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Infection and Immunity, November 2000, p. 6250-6256, Vol. 68, No. 11
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Antigenic Structure of Outer Membrane Protein E of
Moraxella catarrhalis and Construction and Characterization
of Mutants
Timothy F.
Murphy,1,2,3,*
Aimee L.
Brauer,1
Norine
Yuskiw,1 and
Thomas J.
Hiltke1
Division of Infectious Diseases of the
Department of Medicine1 and the
Department of Microbiology,2 State University of
New York at Buffalo, and the Veterans Affairs Western New York
Healthcare System,3 Buffalo, New York 14215
Received 9 June 2000/Returned for modification 25 July
2000/Accepted 9 August 2000
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ABSTRACT |
Outer membrane protein E (OMP E) is a 50-kDa protein of
Moraxella catarrhalis which possesses several
characteristics indicating that the protein will be an effective
vaccine antigen. To study the antigenic structure of OMP E, eight
monoclonal antibodies were developed and characterized. Three of the
antibodies recognized epitopes which are present on the bacterial
surface. Fusion peptides corresponding to overlapping regions of OMP E
were constructed, and immunoblot assays were performed to localize the
areas of the molecule bound by the monoclonal antibodies. These studies identified a surface-exposed epitope in the region of amino acids 80 through 180. To further study the protein, two mutants which lack OMP E
were constructed. In bactericidal assays, the mutants were more readily
killed by normal human serum compared to the isogenic parent strains.
These results indicate that OMP E is involved in the expression of
serum resistance of M. catarrhalis.
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INTRODUCTION |
Moraxella catarrhalis is
an important human respiratory tract pathogen (6, 8, 9, 21,
22). It is the third most common cause of otitis media,
accounting for 15 to 20% of all episodes based on cultures of middle
ear fluid obtained by tympanocentesis (13, 29). M. catarrhalis also causes lower respiratory tract infections, often
called exacerbations, in adults with chronic obstructive pulmonary
disease (COPD) (23, 25). It is difficult to state precisely
the etiology of exacerbations in individual patients; however, one
study estimated that approximately 30% are caused by M. catarrhalis (33). Nosocomial outbreaks of respiratory tract infections caused by M. catarrhalis have been
recognized since the mid-1980s (18, 20, 26-28). Many of
these outbreaks of infections have occurred in respiratory units where
the presence of a susceptible population with underlying lung disease
contributed to the clusters.
In view of the importance of M. catarrhalis as a human
pathogen, there is interest in developing a vaccine to prevent these infections. Two populations would benefit most from such a vaccine. Infants would be immunized in an effort to prevent otitis media, with
particular emphasis on preventing recurrent otitis media in
otitis-prone children. The second population that would benefit from
such a vaccine is adults with COPD.
Outer membrane protein E (OMP E) is a 50-kDa heat-modifiable outer
membrane protein (OMP) which has characteristics that indicate that it
may be an effective vaccine antigen (2, 3). The protein is
abundantly expressed on the bacterial surface as demonstrated by
immunofluorescence assays and flow cytometry with monoclonal antibodies
(MAbs) (3). OMP E is highly conserved among strains of
M. catarrhalis (2, 3). These two features of OMP
E suggest that inducing an immune response to the protein may result in protection from infection.
The present study was undertaken to further characterize the antigenic
structure of OMP E. MAbs were developed and characterized. The regions
of the OMP E molecule bound by the MAbs were identified, and two
mutants which are defective in expression of OMP E were constructed and characterized.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
M.
catarrhalis strain 25240 is from the American Type Culture
Collection. Strain 7169 was recovered by tympanocentesis from the
middle ear fluid of a child with otitis media and was kindly provided
by Howard Faden (Children's Hospital, Buffalo, N.Y.). M. catarrhalis strains were grown on brain heart infusion (BHI) plates or in BHI broth, unless otherwise noted.
SDS-PAGE and immunoblot assay.
Whole-bacterial-cell lysates,
purified outer membrane preparations, purified recombinant OMP E, and
fusion peptides were subjected to sodium dodecyl sulfate-12%
polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot assay as
previously described (3) but with the following
modifications. Immunoblots were blocked with 3% nonfat dry milk for
1 h at room temperature. After 3 washes in buffer A (0.01 M
Tris-0.15 M NaCl [pH 7.4]), immunoblots were incubated with tissue
culture supernatants of MAbs overnight at room temperature. After three
washes, blots were incubated with goat anti-mouse immunoglobulin G
(IgG)-immunoglobulin M conjugated to peroxidase (Boehringer Mannheim,
Indianapolis, Ind.) for 1 h at room temperature. The blots were
then washed, and bands were visualized with
H2O2 and HRP color developer (Bio-Rad
Laboratories, Richmond, Calif.).
