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Infection and Immunity, July 2001, p. 4202-4209, Vol. 69, No. 7
Department of Microbiology and Molecular Biology, Tufts
University School of Medicine,1 and
Division of Gastroenterology, Department of Medicine, New
England Medical Center,2 Boston, Massachusetts
02111, and Department of Veterinary Biosciences, Ohio State
University, Columbus, Ohio 432103
Received 11 January 2001/Returned for modification 20 February
2001/Accepted 3 April 2001
Infection with Helicobacter pylori strains containing
the cag Pathogenicity Island (cag PAI) is
strongly correlated with the development of severe gastric
disease, including gastric and duodenal ulceration,
mucosa-associated lymphoid tissue lymphoma, and gastric carcinoma.
Although in vitro studies have demonstrated that the expression of
genes within the cag PAI leads to the activation of a
strong host inflammatory response, the functions of most cag gene products and how they work in concert to promote
an immunological response are unknown. We developed a transcriptional
reporter that utilizes urease activity and in which nine putative
regulatory sequences from the cag PAI were fused to the
H. pylori ureB gene. These fusions were introduced in
single copies onto the H. pylori chromosome without
disruption of the cag PAI. Our analysis indicated that
while each regulatory region confers a reproducible amount of promoter
activity under laboratory conditions, they differ widely in levels of
expression. Transcription initiating upstream of cag15 and
upstream of cag21 is induced when the respective fusion
strains are cocultured with an epithelial cell monolayer. Results of
mouse colonization experiments with an H. pylori strain carrying the cag15-ureB fusion suggested that this putative
regulatory region appears to be induced in vivo, demonstrating the
importance of the urease reporter as a significant development toward
identifying in vivo-induced gene expression in H. pylori.
Chronic infection with the gastric
pathogen Helicobacter pylori is a significant cause of
worldwide morbidity and mortality. Millions of people annually
experience H. pylori-associated disease that most often
presents as chronic gastritis. However, infection has also been
correlated with gastric and duodenal ulcer disease, gastric
adenocarcinoma (22), and mucosa-associated lymphoma (8, 20). The most severe H. pylori-mediated
disease states are attributable to strains harboring the cag
pathogenicity island (cag PAI) (type I strains). Two groups
independently identified this discrete 40-kb DNA element in different
clinical isolates (1, 8).
Analysis of the cag PAI sequence suggested that it encodes a
putative secretion apparatus with homology to type IV secretion systems
(8, 35), which are involved in the transfer of effector macromolecules into host cells (7, 39). Evidence
supporting such a role for the H. pylori cag PAI includes
the finding that null mutations in several of the genes abolish the
ability of type I strains to elicit interleukin 8 (IL-8) secretion by
gastric epithelial cells (1, 8, 18, 37). This cytokine
signal triggers an inflammatory response that, when chronic,
contributes to epithelial cell death and tissue damage
(34). The ability of type I strains to elicit an IL-8
response strongly supports a role for the cag PAI in chronic
inflammation (8, 9, 10, 14, 27). Additionally, it has been
demonstrated that one of the cag gene products, CagA, is
translocated into host cells, where it becomes modified by tyrosine
phosphorylation (2, 24, 26, 32). Inactivation of several
of the cag genes was shown to abolish both CagA
translocation and tyrosine phosphorylation, suggesting that both events
depend on an intact cag PAI (24, 26, 32).
We are interested in how and to what extent H. pylori
regulates its gene expression, particularly with regard to
establishing infection. An impediment to this type of analysis
for H. pylori has been the lack of sensitive
reporter systems for measuring the gene expression that is required for
establishing infection. Here we describe the development of a new
reporter system for H. pylori that utilizes urease
production as a measure of gene expression. The urease reporter
provides a sensitive and accurate measure of H. pylori gene
expression that can be quantified by an enzymatic assay, Western
analysis, or mRNA determination. We have used this reporter system to
identify and characterize transcriptionally active regions of the
cag PAI in H. pylori. Our results suggest that
the cag PAI is comprised of genes arranged in several
multicistronic units. Each transcriptional unit that we investigated
has a characteristic level of expression in H. pylori cells
grown on laboratory medium. We provide evidence that transcription from
two of these units is upregulated when H. pylori is
cocultured in the presence of a human epithelial cell line.
