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Previous Article | Next Article 
Infection and Immunity, May 2001, p. 3502-3506, Vol. 69, No. 5
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.5.3502-3506.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Characterization of Proteins in the Outer Membrane
Preparation of a Murine Pathogen, Helicobacter
bilis
Zhongming
Ge,1
Peter
Doig,2 and
James G.
Fox1,*
Division of Comparative Medicine,
Massachusetts Institute of Technology,
Cambridge,1 and AstraZeneca R&D Boston,
Waltham,2 Massachusetts
Received 29 December 2000/Accepted 1 February 2001
 |
ABSTRACT |
Helicobacter bilis is a bacterial pathogen associated
with multifocal hepatitis and inflammatory bowel disease in certain strains of mice. This bacterium colonizes the liver, bile, and lower
intestine in mice and has also been isolated from a wide spectrum of
laboratory animals. In this study, proteins present in the outer
membrane preparation (OMP) of four H. bilis strains isolated from a mouse, a dog, a rat, and a gerbil were characterized and compared with that of Helicobacter pylori, a human
gastric pathogen. All four H. bilis strains had similar OMP
protein profiles that were distinct from those of H. pylori. Immunoblotting demonstrated that OMP proteins from
H. bilis and H. pylori have little
cross-reactivity, except for their flagellins. Nine major immunogenic
polypeptides were present in the H. bilis OMPs. By using
two-dimensional sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, five heat-modifiable proteins with molecular masses of
82, 66, 52, 47 and 37 kDa were identified. The N-terminal sequences of
the 46- and 47-kDa OMP proteins had no homology with protein sequences
available in public databases. These results indicate that H. bilis has a conserved, unique OMP protein profile that is
distinct from those of H. pylori.
| |
TEXT |
Helicobacter bilis is a
gram-negative, microaerophilic, spiral bacterium and a member of the
rapidly expanding genus Helicobacter (27). This
microorganism was originally identified from the bile, livers, and
intestines of aged inbred mice and was able to survive in 20% bile in
vitro (10). H. bilis has also been isolated
from dogs, gerbils, rats, and cats (6, 8, 12; J. G. Fox, F. E. Dewhirst, and B. J. Paster, unpublished data). Natural infection by H. bilis has been associated with an
outbreak of colitis in scid mice (23). In
addition, experimental infection by H. bilis in
scid mice and immunodeficient rats induces inflammatory bowel diseases (IBD) and hepatitis (11, 12, 24). Present evidence indicates that H. bilis has pathogenic potential in
a variety of animal species, and it has provided a murine model of
human IBD. Recently, H. bilis DNA was amplified from the
bile and gallbladder tissues of Chileans with chronic cholecystitis (9), suggesting that this bacterium may play a role in the development of hepatobiliary disease in humans.
Gram-negative bacteria possess an outer membrane (OM) that acts as a
selective barrier, controlling the transportation of molecules, such as
antibiotics and nutrients, and ions from the environment into the cell
(13, 19, 21). In this case, a protein class (termed
porins) forms channels and is responsible for facilitating the
selective diffusion of hydrophilic molecules across the OM (3,
13). In addition, the OM has an important role in interacting
with the host, such as in adhesion and/or invasion of host tissues.
Furthermore, many OM proteins from pathogenic bacteria, including
porins, are also involved in the modulation of host immune responses,
either through inflammation, suppression, or avoidance; these proteins
can be important in persistence and/or disease induced by these bacteria.
Given that H. bilis has become an important pathogen in mice
and can be utilized in murine models for studying human IBD, it is
important to characterize the bacterial components contributing to its
colonization, persistence, and pathologic potential. In this study, we
identified the profiles, heat modifiability, and immunogenic components
of the outer membrane preparation (OMP) from H. bilis
strains isolated from four different animal species. In addition, the
profiles and the antigenic cross-reactivities of the OMP proteins of
H. bilis and Helicobacter pylori were compared.
Conservation of OMP protein profiles among H. bilis
strains.
