed adjacent to culture flasks to absorb volatilized radioactivity. At varying times,
15-ml aliquots of the culture were removed and centrifuged at
17,000 × g for 20 min at 4°C to remove bacteria.
Proteins in the broth culture supernatant were precipitated by the
addition of 70% (wt/vol) solid ammonium sulfate. H. pylori
proteins also were radiolabeled in methionine-and-cysteine-free RPMI
1640 medium (GIBCO BRL) containing 10 µg of vancomycin per ml.
Analysis of radiolabeled H. pylori proteins.
Radioactivity in preparations of bacterial cells and preparations of
concentrated supernatant proteins was quantified by liquid scintillation counting (5 to 10 µl per 4 ml of Envirosafe)
(ANOROC Scientific, Hackensack, N.J.). Equal amounts of
radioactivity (0.5 × 105 to 1 × 105
cpm) were loaded onto 12% polyacrylamide gels, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with the method of Laemmli (19). Following
SDS-PAGE, 35S-containing gels were soaked in Entensify (NEN
Dupont) according to the manufacturer's instructions, were
vacuum-dried at 80°C for 1 h, and were exposed to X-ray film
(full-speed blue film; Kodak) with an intensifier screen at 
70°C.
Analysis of extracellular membrane blebs.
To identify
proteins that may be components of extracellular membrane blebs,
H. pylori cultures were centrifuged at 17,000 × g to remove intact bacteria, and the culture supernatant was recentrifuged at 100,000 × g for 60 min at 4°C.
Putative membrane bleb preparations and soluble proteins were analyzed
by SDS-PAGE and silver staining.
N-terminal sequence analysis.
H. pylori proteins were
separated by SDS-PAGE and transferred to polyvinylidene difluoride
(PVDF) paper (Bio-Rad) by electroblotting for 30 min at 50 V in 10 mM
CAPS (3-cyclohexylamino-1-propanesulfonic acid) buffer (pH 11). After
Coomassie blue staining, the bands of interest were excised, and
N-terminal sequence analysis was performed with a PE Biosystems Procise
492 protein sequencing apparatus in the Protein Chemistry Core
Laboratory of Vanderbilt University School of Medicine. N-terminal
amino acid sequences were identified by searching for matches in the
known genome sequence of H. pylori 26695 (34).
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RESULTS |
Time-dependent extracellular release of radiolabeled H. pylori proteins.
To investigate the kinetics and mechanisms
by which H. pylori proteins are released into the
extracellular space, we metabolically labeled H. pylori
proteins during growth in liquid media and analyzed the entry of
radiolabeled proteins into the extracellular space at serial time
points. During growth of H. pylori 60190 in SFBB medium,
bacterial proteins were effectively radiolabeled by the 8-h time point,
but radiolabeled proteins were not detected in any substantial quantity
in the culture supernatant at this time point (Fig.
1). At the 24-h time point, the most
prominent radiolabeled supernatant protein migrated as an 87-kDa band,
which was detected in only trace quantities in the bacterial cell
pellet. Most other supernatant bands detected at the 24-h time point
corresponded to major bands in the bacterial cells. At the 48-h time
point, the supernatant contained numerous dense bands that were also present in intact bacterial cells. The size of the 87-kDa band detected
in the culture supernatant at early time points corresponds to the
known molecular mass of VacA (4, 5). To determine whether
the radiolabeled 87-kDa band indeed represented VacA, wild-type
H. pylori 60190 and an isogenic strain containing an insertion mutation in vacA (8) were each
metabolically labeled, and culture supernatants were analyzed. A
radiolabeled 87-kDa band was detected in supernatant from the wild-type
strain but not from the mutant (Fig. 2).
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FIG. 1.
Metabolic labeling of H. pylori during growth
in broth culture. H. pylori 60190 was inoculated into 50 ml
of SFBB containing 50 µCi of [35S]cysteine and
[35S]methionine per ml (initial OD600, 0.13),
and aliquots were removed after 8, 24, and 48 h of growth at
37°C. Turbidities (judged by OD600) of the culture at
these time points were 0.256, 0.872, and 0.726, respectively. Bacterial
cell pellets and supernatant proteins were prepared and analyzed by
SDS-PAGE and autoradiography, as described in Materials and Methods.