Bacterial strains and plasmids.
H. pylori C57, a
type I clinical isolate, and M6, a mouse-adapted type I isolate, were
donated by Steven Czinn (Case Western Reserve University, Cleveland,
Ohio) and are described in Table 1.
Strain 412 is a derivative of H. pylori C57 in which the
ureB gene has been replaced by a kanamycin resistance (Km)
gene from Tn903. A plasmid carrying the relevant upstream
(1,546 bp) and downstream (945 bp) sequences flanking the Km gene was
constructed from PCR DNA and used to transform strain C57 with
selection for kanamycin resistance. The deletion in this strain extends
from nucleotide 35 in the ureB coding sequence to 9 nucleotides beyond the 3' end of the gene. Strain 472 is a derivative
of strain 412 containing the ureB gene located downstream of
the hpn gene.
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.7.4202-4209.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Differential Gene Expression from Two
Transcriptional Units in the cag Pathogenicity Island of
Helicobacter pylori
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
H. pylori strains used in this study
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(Bethesda
Research Laboratories) and ER1793 (New England Biolabs, Beverly,
Mass.).
Bacterial growth and HEp-2 cell tissue culture.
H.
pylori C57 was maintained on campylobacter agar (Difco, Detroit,
Mich.)-5% defibrinated sheep blood (Binax-NEL, Waterville, Maine)
supplemented with 10 µg of vancomycin (Sigma, St. Louis, Mo.) per ml,
2.5 U of polymyxin B (Sigma) per ml, 5 µg of
trimethoprim-sulfamethoxazole (Elkins Sinn, Cherry Hill, N.J.) per ml,
and, where appropriate, 15 µg of kanamycin (Sigma) per ml and 10 µg
of chloramphenicol (Sigma) per ml. H. pylori broth cultures
were grown in brucella broth (Difco and Gibco BRL, Grand Island, N.Y.)
supplemented with 5% horse serum (Gibco BRL). All H. pylori
cultures were incubated in GasPak jars (BBL, Becton Dickinson Labware,
Lincoln, N.J.) with Campypaks (BBL) to generate a microaerobic
environment. E. coli DH5 and ER1793 were grown on L
medium. The human laryngeal carcinoma cell line HEp-2 was cultured in
RPMI 1640 medium containing 5% fetal calf serum and glutamine (Gibco
BRL). Cells were maintained at 37°C in an atmosphere of 5%
CO2.
Urease assay. Urease activity was determined by the phenol-hypochlorite method (Sigma) (3, 16, 17). For this, confluent cells from plate cultures of H. pylori strains were suspended in phosphate-buffered saline (PBS), washed once, and resuspended in PBS. Equivalent numbers of cells from each strain, as determined by measurement of the optical density at 600 nm, were pelleted and resuspended in ice-cold water containing 1 mM phenylmethylsulfonyl fluoride. Samples were vortexed vigorously for 1 min, incubated on ice for 15 min, and revortexed. After centrifugation, supernatants were added to a buffer solution of urea (70 mg/ml) in 5 mM NaH2PO4 (pH 6.8) and incubated at room temperature. Samples were removed at 0, 5, 15, 60, and 120 min, added to phenol nitroprusside and alkaline hypochlorite, and incubated at room temperature for 15 min. Color development was measured with a spectrophotometer as the optical density at 625 nm. Urease activity is reported as nanomoles of urea hydrolyzed per minute per microgram of total protein, as determined by the Bradford assay (Bio-Rad).
Construction of cag-ureB fusions.