H. bilis strains, including ATCC 51630, from a
mouse (10); MIT95-477, from a dog; MIT96-5983, from a
gerbil; and MIT96-151N3, from a rat, were used in this study. All of
these H. bilis strains were isolated at the Division of
Comparative Medicine, Massachusetts Institute of Technology. In
addition, H. pylori strains J99 (1), AH244, and
Sydney SS1 (18) were used for the preparation of H. pylori OMP. H. bilis and H. pylori were
grown on sheep blood agar (Remel, Lenexa, Kans.) for 3 to 4 days under
microaerobic conditions (10% CO2, 10% H2,
80% N2). The H. pylori and H. bilis cells were collected and washed three times with 20 ml of 20 mM Tris-HCl (pH. 8.0). The OM proteins were prepared as described previously (5). Briefly, the cells were sonicated on ice
(eight times for 30 s each time at intervals of 1 min). Unbroken
cells and debris were removed by centrifugation at 4,000 × g for 10 min at 4°C. The supernatants were then centrifuged at
45,000 × g for 20 min at 4°C, and the pellets were
washed three times with 20 mM Tris-HCl (pH. 8.0). The pellets were
suspended in 0.6% N-lauroyl sarcosine-20 mM Tris-HCl (pH.
8.0) to a final concentration of 1 mg of protein/ml and incubated at
room temperature for 20 min. The OMPs were collected by centrifugation
at 45,000 × g for 20 min at 4°C. The resulting
pellets were dissolved in 20 mM Tris-HCl (pH. 8.0), and the protein
concentration was determined by a bicinchoninic acid protein assay
(Pierce, Rockford, Ill.) employing bovine serum albumin as a standard.
For comparing the OMP profiles among these helicobacters, 10 µg of
OMPs were mixed with 2× sample buffer (100 mM Tris-HCl, pH 6.8, 200 mM
dithiothreitol, 4% sodium dodecyl sulfate [SDS] 0.2% bromophenol
blue, 20% glycerol) and heated at 100°C for 5 min. The samples were
centrifuged at 10,000 × g for 10 min. The OMPs of the
H. bilis and H. pylori strains were electrophoretically separated on SDS-10 or 12% polyacrylamide gels
and visualized with Coomassie blue.
In the OMP samples from four H. bilis strains (ATCC 51630, MIT96-5983, MIT95-477, and MIT96-151N3), approximately 10 polypeptides with apparent molecular masses ranging from 16 to 80 kDa were visualized by Coomassie blue staining on an SDS-12% polyacrylamide gel (Fig. 1, lanes 1 to 4). A conserved
pattern of polypeptides was revealed for the four H. bilis
strains, with strain-specific differences evident in minor
polypeptides. Five of the OM polypeptides, with apparent molecular
masses of 16, 18, 22, 36, and 52 kDa, were major components in all
strains. An additional two polypeptides with apparent molecular masses
of 35 and 44 kDa were also present within the OMPs of all four H. bilis strains, but their expression levels differed significantly
between strains. The relative proportion of the OMP proteins made up of
a 45-kDa polypeptide in strains MIT95-477 and MIT9615-1N3 (Fig. 1,
lanes 2 and 4) was much greater than in strains ATCC 51630 and
MIT96-5983 (lanes 1 and 3), whereas the content of an ~35-kDa
polypeptide in ATCC 51630 and MIT95-477 (lanes 1 and 2) was higher than
in MIT96-5983 and MIT96-151N3 (lanes 3 and 4).
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FIG. 1.