Lanes represent bacterial cells from 8- (a), 24- (b), and 48-h (c) time
points and supernatant proteins from 8- (d), 24- (e), and 48-h (f) time
points. An 87-kDa band constituted the major extracellular protein at
the 24-h time point (arrow). Molecular mass in kilodaltons is shown on
the left.
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FIG. 2.
Secretion of H. pylori VacA. H. pylori 60190 (wild-type strain) and an isogenic
vacA-null mutant (60190-v2) were metabolically labeled with
50 µCi of [35S]cysteine and
[35S]methionine per ml of SFBB medium for 30 h.
Bacterial cell pellets and supernatant proteins were prepared and
analyzed by SDS-PAGE and autoradiography. Lane a, H. pylori
60190 supernatant proteins; lane b, H. pylori 60190-v2
supernatant proteins. An 87-kDa band corresponding to VacA is present
in lane a (arrow) but not in lane b. Molecular mass in kilodaltons is
shown on the left.
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These data indicate that VacA enters the extracellular space at
relatively early time points via a selective secretory process, whereas
most other H. pylori proteins enter the extracellular space
at later time points via a less selective process. Scintillation counting indicated that culture supernatant proteins from a 72-h broth
culture contained four- to fivefold higher levels of radioactivity (in
counts per minute) than supernatant proteins from a 24-h culture, whereas there was no substantial increase during this time period in
the radioactivity of bacterial cells (Table
1). In addition, the turbidity (judged by
OD600) of 72-h cultures was about 30% less than the
maximum turbidity measured at earlier time points (Table 1). These data
suggest that H. pylori releases proteins into the
extracellular space as a result of autolysis.
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TABLE 1.
Incorporation of 35S into H. pylori cells and release of 35S-labeled proteins into
the extracellular spacea
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Effect of culture medium composition on VacA secretion and
autolysis.
We next sought to determine whether rates of VacA
secretion and autolysis were dependent upon the composition of the
liquid culture medium. Two different media (SFBB as described above and methionine-and-cysteine-free RPMI 1640 medium [GIBCO BRL]) were inoculated simultaneously with H. pylori 60190, and proteins
were metabolically radiolabeled. The efficiency of metabolic labeling of bacterial cells was about sevenfold greater in RPMI medium than in
SFBB medium (data not shown). Diminished incorporation of
[35S]methionine and [35S]cysteine into
proteins of bacteria grown in SFBB medium may be attributable to the
competitive effects of nonradioactive methionine and cysteine in the
SFBB medium. Analysis of radiolabeled proteins in supernatant from
SFBB-based cultures showed results similar to those in the first
experiment, i.e., evidence of VacA secretion within 16 h and
increasing autolysis at the 48-h time point (Fig. 3, top panel). In contrast, VacA did not
constitute a prominent component of the supernatant proteins from RPMI
medium-based cultures (Fig. 3, lower panel). A comparison of
supernatant protein profiles with bacterial cell protein profiles
indicates that substantial autolysis occurred within 16 h of
inoculation of H. pylori into RPMI medium. Additional
experiments indicated that radiolabeled proteins were released and
detectable by autoradiography within 1 h of the addition of
H. pylori to RPMI medium (data not shown).
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FIG. 3.
Effect of culture medium composition on VacA secretion
and autolysis. H. pylori 60190 was inoculated simultaneously
into 50 ml of SFBB and 50 ml of methionine-and-cysteine-free RPMI
medium and metabolically labeled with [35S]cysteine and
[35S]methionine. Aliquots were removed after 16, 24, and
48 h at 37°C. Turbidities (judged by OD600) of the
cultures at 0, 16, 24, and 48 h were as follows: SFBB, 0.130, 0.585, 0.736, and 0.532; RPMI, 0.130, 0.045, 0.047, and 0.034. Bacterial cell pellets and broth culture supernatant proteins were
prepared and analyzed by SDS-PAGE and autoradiography, as described in
Materials and Methods. Upper panel, culture from SFBB; lower panel,
culture from methionine-and-cysteine-free RPMI medium. Lanes represent
bacterial cells from 16- (a), 24- (b), and 48-h (c) time points and
culture supernatant proteins from 16- (d), 24- (e), and 48-h (f) time
points. Molecular mass in kilodaltons is shown on the left.