Several of the
ORFs in the cag PAI DNA (35) are preceded by
noncoding sequences of several hundred nucleotides, which we thought
likely to contain sequences required for their expression. Nine of
these putative promoter regions were analyzed using the ureB
reporter. A portion (500 to 1,000 bp) of sequence located directly
upstream of the ATG start codon of the nine selected cag
genes was PCR amplified from the clinical isolate Alston. The primers
used for the amplifications are listed as pairs using the following
notation: primer designation (locus amplified, 5' TIGR [The Institute
for Genomic Research] genome coordinate) (35)
ure1 ( ureA, 78718) and ure2 (ureB, 80249);
ure3 (ureI, 83814) and ure4 (ureI,
84848); ure5 (ureB, 77277) and ure6
(ureB, 75521); 520-5 (cag1, 546343) and
520-3 (cag1, 547345); 530-5 (cag10,
564951) and 530-3 (cag10, 563959); 531-5 (cag11, 563446) and 531-3 (cag11, 564405); 534-5 (cag13, 567653) and 534-3 (cag13, 566676); 535-5 (cag14, 568449)
and 535-3 (cag14, 567487); 536-5 (cag15,
569258) and 536-3 (cag15, 568278); 537-5 (cag16, 567734) and 537-3 (cag16, 568771); 542-5 (cag21, 575199) and 542-3 (cag21, 574242); and 546-5 (cag25,
579087) and 546-3 (cag25, 580087). All primers
were 20 to 25 nucleotides long and corresponded exactly to the
published sequence (35).
Assay of adhesion of H. pylori strains to HEp-2 cells. HEp-2 cells were seeded in 100-mm tissue culture dishes (Corning) at densities of 105 to 106 cells/dish. After a 24-h incubation period, the cells were washed twice with PBS and infected with 5 × 109 CFU of H. pylori strain C57, 412, 472, or 585 or one of the cag-ureB fusion strains. H. pylori cultures used for infections were harvested in PBS after 16 h of growth on campylobacter agar plates, pelleted, and then resuspended in RPMI 1640 medium to 109 CFU/ml. Negative controls included uninfected HEp-2 cells and samples of each cag-ureB fusion strain in the absence of a monolayer. Incubations were carried out for 8 h in a 5% CO2 atmosphere at 37°C. Following this step, supernatants of all samples were removed for microscopic examination, determination of viable plate counts, IL-8 analysis, determination of urease activity, and Western analysis.
Assay of IL-8 induction by HEp-2 cells. IL-8 analysis was performed at the laboratory of David Acheson (New England Medical Center, Boston, Mass.). The analysis was carried out with an enzyme-linked immunosorbent assay as described by Sharma et al. (27). Following the adhesion assay, 300-µl aliquots of the supernatants overlaying the epithelial cell monolayer were removed and centrifuged to remove H. pylori cells. The resulting culture fluid was assayed for IL-8 using an enzyme-linked immunosorbent assay kit (Endogen, Woburn, Mass.) in accordance with the manufacturer's instructions.
Colonization ability of cag-ureB fusions in mice. For mouse colonization, the cag-ureB constructs were transformed into M6, the mouse-adapted strain, in the following manner. Chromosomal DNA was isolated from strain 412 (Qiagen, Valencia, Calif.). The recipient strain, H. pylori M6, was grown overnight in brucella broth with 10% fetal calf serum (BBH10) under standard conditions. Cultures containing 108 to 109 CFU/ml were diluted 1:100 in 10 ml of BBH10 and incubated for up to 6 h, after which 20 to 50 ng of 412 chromosomal DNA was added; incubation was continued overnight. Each culture was then diluted 1:4 in BBH10 containing 20 µg of kanamycin per ml and grown overnight. Aliquots (1 ml) were plated on 5% sheep blood agar plates containing 20 µg of kanamycin per ml and incubated for 4 to 5 days. Several candidate colonies were chosen, and replacement of ureB with the kanamycin cassette was verified by PCR. One of the candidates, which was also shown to be urease negative, was selected and called 412M.