Purification of recombinant OMP E.
Recombinant OMP E was
expressed in plasmid pESA, which was derived from pRSET (Invitrogen,
San Diego, Calif.) and expresses the full-length mature OMP E protein
with six histidines on the amino terminus (3). The protein
was purified by elution from Talon resin (Clontech, Palo Alto, Calif.)
as described previously (3).
Cloning of ompe fragments into pGEX4T.
Peptides
corresponding to selected regions of the gene which encodes OMP E were
expressed as fusion proteins by using the pGEX4T plasmid expression
system (24, 31). Oligonucleotides were designed with
BamHI and EcoRI sites so that DNA fragments amplified by PCR were cloned directionally into pGEX4T which had been
cut with BamHI and EcoRI. PCR products
corresponding to specific regions of ompe were ligated into
pGEX4T with T4 DNA ligase (Ligation Express; Clontech). Plasmids were
electroporated into Escherichia coli HB101 and plated.
Clones selected for further study were confirmed by sequencing the
entire insert.
Purification of GST fusion proteins.
Fusion proteins with
glutathione-S-transferase (GST) were purified by elution
from glutathione-Sepharose essentially as previously described
(10).
Development of MAbs.
The eight MAbs were developed from two
different fusions. MAbs 1B3 and 9G10d were described previously
(3). The remaining six MAbs were developed in BALB/c mice
using the following schedule of immunization: days 0 and 14, 50 µg of
purified recombinant OMP E emulsified with incomplete Freund's
adjuvant administered subcutaneously; day 28, ~108 CFU of
M. catarrhalis 25240 suspended in phosphate-buffered saline administered intraperitoneally. On day 31, the mice were euthanized, and the spleens were removed and perfused to collect splenocytes.
To perform the fusion, splenocytes were fused with SP2/0-Ag-14
plasmacytoma cells by a modification of the procedure of Kennett (12) and our own previously described methods
(30). After clones producing antibodies were identified, the
hybridomas were cloned by limiting dilution, and the isotypes were
determined by using the mouse MonoAB ID kit (Zymed, San Francisco,
Calif.). MAbs were purified by elution from protein G.
Flow cytometry.
Flow cytometry was performed by using a
Becton Dickinson FACScan to determine whether antibodies recognized
epitopes which were present on the intact bacterial cell as previously
described (3).
Construction of mutants.
A PstI site was
engineered into ompe in pESA with one-step overlap extension
PCR as follows (15, 32). Primers were designed to amplify
the 5' 423 bp of ompe with a BamHI site at the 5'
end and a PstI site at the 3' end (Table
1). A second set of primers was designed
to amplify the remaining 3' end of ompe with a
PstI site at the 5' end and an EcoRI site at the
3' end. A one-step overlap extension PCR that used the two PCR products
above as template and primers corresponding to the 5' and 3' ends of
ompe was performed with VentI DNA polymerase. The
resulting 1.38-kb PCR product was ligated back into pESA to yield a
plasmid with ompe that had a PstI site. The
aphA-3 nonpolar kanamycin cassette from pUC18K
(19) was amplified by PCR using primers with PstI sites (Table 1) and ligated into the PstI site in
ompe in the above described plasmid. The resultant
kanamycin-resistant plasmid pESA::kan was prepared for
electroporation.
M. catarrhalis strains 25240 and 7169 were subjected to
electroporation by using the method of Helminen et al. (
11)
and
Luke et al. (
17). The strains were electroporated with 1 µg
of whole pESA::kan, linearized pESA::kan cut
with
ScaI, and the
purified insert in separate
electroporation experiments. Bacteria
were plated on BHI plates
containing 20 µg of kanamycin per ml.
Colonies were picked and
characterized as described in
Results.
Southern blot assays.
DNA was transferred from agarose gels
by vacuum transfer (Hoefer Trans-Vac TE 80) to Hybond-N+ membrane
(Amersham). The blots were probed with oligonucleotides labeled by
random priming using the Phototope Kit (New England Biolabs). Blots
were developed using the Phototope Star detection kit (New England Biolabs).
Pulsed-field gel electrophoresis.