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Further studies were carried out to investigate and compare the
physiological condition of the bacteria in the two different media. The
turbidity (judged by OD600) of H. pylori
cultures in SFBB medium increased over the first 24 h, whereas
there was a progressive decrease in the OD of the culture in RPMI
medium (Fig. 4, top panel). In addition,
the number of CFU remained stable in SFBB, but steadily decreased in
RPMI medium (Fig. 4, bottom panel). Thus, during culture in SFBB
medium, bacterial growth seems to be balanced by a loss of bacterial
viability, whereas during culture in RPMI medium, no bacterial growth
is detectable. However, the capacity of H. pylori to
incorporate [35S]methionine and
[35S]cysteine into newly synthesized proteins indicates
that the bacteria are metabolically active in both types of media.
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FIG. 4.
Effect of culture medium composition on H. pylori growth and viability. H. pylori 60190 was
inoculated simultaneously into 50 ml of SFBB and 50 ml of
methionine-and-cysteine-free RPMI medium. Aliquots were removed at 0, 6, 24, and 48 h for measurement of turbidity (judged by
OD600) (top panel) and viable bacterial density (CFU per
ml) (bottom panel). Measurements of CFU per ml represent the means of
quadruplicate determinations; standard deviations were smaller than the
symbol size at all time points. Closed symbols, SFBB; open symbols,
RPMI medium.
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Extracellular release of H. pylori superoxide
dismutase.
In an effort to identify novel proteins other than VacA
that might be specifically secreted into the extracellular space, we
carefully compared the SDS-PAGE protein profiles of H. pylori cells and extracellular proteins. A 25-kDa band was
identified, which appeared to be overrepresented in supernatant
compared to intact bacterial cells from RPMI-based cultures (Fig.
5). N-terminal sequence analysis of this
band yielded the sequence MFTLRELPFA, which coincides with the sequence
of the N terminus of H. pylori superoxide dismutase (SodB)
(25, 31).
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FIG. 5.
Analysis of proteins released into the extracellular
space during incubation of H. pylori in RPMI medium.
H. pylori 60190 was grown for 24 h in 500 ml of SFBB,
and the bacterial cells were then pelleted, washed in RPMI medium, and
inoculated into 50 ml of methionine-and-cysteine-free RPMI medium.
After incubation at 37°C for 24 h, the culture was centrifuged,
and both cells and concentrated supernatant proteins (10 µg of each
protein) were separated by SDS-PAGE, electroblotted to PVDF paper, and
stained with Coomassie blue. The indicated 25-kDa band in the
supernatant (arrowhead) was excised, and N-terminal sequence analysis
yielded a sequence (MFTLRELPFA) that coincides with the sequence of the
N terminus of superoxide dismutase (25, 31). Lane a,
H. pylori cells; lane b, H. pylori supernatant
proteins. Molecular mass in kilodaltons is shown on the left.
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Role of membrane vesicles in extracellular protein release.
In
addition to undergoing bacterial autolysis, H. pylori may
release proteins in the form of membrane vesicles or blebs (12, 17, 30). To identify proteins that may be components of such structures, H. pylori was incubated in
methionine-and-cysteine-free RPMI medium for 24 h as described
above, and then the culture was centrifuged at 17,000 × g to remove intact bacteria. To pellet membranous material, the
culture supernatant then was recentrifuged at 100,000 × g for 60 min. As shown in Fig. 6,
several proteins were pelleted by ultracentrifugation (lane c), but
most supernatant proteins remained soluble (lane b). This indicates
that most H. pylori proteins released into the extracellular
space are not components of membrane vesicles or blebs. The protein
compositions of putative membrane vesicle preparations from RPMI and
SFBB media were similar (Fig. 7). One of
the most prominent extracellular proteins pelleted by centrifugation at
100,000 × g migrated at 19 kDa (Fig. 6 and 7).