412M was used as the recipient for chromosomal DNA isolated from the 585 derivatives containing the cag-ureB fusions and was transformed exactly as described above. Colonies from each transformation were pooled and stored at
70°C in brucella broth
with 15% glycerol until mouse inoculation. Urease activity was assayed
in vitro as previously described (11).
Female 4- to 6-week-old Helicobacter-free C57BL/6 mice from
Jackson Laboratory were orally inoculated with 107 to
108 rapidly motile, mid-log-phase H. pylori cells grown in BBH10. Mice were sacrificed 2 or 8 weeks
after infection, and the number of CFU per gram of gastric
mucosa was determined by plate dilution.
Protein preparation. H. pylori whole cells and their supernatants incubated in the absence of an epithelial cell monolayer were transferred to 2-ml Microfuge tubes (USA Scientific, Ocala, Fla.) and precipitated overnight with trichloroacetic acid (10% final concentration). Precipitates were collected by centrifugation, washed with acetone, air dried, and resuspended in equal volumes of PBS and boiling buffer.
The supernatants of samples containing both H. pylori cells and epithelial cells were aspirated, and the monolayers were washed twice with PBS to remove any nonadherent H. pylori cells. One milliliter of lysis buffer, made with 9 ml of PBS, 0.9 ml of 0.05% Triton X-100 (Sigma), and one protease inhibitor cocktail tablet (Roche Molecular Biochemicals, Mannheim, Germany), was added to the adherent H. pylori and epithelial cells; the mixture was incubated at room temperature for 10 min. The lysed monolayers were scraped from the plates into Corex tubes and vortexed vigorously several times. One-twentieth of the sample volumes was removed to determine urease activity, and 1/50 of the sample volumes was used to measure protein concentrations. The samples were placed on ice and sonicated with three 30-s pulses using a 50% duty cycle, followed by centrifugation at 12,000 rpm (Eppendorf microcentrifuge 5415C) for 5 min at 4°C. Supernatants, containing the cytoplasmic fractions, were mixed with 4 ml of methanol 1 ml of chloroform and collected by centrifugation at 7,500 rpm (Eppendorf microcentrifuge 5415C) for 1 min at 4°C. The resulting pellets were resuspended in equal volumes of PBS and boiling buffer. All protein samples were stored at20°C.
Western analysis. Samples, each containing protein from approximately 107 CFU of H. pylori, were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to Immobilon-P membranes (Millipore, Bedford, Mass.), and blocked overnight at 4°C in PBS-5% milk. Membranes were probed with monoclonal anti-UreB antibody (1:4,000), donated by Harry Kleanthous (Oravax, Cambridge, Mass.), and a polyclonal antibody raised against H. pylori isocitrate dehydrogenase (1:400), donated by LiLi Huang (St. Elizabeth's Hospital, Boston, Mass.), to normalize for loading differences. The secondary antibodies, goat anti-rabbit immunoglobulin (IgG) and rabbit anti-mouse IgG (1:1,500), were both conjugated to horseradish peroxidase (Zymed, San Francisco, Calif.). Proteins were detected using an ECL kit (Amersham Pharmacia, Uppsala, Sweden) in accordance with the manufacturer's recommendations.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Use of urease as a reporter of gene expression.
A derivative
of H. pylori C57 with a deletion of the ureB
gene, which encodes one of the two subunits of urease, was constructed to determine if urease activity could be used as a reporter of promoter
activity in H. pylori. This strain, 412, was devoid of urease activity (Table 2). Transformation
of strain 412 with plasmid pHPNureB, carrying a promoterless
ureB gene flanked by sequences homologous to the
hpn locus, yielded recombinants with the ureB
gene integrated immediately downstream of the hpn coding sequence on the chromosome. Since hpn is highly expressed in
H. pylori (J. V. Gilbert, unpublished observation), we
expected that transcription from the hpn promoter would
drive the expression of ureB in this strain, complementing
the urease defect. The recombinants produced high levels of urease
(Table 2). Primer extension analysis of RNA isolated from one of the
recombinants, strain 472, indicated that ureB was expressed
from the hpn promoter (results not shown). These data
indicate that the presence of ureB at the hpn
locus efficiently complements the urease defect in strain 412. Plasmid pHPN was specifically designed to insert desired sequences at the
neutral hpn locus. This plasmid is unable to replicate
in H. pylori and, as a result of double-crossover events,
yields recombinants containing the desired gene (ureB in
this case) and lacking plasmid sequences. Thus, it is a convenient tool
for complementation analysis and evaluation of the expression of genes
present in single copies on the chromosome. It is particularly useful
for H. pylori, which has few, if any, stable plasmid
vectors.