Strains were subjected to
pulsed-field gel electrophoresis as described previously
(14). Genomic DNA was cut individually with both
SpeI and NheI.
Bactericidal assays.
Bactericidal assays were performed as
described by Chen et al. (7). The complement source was
normal human serum which was adsorbed with protein G to remove IgG. A
pool of serum from healthy adults was heat inactivated by heating in a
56°C water bath for 30 min. Bacteria were grown on Mueller-Hinton
plates for bactericidal assays.
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RESULTS |
Characterization of MAbs.
Eight MAbs to OMP E were developed
(Table 2). Two have been reported
previously (3). To assess the antigenic specificity of the
MAbs, they were assayed in an immunoblot assay with whole-cell lysates, outer-membrane preparations, and purified, recombinant OMP
E. All eight recognized OMP E. Figure 1
shows that MAbs 5B3 and 14E10 preferentially recognize the
non-heat-modified form of OMP E (~35 kDa), whereas the other six MAbs
recognized both the heat-modified (~50-kDa) and non-heat-modified
forms of OMP E (some data not shown).
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FIG. 1.
Immunoblot assay. All lanes contain a whole bacterial
cell lysate of M. catarrhalis 25240. Lanes H, lysate
incubated at 100°C in sample buffer containing  -mercaptoethanol;
lanes U, lysate incubated at room temperature in sample buffer in the
absence of -mercaptoethanol. MAbs are noted at the top and molecular
mass markers are noted in kilodaltons on the left.
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Flow cytometry was performed to determine whether the MAbs recognized
epitopes which were present on the surface of the intact
bacterium.
MAbs 1B3, 9G10d, and 5B4 were reactive in flow cytometry,
indicating
that they recognize surface epitopes. The remaining
5 MAbs were
nonreactive in flow cytometry, indicating that they
recognized epitopes
which are buried in the outer membrane and
not available on the
bacterial surface (Table
2). The three MAbs
which recognized
surface-exposed epitopes (all subclass IgG1)
had no bactericidal
activity when tested in bactericidal
assays.
To begin to elucidate the regions of the OMP E molecule which are
recognized by the MAbs, overlapping fusion peptides corresponding
to
regions of OMP E were constructed as depicted in Fig.
2. The
GST fusion peptides were purified
and subjected to immunoblot
assay. Figure
3 shows that MAb 5B4 recognized an
epitope in amino
acids 80 through 180 of OMP E and MAb 9E3 recognized
an epitope
in amino acids 240 through 340. Figure
2 summarizes the
results
of immunoblot assays of the overlapping fusion peptides with
all
eight MAbs. The observation that MAbs 1B3, 9G10d, and 5B4 were
reactive in flow cytometry (Table
2) along with the results of
immunoblot assays with the fusion peptides which span the OMP
E
molecule (Fig.
2) allowed us to conclude that a surface-exposed
epitope
is present in the region of amino acids 80 through 180.
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FIG. 2.
Schematic diagram depicting five fusion protein
constructs of OMP E. Numbers correspond to amino acids in the mature
protein in each construct. The reactivity of eight MAbs to OMP E with
each fusion protein in immunoblot assays is noted on the right.
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FIG. 3.
Immunoblot assays with MAbs 5B4 and 9E3. Lanes a through
e contain GST fusion proteins corresponding to amino acids (aa) of OMP
E as follows: a, aa 1 through 100; b, 80 through 180; c, 160 through
260; d, 240 through 340; e, 320 through 435. Lane  , empty; lane rE,
recombinant OMP E. Molecular mass standards are noted in kilodaltons on
the left. Lane d in the lower panel contains some proteolytic
degradation of the fusion protein.
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Construction and characterization of mutants.
Electroporation
of M. catarrhalis 25240 with whole pESA::kan,
which contains ompe with a kanamycin cassette inserted in
the central portion of the gene, yielded 30 to 40 colonies, whereas electroporation of 25240 with a linearized plasmid and an isolated insert yielded no colonies. By contrast, strain 7169 yielded colonies following electroporation with all three forms of DNA including whole
plasmid, linearized plasmid, and isolated insert. Two clones were
picked and characterized further. Strain 25240E
was from
an electroporation with whole pESA::kan, and
7169E was from an electroporation with the isolated
insert of pESA::kan.
To confirm the insertion of the kanamycin cassette into
ompe, genomic DNA from the mutants and the isogenic parent
strains
was subjected to PCR analysis. PCR amplification of
ompe was performed
with primers corresponding to the 5' and
3' ends of the gene.