N-terminal sequence analysis of 19-kDa bands derived from RPMI- and
SFBB-based culture supernatants yielded the same sequence (MLSKDIIKLL),
which coincides with the sequence of the N terminus of non-heme
iron-containing ferritin (Pfr) (9, 13).
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FIG. 6.
Analysis of potential membrane bleb-associated proteins
in H. pylori supernatant. H. pylori 60190 was
grown for 24 h in 100 ml of SFBB, pelleted, washed, and then
resuspended and incubated in 25 ml of methionine-and-cysteine-free RPMI
medium for 24 h at 37°C. The culture was centrifuged at
17,000 × g for 20 min to remove intact bacteria, and
the supernatant was then recentrifuged at 100,000 × g
for 1 h. Proteins (5 µg/lane) were separated by SDS-PAGE
followed by visualization with silver staining. Lane a, bacterial cells
pelleted by 17,000 × g centrifugation; lane b,
proteins remaining in supernatant after centrifugation at
100,000 × g; and lane c, proteins present in
supernatant after 17,000 × g centrifugation and
subsequently pelletted by centrifugation at 100,000 × g. Molecular mass in kilodaltons is shown on the left.
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FIG. 7.
Analysis of proteins in putative membrane bleb fractions
isolated from H. pylori broth culture supernatants. H. pylori 60190 was incubated in methionine-and-cysteine-free RPMI
medium or in SFBB medium for 24 h at 37°C. Cultures were
centrifuged at 17,000 × g for 20 min to remove intact
bacteria, and supernatants were then recentrifuged at
100,000 × g for 1 h. Proteins in intact bacterial
cells and putative membrane bleb fractions then were separated by
SDS-PAGE, electroblotted to PVDF paper, and stained with Coomassie
blue. Lane a, bacterial cells from RPMI medium-based culture; lane b,
putative membrane bleb components from RPMI medium-based culture; lane
c, bacterial cells from SFBB-based culture; and lane d, putative
membrane bleb components from SFBB-based culture. N-terminal sequence
analysis of a 19-kDa band (arrow) yielded a sequence (MLSKDIIKLL) that
coincides with the sequence of the N terminus of non-heme
iron-containing ferritin (Pfr) (9, 13). Molecular mass in
kilodaltons is shown on the left.
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DISCUSSION |
In summary, these results indicate that H. pylori
proteins can enter the extracellular space via at least three different mechanisms. (i) An early, selective appearance of radiolabeled VacA in
culture supernatant relative to other proteins, combined with its
preferential localization in supernatant rather than the cell pellet,
provides convincing evidence that VacA is specifically secreted. This
conclusion is consistent with the structural organization of VacA,
which is similar to that of the immunoglobulin A protease family of
secreted proteins (8, 28, 33). (ii) Nonselective entry of
H. pylori proteins into culture supernatant, concomitant with a gradual decrease in the OD of broth cultures and loss of bacterial viability, is attributed to bacterial autolysis. The occurrence of autolysis in H. pylori has been proposed
previously based on ultrastructural evidence of bacterial membrane
fragmentation (26) and may account for the surface
localization of proteins such as urease and HspB (11, 26).
(iii) Several previous studies have reported that H. pylori
releases membrane vesicles or blebs (12, 17, 30). Many
extracellular proteins that are sedimented by ultracentrifugation are
likely to be components of such membranous structures (17).
However, the 19-kDa protein (Pfr) is primarily a cytoplasmic protein
(9, 13), and therefore, we speculate that it is pelleted in
the form of paracrystalline inclusions (9, 13) released from
autolysed bacteria rather than as a component of membranous blebs.