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Analysis of cag promoters using cag-ureB fusions. The cag genes present on the cag PAI of type I H. pylori strains play a key role in interactions between the bacterium and its host. We wished to test the possibility that some of these genes might be induced when cocultured with eukaryotic cells. Since little information was available on the location of promoter sequences within the cag PAI region, we used the ureB reporter system to identify cag sequences with promoter activity. DNA containing 500 to 1,000 bp of sequence upstream from a number of the cag genes was tested for promoter activity. Nine cag PAI DNA sequences containing putative promoters were cloned into p342 in the correct orientation with respect to ureB, and the resulting plasmids were used for transformation of strain 412. The transformants were merodiploid for the cloned cag sequences and had an intact cag PAI. While each fusion strain gave a reproducible level of urease activity when grown on laboratory medium, the amount of urease activity among the fusions varied widely, ranging from 0.16 to 13.1 nmol of urea hydrolyzed/min/µg of total protein. Of the nine regions examined by this approach, four showed promoter activity above that of strain 585, as measured by the hydrolysis of urea (Table 2). The cag1-ureB fusion produced approximately 50% of the urease activity of parent strain C57, while the cag10-, cag21-, and cag25-ureB fusions produced between 10 and 15% of the parental urease activity. These results indicate that these cag DNA fragments contain promoters that give rise to substantial promoter activity. The urease activities in strains carrying the cag15- and cag16-ureB fusions were similar to that of strain 585. However, the urease activities in strains carrying the cag13- and cag14-ureB fusions and particularly the strain containing the cag11-ureB fusion, were markedly below that of strain 585.
Coculturing with an epithelial cell monolayer influences
cag-ureB expression.
Figure
2 shows the results of a Western blot
analysis performed on protein extracts isolated from
cag-ureB fusion strains cocultured with epithelial cells as
well as the same fusion strains cultured in medium alone. The
expression of all but two of the cag-ureB fusions was
unaltered under these conditions. The two exceptions, strain 585 containing the cag15-ureB fusion
[585(cag15-ureB)] and strain
585(cag21-ureB), showed an approximately 14-fold
increase in UreB protein levels upon coculturing with epithelial cells relative to the level strains cultured in RPMI 1640 medium alone (Fig.
2). To confirm that the fusion strains interacted with the epithelial
cells, we measured IL-8 levels produced by the epithelial cells. High
levels of IL-8 were produced in all cases, indicating that the
cag PAI was intact and that some portion of the H. pylori population interacted with the epithelial cell monolayer
(data not shown). The levels of IL-8 produced from uninfected
monolayers were not above background, and no IL-8 was detected in
supernatants collected from the cag-ureB fusion strains
alone (data not shown). This differential expression of ureB
suggests that the promoter activities of the cag15- and
cag21-ureB fusions are responsive to environmental cues
experienced during coculturing with an epithelial cell monolayer.
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Colonization ability of cag-ureB fusions in mice.