Figure
4 shows that
the parent strains yielded amplicons of 1,299
bp, which corresponds to
the predicted size of
ompe plus restriction
sites in the
primers. The mutant strains yielded amplicons of
2,170 bp, which
corresponds to the sum of
ompe and the kanamycin
cassette.
PCR amplification with primers corresponding to the
kanamycin cassette
resulted in PCR products of 871 bp, the predicted
size of the kanamycin
cassette plus restriction sites in the primers
and in the mutants and
no PCR product in the parent strains (Fig.
4).
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FIG. 4.
Agarose gel stained with ethidium bromide. PCRs were
performed using primers to amplify ompe and the kanamycin
cassette (Kan) as noted at the top of the gel. Templates for PCRs were
genomic DNA from M. catarrhalis strains as follows: lanes a,
7169; b, 7169E; c, 25240; d, 25240E.
Molecular size markers are noted in kilobases at the left.
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To further confirm the insertion of the kanamycin cassette into
ompe, genomic DNA was isolated from the mutant and parent
strains, cut with
NdeI, and subjected to agarose gel
electrophoresis
and Southern blot assay. Probing the blot with an
oligonucleotide
corresponding to
ompe yielded a band of ~5
kb in the parent strains
and a band of ~5.8 kb in the mutants (data
not shown). Probing
separately with an oligonucleotide corresponding to
the kanamycin
cassette revealed a band of ~5.8 kb in the mutants and
no band
in the parent strains. These results indicated that the
kanamycin
cassette is inserted into
ompe.
The nucleotide sequences of the entire PCR-amplified
ompe
with kanamycin cassettes from the mutants were determined. The sequence
of
ompe from strain 7169 was identical to
ompe
from strain 25240
(GenBank accession number
L31788). In addition, the
downstream
sequences were in frame with the kanamycin cassette,
confirming
that these mutations were
nonpolar.
To further confirm that the mutants are isogenic, strains
25240E
, 7169E
, and their respective parent
strains 25240 and 7169 were subjected
to pulsed-field gel
electrophoresis. Digestion of DNA individually
with
SpeI and
NheI produced banding patterns which were identical
in
corresponding mutant and parent strains (data not
shown).
Expression of OMP E in mutant and parent strains.
To assess
the expression of OMP E in the mutant and parent strains, whole-cell
lysates and outer membrane preparations were subjected to SDS-PAGE and
immunoblot assays. Figure 5 shows that the OMP E band in outer-membrane preparations was absent in the mutants, but the banding patterns were otherwise identical with those
of the respective parent strains. Immunoblot assays with MAbs 1B3 (Fig.
5) and 9E3 (data not shown) confirmed the absence of expression of OMP
E in the mutants. MAb 1B3 recognized an epitope corresponding to a
region upstream of the kanamycin cassette while MAb 9E3 recognized an
epitope corresponding to a region downstream of the kanamycin cassette.
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FIG. 5.
Left panel: Coomassie blue-stained sodium dodecyl
sulfate gel of outer membranes. Lanes: a, 7169; b, 7169E;
c, 25240; d, 25240E. Arrows denote OMP E. Right panel:
immunoblot assay with MAb 1B3. Lanes a through d as per left panel.
Molecular mass markers are noted in kilodaltons.
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The mutant strains and their respective parent strains were subjected
to flow cytometry with MAb 1B3, which recognizes an
epitope on the
surface of the intact bacterial cell (
3). Figure
6 shows that MAb 1B3 recognized an
abundantly expressed epitope
on the bacterial surface of the parent
strain 25240. The mutant
strain 25240E
did not fluoresce
in flow cytometry, confirming the absence of
expression of this epitope
on the bacterial surface. Strains 7169
and 7169E
gave
identical results in that the parent strain was strongly
positive while
the mutant was negative in flow cytometry.
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FIG. 6.
Results of flow cytometry with MAb 1B3 and strains 25240 and 25240E as noted. x axis, fluorescence in
arbitrary units; y axis, number of cells.
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Growth characteristics of mutants.
Mutants and parent strains
had identical microscopic appearances when subjected to Gram stain. The
rate of growth of the OMP E mutants in BHI broth was monitored in
parallel with the respective parent strains. Figure
7 shows that the mutant
25240E grew slightly more slowly in BHI broth compared to
the parent strain. When growth was continued overnight, the optical
density of the stationary-phase culture of the mutant remained lower
than that of the parent strain. The growth rate of the mutant
7169E was lower than that of its parent strain, but the
difference was less pronounced than that of 25240E and
25240.