An unexpected finding was that a 25-kDa protein corresponding to
superoxide dismutase seemed to be selectively released into the
extracellular space. In accordance with this observation, Spiegelhalder
et al. demonstrated that superoxide dismutase is localized on the
surface of H. pylori (31). H. pylori
superoxide dismutase does not contain an N-terminal signal sequence,
and there are no contiguous genes in the genome of H. pylori
26695 (34) that encode putative proteins involved in
secretory pathways. Thus, at present, there is no obvious explanation
for the selective release of H. pylori superoxide dismutase
into broth culture supernatant, but a specific secretion pathway must
be considered. Similarly, a recent study concluded that H. pylori urease, HspA, and HspB are selectively released into the
extracellular space, despite the absence of any known specific
secretion pathways for these proteins (35).
In this study, we noted that the rate of H. pylori autolysis
is increased when organisms are incubated in RPMI medium compared to
SFBB. Conversely, VacA secretion was more prominent in SFBB. SFBB is a
rich culture medium prepared from animal tissue, yeast extract, and
casein, whereas RPMI medium is comparatively sparse in nutrient
content. In particular, the RPMI medium used in this study was free of
alanine and methionine, both of which are thought to be essential amino
acids for H. pylori growth (24, 27). Thus, both
autolysis and VacA secretion may be regulated phenomena that are
influenced either by the composition of the bacterial culture medium or
by the growth phase of the organism.
Autolysis of H. pylori potentially may serve several useful
functions. One model suggests that the autolytic release of H. pylori proteins is a mechanism for evading host defenses, either by modifying surface properties of viable organisms or by overwhelming host defenses with decoy antigens (26). Another model
suggests that released proinflammatory H. pylori proteins
enter the gastric mucosa, stimulate an inflammatory response, and
thereby provide H. pylori with an enhanced supply of
nutrients (2). Finally, autolysis is an effective mechanism
for releasing bacterial DNA, which can be taken up by viable, naturally
competent organisms and used to generate recombinant alleles. Genetic
recombination seems to have played an important role in the evolution
of H. pylori (14, 32) and presumably provides a
mechanism for generating diversity that is more rapid than mutation
alone (1). Thus, autolysis may be an important adaptation
that facilitates persistence of H. pylori in the gastric
mucus layer.
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ACKNOWLEDGMENTS |
This work was supported in part by the National Institutes of
Health (AI 39657) and the Medical Research Service of the Department of
Veterans Affairs. Protein sequencing facilities are supported by
funding to the Vanderbilt Cancer Center (P30 CA68485).
We thank M. H. Forsyth for helpful discussions.
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FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Infectious Diseases, A3310 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232-2605. Phone: (615) 322-2035. Fax: (615) 343-6160. E-mail:
COVERTL{at}ctrvax.vanderbilt.edu.
Editor:
D. L. Burns
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REFERENCES |
| 1.
|
Berg, D. E.,
P. S. Hoffman,
B. J. Appelmelk, and J. G. Kusters.
1997.
The Helicobacter pylori genome sequence: genetic factors for long life in the gastric mucosa.
Trends Microbiol.
5:468-474[Medline].
|
| 2.
|
Blaser, M. J.
1992.
Hypotheses on the pathogenesis and natural history of Helicobacter pylori-induced inflammation.
Gastroenterology
102:720-727[Medline].
|
| 3.
|
Cao, P.,
M. S. McClain,
M. H. Forsyth, and T. L. Cover.
1998.
Extracellular release of antigenic proteins by Helicobacter pylori.
Infect. Immun.
66:2984-2986[Abstract/Free Full Text].
|
| 4.
|
Cover, T. L.
1996.
The vacuolating cytotoxin of Helicobacter pylori.
Mol. Microbiol.
20:241-246[Medline].
|
| 5.
|
Cover, T. L., and M. J. Blaser.
1992.
Purification and characterization of the vacuolating toxin from Helicobacter pylori.
J. Biol. Chem.
267:10570-10575[Abstract/Free Full Text].
|
| 6.
|
Cover, T. L., and M. J. Blaser.
1996.
Helicobacter pylori infection, a paradigm for chronic mucosal inflammation: pathogenesis and implications for eradication and prevention.
Adv. Intern. Med.
41:85-117[Medline].
|
| 7.
|
Cover, T. L.,
P. I. Hanson, and J. E. Heuser.
1997.
Acid-induced dissociation of VacA, the Helicobacter pylori vacuolating cytotoxin reveals its pattern of assembly.