To determine if the increase in ureB expression from the
cag15-ureB and cag21-ureB fusion strains had in
vivo significance, mouse-adapted strains (from strain M6; designated
with the suffix "M") containing these fusions were constructed and
tested for their ability to colonize mice. The results (Table
3) indicated that while fusion strains
585M(cag1-ureB) and 585M(cag15-ureB) were able to
colonize mice, they achieved a cell density that was 10-fold lower than
that of the parent strain, M6, under the conditions used. These two
strains differ widely in their levels of in vitro urease activity,
expressing 50 and 5% of the activity seen in M6, respectively. Strain
585M(cag21-ureB), which produces 11% of the parental level
of urease, twofold more than the in vitro level of expression of strain
585M(cag15-ureB), failed to colonize mice. Although
it is not known how much urease activity is required for colonization,
these results suggest that the in vitro expression of
cag15-driven ureB is not equivalent to its in
vivo expression.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The ureB expression vector developed in this work
offers several advantages for the analysis of gene expression in
H. pylori. Fusions of genes to ureB,
constructed using this vector, are readily inserted in single copies at
a neutral site on the H. pylori chromosome, thus avoiding
polar effects on downstream genes. The normal copy of the gene being
examined remains intact. The production of urease in the
ureB reporter strain is absolutely dependent on the
expression of ureB from a promoter(s) located within
the sequence fused upstream of ureB; thus, the levels of
urease activity provide a direct measure of the strength of a
promoter fused to the ureB reporter. Perhaps the most unique
feature of the urease expression system is that it can be used as a
selectable trait, both in vitro and in vivo. Urease is the most
abundant protein synthesized by H. pylori, and it has one of
the lowest reported Km values (0.17 mM) of any
urease (23), enabling it to function efficiently under
conditions of low substrate concentrations. Several well-established assays facilitate quick and easy quantitative measurements of enzyme
activity (3, 17).
Little is known about the process of transcriptional regulation in
H. pylori, particularly in relation to pathogenesis
(5, 15, 25, 28, 29, 30, 31, 33). Since most H. pylori isolates from patients with gastric ulcers and cancers are
classified as cagA-expressing type I strains, we used the
urease reporter system to analyze gene expression in the cag
PAI, 40-kb region of DNA which contains approximately 27 ORFs and which
plays a key role in interactions between H. pylori and the
gastric epithelium. Based on the known ORF sequence and organization
within the cag PAI (Fig. 3),
we selected nine noncoding intergenic DNA sequences that we considered
likely to contain cag gene promoters for analysis using the urease reporter. One of the resulting
cag-ureB fusion strains (cag1-ureB) gave
high-level expression, while others (cag15-, cag16-,
cag21-, and cag25-ureB) gave
intermediate levels of expression. However, three strains
(cag11-, cag13-, and cag14-ureB) gave levels of
activity that were below that of strain 585. While these results indicate that the cloned fragments either lack a promoter or have a
promoter that is not expressed under the growth conditions used, they
may also imply that these sequences encode transcriptional terminators,
which would suppress the background urease expression seen in strain
585.
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In an effort to identify DNA features that might be important for
transcribing cag DNA, we compared the nine
cag sequences with H. pylori promoters that
had been previously mapped by primer extension or RNase I protection
(Fig. 4). The cag sequences
contain putative 10 and 35 regions, as well as other motifs that
have been associated with promoter activity in H. pylori.
Although five of the nine sequences that we examined have obvious
homologies to both 10 and 35 sequences, the number of nucleotides
between these putative sites varies; this variation may account for the observed differences in ureB expression. Shirai et al.
(28) have reported that maximal levels of gene expression
in H. pylori occur when spacing between the 10 and 35
sites is 17 or 18 nucleotides. In fact, the cag1 sequence,
which gave the highest levels of urease activity when fused to
ureB, contains highly conserved 10 and 35 sequences
separated by 18 nucleotides (Fig. 4). All of the potential
cag regulatory sequences identified here are 100% identical to the corresponding sequences present in the cag PAI of
H. pylori strain J99. This finding suggests that phenotypic
differences that these strains may show with regard to the
cag PAI cannot obviously be attributed to differences in
promoter structure.
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Expression from all but two of the transcriptionally active
cag regions was unaffected by interaction with epithelial
cell monolayers. We conclude that most of the cag genes that
we examined are expressed at some constitutive level under the
conditions tested. The two exceptions were the fusions present in
strains 585(cag15-ureB) and 585(cag21-ureB).