OMP profiles of H. bilis and H. pylori strains on an SDS-10% polyacrylamide gel. H. bilis strains: lane 1, ATCC 51630; lane 2, MIT95-477; lane 3, MIT96-5983; lane 4, MIT96-151N3. H. pylori strains: lane 5, J99; lane 6, AH244; lane 7, SydneySS1. Molecular mass markers are on
the left.
|
|
The OMP protein profiles of these H. bilis strains were
compared to those of three H. pylori strains: J99
(1), AH244, and SydneySS1 (18). Both H. pylori J99 and SydneySS1 are positive for cagA and
cagE (also called picB), parts of the H. pylori pathogenicity island (1, 18), whereas AH244
does not contain these two genes (Z. Ge and J. G. Fox, unpublished
data). However, the OMP protein profiles in H. pylori
strains J99 and AH244 (Fig. 1, lanes 5 and 6) were similar but distinct
from that of strain SydneySS1 (lane 7). There was little similarity
between the OMP protein profiles of selected H. bilis
strains and those of the three H. pylori strains. H. pylori strains AH244 and J99 had more major OMP species (~10)
than H. bilis (~5). Interestingly, the majority of the
H. pylori OMP proteins had masses ranging from 50 to 100 kDa, whereas four of the five major H. bilis OMP proteins
had apparent molecular masses of 17 to 36 kDa.
Identification of HMPs in OMP.
The OMP of the H. bilis type strain, ATCC 51630, was used to identify
heat-modifiable proteins (HMPs). Twenty-five micrograms of the OMP were
mixed with an equal volume of 2× sample buffer, incubated at room
temperature for 20 min, and then separated by SDS-polyacrylamide gel
electrophoresis (PAGE) using a 10% acrylamide gel. The lane containing
the separated OMP proteins was excised, placed in 2× sample buffer,
and heated at 100°C for 10 min. The gel strip was then overlaid on an
SDS-10% polyacrylamide gel, and the polypeptides were separated
electrophoretically. The polypeptides were visualized with Coomassie blue.
When the heat-denatured and the unboiled OM polypeptides were compared
on an SDS-12.5% polyacrylamide gel, three polypeptides in the
heat-denatured sample, but not in the unboiled sample, could be
identified (corresponding to apparent molecular masses of 35, 44, and
82 kDa) (Fig. 2A). Subsequently, OMP
proteins with heat-modifiable electrophoretic mobility were detected by
two-dimensional SDS-PAGE. Five polypeptides, which were shown to
display slower electrophoretic mobility in the second dimension (Fig.
2B, above the diagonal banding patterns), were identified as HMPs.
These proteins were designated OmpA (~82 kDa), OmpB (~66 kDa), OmpC (~52 kDa), OmpD (~47 kDa), and OmpE (~37 kDa) (Fig. 2B). Several polypeptides migrated with higher apparent molecular masses in the
second-dimensional gel (below the diagonal banding patterns) and likely
represent monomers of heat-dissociated high-molecular-weight complexes.
Two such polypeptides with apparent molecular masses of 35 and 44 kDa
corresponded to the two bands present only in the heated samples in
one-dimensional SDS-PAGE (Fig. 2B).
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FIG. 2.
OMPs of H. bilis strain ATCC 51630 in first-
(A) and second- (B) dimensional SDS-polyacrylamide gels (10%). Five
OMPs with heat modifiability are indicated by arrows (A to E). The
numbers 1 and 2 denote the heat-dissociated polypeptides from
high-molecular-weight complexes. RT and H stand for room temperature
and heated, respectively. Molecular mass markers are on the left.
|
|
For peptide sequencing of two OMP proteins, 20 µg of the OMP of
H. bilis ATCC 51630 were separated on SDS-10% PAGE and
transferred onto a polyvinylidene difluoride membrane (Bio-Rad
Laboratories, Hercules, Calif.). The polypeptides were visualized with
Ponceau S solution, and the polypeptide bands of interest were excised with a razor. N-terminal amino acid sequencing was performed by Edman
degradation using an Applied Biosystems amino acid sequencer. Two of
these OMP polypeptides from the unheated sample, which had approximate
molecular masses of 46 and 47 kDa, were sequenced. The N-terminal
sequences, AKLSDVINNASIDNF for the 46-kDa protein and
KNGADIMEWALQDN for the 47-kDa protein, were not similar to any amino acid sequences available in the public databases Swissprot and Trembl.
Antigenicity of the OMP proteins of H. bilis.