J. Cell Biol.
138:759-769[Abstract/Free Full Text].
|
| 8.
|
Cover, T. L.,
M. K. R. Tummuru,
P. Cao,
S. A. Thompson, and M. J. Blaser.
1994.
Divergence of genetic sequences for the vacuolating cytotoxin among Helicobacter pylori strains.
J. Biol. Chem.
269:10566-10573[Abstract/Free Full Text].
|
| 9.
|
Doig, P.,
J. W. Austin, and T. J. Trust.
1993.
The Helicobacter pylori 19.6-kilodalton protein is an iron-containing protein resembling ferritin.
J. Bacteriol.
175:557-560[Abstract/Free Full Text].
|
| 10.
|
Dunn, B. E.,
H. Cohen, and M. J. Blaser.
1997.
Helicobacter pylori.
Clin. Microbiol. Rev.
10:720-741[Abstract].
|
| 11.
|
Dunn, B. E.,
N. B. Vakil,
B. G. Schneider,
M. M. Miller,
J. B. Zitzer,
T. Peutz, and S. H. Phadnis.
1997.
Localization of Helicobacter pylori urease and heat shock protein in human gastric biopsies.
Infect. Immun.
65:1181-1188[Abstract].
|
| 12.
|
Fiocca, R.,
V. Necchi,
P. Sommi,
V. Ricci,
J. Telford,
T. L. Cover, and E. Solcia.
1999.
Release of Helicobacter pylori vacuolating cytotoxin by both a specific secretion pathway and budding of outer membrane vesicles. Uptake of released toxin and vesicles by gastric epithelium.
J. Pathol.
188:220-226[Medline].
|
| 13.
|
Frazier, B. A.,
J. D. Pfeifer,
D. G. Russell,
P. Falk,
A. N. Olsen,
M. Hammar,
T. U. Westblom, and S. J. Normark.
1993.
Paracrystalline inclusions of a novel ferritin containing nonheme iron, produced by the human gastric pathogen Helicobacter pylori: evidence for a third class of ferritins.
J. Bacteriol.
175:966-972[Abstract/Free Full Text].
|
| 14.
|
Go, M. F.,
V. Kapur,
D. Y. Graham, and J. M. Musser.
1996.
Population genetic analysis of Helicobacter pylori by multilocus enzyme electrophoresis: extensive allelic diversity and recombinational population structure.
J. Bacteriol.
178:3934-3938[Abstract/Free Full Text].
|
| 15.
|
Harris, P. R.,
H. L. T. Mobley,
G. I. Perez-Perez,
M. J. Blaser, and P. D. Smith.
1996.
Helicobacter pylori urease is a potent stimulus of mononuclear phagocyte activation and inflammatory cytokine production.
Gastroenterology
111:419-425[Medline].
|
| 16.
|
Hawrylik, S. J.,
D. J. Wasilko,
S. L. Haskell,
T. D. Gootz, and S. E. Lee.
1994.
Bisulfite or sulfite inhibits growth of Helicobacter pylori.
J. Clin. Microbiol.
32:790-792[Abstract/Free Full Text].
|
| 17.
|
Keenan, J. I.,
R. A. Allardyce, and P. F. Bagshaw.
1998.
Lack of protection following immunisation with H. pylori outer membrane vesicles highlights antigenic differences between H. felis and H. pylori.
FEMS Microbiol. Lett.
161:21-27[Medline].
|
| 18.
|
Labigne, A., and H. deReuse.
1996.
Determinants of Helicobacter pylori pathogenicity.
Infect. Agents Dis.
5:191-202[Medline].
|
| 19.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[Medline].
|
| 20.
|
Mai, U. E. H.,
G. I. Perez-Perez,
J. B. Allen,
S. M. Wahl,
M. J. Blaser, and P. D. Smith.
1992.
Surface proteins from Helicobacter pylori exhibit chemotactic activity for human leukocytes and are present in gastric mucosa.