Interestingly, although the putative cag15-ureB promoter
region contains conserved 10 and 35 sequences separated by 18 nucleotides, it showed low promoter activity when
585(cag15-ureB) was grown under laboratory conditions. However, coculturing of this strain with epithelial cells resulted in a
14-fold increase in ureB expression from this putative
promoter (Fig. 2). A similar observation was made for fusion
strain 585(cag21-ureB), distinguishing the H. pylori
cag PAI as an example of a type IV secretion system in which gene
expression appears to be induced upon interaction with eukaryotic cells.
To determine if these observations had in vivo significance, we transferred the cag15- and cag21-ureB fusions into the mouse-adapted strain M6 and tested the ability of the M6 derivatives to colonize mice. Because of the low level of urease that these fusion strains produced during growth in bacteriological medium (Table 2) and because urease is essential for colonization (11, 12, 13, 19, 21, 23, 36), we expected that these strains would fail to colonize. Surprisingly, fusion strain 585M(cag15-ureB) colonized as well as 585M(cag1-ureB), another fusion strain, which produced much higher levels of urease. On the other hand, fusion strain 585M(cag21-ureB), which showed a similar increase in expression upon interaction with epithelial cells, failed to colonize mice (Table 3). The discrepancy among these observations underscores the significant differences between in vitro and in vivo situations, raising the intriguing possibility that the putative cag15 promoter may be induced in vivo.
cag15 does not appear to be part of an operon, based on its location within the cag gene cluster (Fig. 3). While there is a segment of DNA upstream of cag21 with promoter activity, the intergenic spacing between cag21 and the upstream coding sequence of cag22 is small, and the possibility exists that cag21 is also influenced by putative promoters located further upstream. The predicted protein product of cag15 (8) has low-level homology to a fimbrial assembly protein, FimB, from Dichelobacter nodosus and a type IV prepilin peptidase, encoded by pilD, from Pseudomonas aeruginosa. The homology that Cag15 shares with PilD and FimB includes putative transmembrane regions. However, the critical residues in PilD that have been shown to be required for peptidase activity are not conserved in Cag15, suggesting that Cag15 probably does not act as a peptidase. Cag21 shares low-level homology with a flagellar motor switch protein, FliM, from Caulobacter crescentus and the toxin-coregulated pilus biosynthesis protein D from Vibrio cholerae.
Mutations in cag15 have not been reported. Experiments to delete cag15 in order to determine its effects on the epithelial cell induction of IL-8 or CagA secretion are currently under way. A null mutation has been made in cag21, and the resulting strain is unable to elicit IL-8 secretion or translocate CagA into a host cell. Although the roles of the Cag15 and Cag21 proteins in Cag function are presently unknown, their homologies with proteins involved in pilin-like biogenesis are intriguing and raise the possibility that there is an as-yet-unidentified pilin structure in type I H. pylori strains that contributes to the pathogenesis of the organism.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge the technical support provided by Anne Kane and her staff at the GRASP Digestive Disease Center at New England Medical Center, Boston, Mass., which is supported by a grant from the National Institutes of Health (NIDDK grant P30DK34928). Joanne V. Gilbert is supported by NIH grant DK-3702. Support for K. A. Eaton is provided in part by Public Health Service grants R01 AI43643 and R29 DK-45340 from the NIH.
We also thank Dorothy Fallows, D. Scott Merrell, Matthew Waldor, and Anne Kane for critical review of the manuscript and thoughtful scientific discussions.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Molecular Biology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-6758. Fax: (617) 636-3307. E-mail: andrew.wright{at}tufts.edu.
Present address: Department of Microbiology and Immunology,
Stanford University School of Medicine, Stanford, CA 94305.
Editor: B. B. Finlay
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, July 2001, p. 4202-4209, Vol. 69, No. 7
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.7.4202-4209.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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