We
investigated the in vivo immunogenicities of the H. bilis
OMP proteins as well as the immunogenic cross-reactivity between the
OMP proteins of H. bilis and those of H. pylori.
Anti-H. bilis and anti-H. pylori polyclonal
antibodies were obtained from mice experimentally infected with
H. bilis strain ATCC 51630 for 24 weeks and from rabbits
immunized by cell lysate of strain J99, respectively. For
immunoblotting, 2 µg of the respective OMPs was separated on an
SDS-12% PAGE minigel apparatus (Bio-Rad, Hercules, Calif.) and then
transferred to a nitrocellulose membrane (MSI Westborough, Mass.) in a
transblotting buffer (30 mM glycine, 48 mM Tris, 0.0375% [wt/vol]
SDS, 20% methanol). Immunoblotting with either anti-H.
bilis serum (1 in 2,000 dilution) or anti-H. pylori
serum (1 in 10,000 dilution) was performed with the ECL system
(Amersham Life Science Inc., Arlington Heights, Ill.) according to the
manufacturer's instructions. Reactive polypeptides were visualized on
X-ray film (Kodak, Rochester, N.Y.).
Nine major polypeptide bands with apparent molecular masses of 20, 21, 24, 27, 30, 36, 38, 52, and 80 kDa reacted to the antisera (Fig.
3A, lanes 1 to 4). These immunogenic OMP
proteins were highly conserved among the four H. bilis
strains. In contrast, whole-cell lysate of the H. bilis type
strain, ATCC 51630, contains numerous immunogenic polypeptides (lane
9).
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FIG. 3.
Immunoblotting of OMPs from selected H. bilis
(Hb) and H. pylori (Hp) strains with
the anti-H. bilis (A) and anti-J99 (B) sera. H. bilis strains: lane 1, ATCC 51630; lane 2, MIT95-477; lane 3, MIT96-5983; lane 4, MIT96-151N3. H. pylori strains: lane 5, J99; lane 6, AH244; lane 7, SydneySS1. Cell lysates: lane 8, H. pylori J99; lane 9, H. bilis ATCC 51630. FlaA from
H. bilis and H. pylori is denoted by arrows.
Molecular mass markers are on the left.
|
|
Except for a 52-kDa polypeptide of H. bilis and a 54-kDa
polypeptide of H. pylori, there was little intraspecies
antigenic cross-reactivity among the OMP proteins (Fig. 3, lanes 1 to
7) or cell lysates (lanes 8 and 9) when either the anti-H.
bilis (Fig. 3A) or the anti-H. pylori (Fig. 3B)
whole-cell sera were used. This 52-kDa polypeptide in H. bilis OMPs reacted specifically with the monoclonal antibodies to
H. pylori FlaA (16). The N-terminal amino acid
sequence of the first 15 residues (AFQVNTNINALNAHV) was
identical to that of Helicobacter felis FlaA
(15) and different by one conservative substitution of Met
for a Leu at position 11 in H. pylori, further confirming
the identity of this protein as an FlaA homolog.
In addition, both monoclonal and polyclonal antibodies to various OM
proteins found in H. pylori were screened. These included monoclonal antibodies specific for HopA, HopC, HopE, JHP438, and JHP117
(5), as well as polyclonal antisera specific for HopB. None of these reacted with either OMs or whole-cell lysates of H. bilis (data not shown).
Due to its likely significance in disease, the composition of the OM of
H. pylori, the type species of the genus
Helicobacter, has received considerable attention with
respect to polypeptide composition and function. The genome of this
pathogen encodes a unique protein family comprising 32 OM proteins
(1, 14, 25). The members of this protein family, including
BabA (15), AlpAB (20), and HopZ
(22), have been reported to be necessary for the adhesion
of H. pylori to human gastric tissues. Another three members
(HopA, HopD, and HopE), as well as HopB and HopC, have been shown to be
heat-modified porin proteins (4, 7). It has been proposed
that these porins may play a role in H. pylori antibiotic
susceptibility and resistance (7, 14). In addition, a
30-kDa OM protein purified from H. pylori was reported to
induce the release of cytokines (interleukin 3 [IL-3], IL-4, IL-6,
IL-8, interferon gamma, and tumor necrosis factor alpha) from human lymphocytes and monocytes in vitro. This suggested that the surface components of H. pylori, particularly porins, could play an
important role in causing the inflammatory and immune responses in the
host, possibly leading to gastritis (26).