J. Exp. Med.
175:517-525[Abstract/Free Full Text].
|
| 21.
|
Mai, U. E. H.,
G. I. Perez-Perez,
L. M. Wahl,
S. M. Wahl,
M. J. Blaser, and P. D. Smith.
1991.
Soluble surface proteins from Helicobacter pylori activate monocytes/macrophages by lipopolysaccharide-independent mechanism.
J. Clin. Investig.
87:894-900.
|
| 22.
|
Mobley, H. L.
1997.
Helicobacter pylori factors associated with disease development.
Gastroenterology
113(Suppl. 6):S21-S28[Medline].
|
| 23.
|
Montecucco, C.
1998.
Protein toxins and membrane transport.
Curr. Opin. Cell Biol.
10:530-536[Medline].
|
| 24.
|
Nedenskov, P.
1994.
Nutritional requirements for growth of Helicobacter pylori.
Appl. Environ. Microbiol.
60:3450-3453[Abstract/Free Full Text].
|
| 25.
|
Pesci, E. C., and C. L. Pickett.
1994.
Genetic organization and enzymatic activity of a superoxide dismutase from the microaerophilic human pathogen, Helicobacter pylori.
Gene
143:111-116[Medline].
|
| 26.
|
Phadnis, S. H.,
M. H. Parlow,
M. Levy,
D. Ilver,
C. M. Caulkins,
J. B. Connors, and B. E. Dunn.
1996.
Surface localization of Helicobacter pylori urease and a heat shock protein homolog requires bacterial autolysis.
Infect. Immun.
64:905-912[Abstract].
|
| 27.
|
Reynolds, D. J., and C. W. Penn.
1994.
Characteristics of Helicobacter pylori growth in a defined medium and determination of its amino acid requirements.
Microbiology
140:2649-2656[Abstract].
|
| 28.
|
Schmitt, W., and R. Haas.
1994.
Genetic analysis of the Helicobacter pylori vacuolating cytotoxin: structural similarities with the IgA protease type of exported protein.
Mol. Microbiol.
12:307-319[Medline].
|
| 29.
|
Scott, D. R.,
D. Weeks,
C. Hong,
S. Postius,
K. Melchers, and G. Sachs.
1998.
The role of internal urease in acid resistance of Helicobacter pylori.
Gastroenterology
114:58-70[Medline].
|
| 30.
|
Sommi, P.,
V. Ricci,
R. Fiocca,
V. Necchi,
M. Romano,
J. L. Telford,
E. Solcia, and U. Ventura.
1998.
Persistence of Helicobacter pylori VacA toxin and vacuolating potential in cultured gastric epithelial cells.
Am. J. Physiol.
275:G681-G688[Abstract/Free Full Text].
|
| 31.
|
Spiegelhalder, C.,
B. Gerstenecker,
A. Kersten,
E. Schiltz, and M. Kist.
1993.
Purification of Helicobacter pylori superoxide dismutase and cloning and sequencing of the gene.
Infect. Immun.
61:5315-5325[Abstract/Free Full Text].
|
| 32.
|
Suerbaum, S.,
J. Maynard Smith,
K. Bapumia,
G. Morelli,
N. H. Smith,
E. Kunstmann,
I. Dyrek, and M. Achtman.
1998.
Free recombination within Helicobacter pylori.
Proc. Natl. Acad. Sci. USA
95:12619-12624[Abstract/Free Full Text].
|
| 33.
| Telford, J. L., 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, and R. Rappuoli. 1994. Gene structure of the Helicobacter
pylori cytotoxin and evidence of its key role in gastric disease.
179:1653-1658.
|
| 34.
|
Tomb, J.-F.,
O. White,
A. R. Kerlavage,
R. A. Clayton,
G. G. Sutton, et al.
1997.
The complete genome sequence of the gastric pathogen Helicobacter pylori.
Nature
388:539-547[Medline].
|
| 35.
|
Vanet, A., and A. Labigne.
1998.
Evidence for specific secretion rather than autolysis in the release of some Helicobacter pylori proteins.
Infect. Immun.
66:1023-1027[Abstract/Free Full Text].
|
Infection and Immunity, October 1999, p. 5247-5252, Vol. 67, No. 10
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