In this study, the OMP proteins conserved among selected H. bilis isolates showed little similarity to those of three H. pylori strains. The differences in the OM compositions of the two
Helicobacter species were further demonstrated by the lack
of cross-reactivity with the heterologous species of sera generated to
either species as well as the lack of cross antigenicity between the
H. bilis OMP proteins and several H. pylori OM
proteins (HopA, HopC, HopE, JHP438, and JHP117). A notable exception
was the flagellin present in the OMPs of H. bilis. The
difference between the OMP protein profiles of the two species as well
as the apparent similarities among these selected H. bilis
strains may reflect the unique adaptations of this species to its
particular gastrointestinal niche. The differences may also indicate
that the two microorganisms have different pathogenic mechanisms.
In order to examine the immunorecognition of OM components, sera were
obtained from mice infected by H. bilis. Nine OMP proteins with apparent molecular masses ranging from 20 to 80 kDa were found to
have strong immunogenicity in vivo. Interestingly, these polypeptides
were highly conserved among the four H. bilis strains isolated from mice, gerbils, rats, and dogs, indicating that they could
play an essential role in H. bilis colonization, survival, and pathogenesis. It should be noted that except for the 52-kDa reactive polypeptide, FlaA, the identities and biological functions of
the immunogenic polypeptides are unknown. Also, it is unclear at
present how these OMP proteins induce the production of the corresponding antibodies in their hosts; possible mechanisms include their surface-exposed epitopes, the secreted antigens, or the release
of OMP proteins from lysed cells.
Putative porins in the OM of H. bilis were investigated. The
sizes of the porins determined by SDS-PAGE are characteristically heat
modifiable, migrating more slowly after treatment at 100°C in SDS.
This heat modifiability of the porins is due to the presence of
extensive 
-sheets in their secondary structures (2, 7). Based on this feature, five porins from H. pylori,
designated HopA (48 kDa), HopB (49 kDa), HopC (50 kDa), HopD (67 kDa),
and HopE (31 kDa), were identified and characterized (4,
7) as members of a larger, functionally important family of
proteins. In this study, five HMPs with the apparent molecular masses
of 82 (OmpA), 66 (OmpB), 52 (OmpC), 47 (OmpD), and 37 (OmpE) kDa were
identified in the OMP of H. bilis. H. bilis OmpB, -C, -D, and -E possessed apparent molecular masses in the range of the H. pylori porins as well as typical bacterial porin sizes (25 to 50 kDa) (4, 7); however, the H. bilis proteins had
no cross-reactivity with HopA to -C and HopE, suggesting they are unique. Further investigation of the pore-forming abilities of these
HMPs is required in order to elucidate whether they act as porins in
vivo. Such studies may lead to the identification of factors that have
immunomodulatory activity and thereby play a role in inflammatory
processes. Their characterization may also reveal candidates for the
development of a vaccine against H. bilis infection.
| |
ACKNOWLEDGMENTS |
These studies were supported by NIH R01 CA 67529 and NIH R01 DK
52413 and a grant from AstraZeneca.
We thank Gejing Deng for amino acid sequence analysis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Comparative Medicine, Massachusetts Institute of Technology, 16-825, 77 Massachusetts Ave., Cambridge, MA 02139. Phone: (617) 253-1735. Fax:
(617) 258-5708. E-mail: jgfox{at}mit.edu.
Editor:
: D. L. Burns
| |
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Infection and Immunity, May 2001, p. 3502-3506, Vol. 69, No. 5
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.5.3502-3506.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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