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FIG. 7.
Growth curves in BHI broth. x axis, time in
hours; y axis, optical density (O.D.) at a wavelength of 600 nm. The two curves marked "25240E-" represent the mutant grown
individually in BHI with kanamycin ( ) and BHI broth alone ( ).
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The growth rate of the mutant 25240E
was identical in the
presence and absence of kanamycin (Fig.
7). Colony counts were
performed
by plating aliquots of broth cultures of the mutants grown in
the absence of kanamycin on both BHI plus kanamycin plates and
BHI
plates. The colony counts were identical, indicating that
individual
bacterial cells of the mutant retained expression of
kanamycin
resistance while growing in broth in the absence of
kanamycin.
Serum resistance.
To assess the sensitivity of the mutants to
complement-mediated killing by human serum, the mutants and their
respective parent strains were tested in bactericidal assays with
various dilutions of pooled normal human serum. Figure
8 shows that mutant 7169E
was more readily killed by normal human serum at essentially all
dilutions tested compared with its isogenic parent strain 7169. The
assay was repeated three times, and the results of all three were
similar in showing that 7169E was more sensitive to
killing by normal human serum. M. catarrhalis 25240E was more serum sensitive than its parent strain
25240; however, the difference was less striking.
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FIG. 8.
Results of bactericidal assays. x axis,
dilution of normal human serum; y axis, percent killing at
60 min. Error bars represent results of duplicate plates from colony
counts.
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DISCUSSION |
In this report, to begin to define the antigenic structure
of OMP E, MAbs were developed, and the regions on OMP E to which the
antibodies bound were identified using immunoassays with fusion peptides constructed to correspond to regions of the protein. Several
different epitopes were represented by the eight MAbs (Fig. 2). Three
of the antibodies (1B3, 9G10d, and 5B4) bound to a surface-exposed
epitope which is located between amino acids 80 and 180. MAbs 5B3 and
14E10 bound to epitopes which were denatured by reduction because they
recognized OMP E exclusively in its nonreduced form.
In previous work, proteinase K treatment of intact M. catarrhalis cells resulted in the cleavage of the amino-terminal
184 amino acids of OMP E and loss of reactivity by MAbs 1B3 and 9G10d (3). These data led to the conclusion that the epitope
recognized by MAbs 1B3 and 9G10d was located in the 184 amino acids at
the amino terminus of OMP E. However, this conclusion was based on indirect evidence, i.e., loss of reactivity with the MAbs. Construction of fusion peptides and analysis of these constructs with the MAbs in
the present study confirm this finding with direct evidence and extend
the observation by further localizing this surface-exposed epitope. The
identification of a surface epitope on OMP E will facilitate more
detailed studies of the human antibody response to OMP E. For example,
it will be important to determine what proportions of antibodies
in human samples are directed at surface epitopes compared to
epitopes buried in the outer membrane, since antibodies which mediate
protection from infection are likely to be directed to surface-exposed epitopes.
Mutants deficient in the expression of OMP E were constructed and
characterized. Several approaches were used to ensure that these
mutants were isogenic. A nonpolar kanamycin cassette was used, and
sequence analysis confirmed that the cassette is in frame with
downstream sequences. Pulsed-field gel electrophoresis, PCR analysis,
Southern blot analysis, and SDS-PAGE of outer membrane preparations all
support the conclusion that the mutants differ only in expression of
OMP E. These mutants will be important tools in a variety of
experiments, such as controls in immunoassays; the availability of the
mutants also will help elucidate the function of OMP E for the bacterium.
Mutants defective in expression of eight OMPs have been constructed in
M. catarrhalis thus far. Six of these are involved in
acquisition of iron, including CopB, TbpA, TbpB, LbpA, LbpB, and Orf3
(5, 11, 16, 17). In addition, mutants lacking expression of
the high-molecular-weight proteins UspA1 and UspA2 have been
constructed (1). OMP E mutants showed a slightly reduced
rate of growth in broth (Fig. 7), whereas the previously reported
mutants appeared to grow at rates similar to their respective parent
strains in broth which includes sources of iron, such as BHI. OMP E
shares borderline homology (49.1% similarity, 25.6% identity) with
E. coli FadL, which is involved in uptake of long-chain fatty acids (4). The observation that a mutation in OMP E
results in a reduced rate of growth is compatible with OMP E being
involved in uptake of nutrients. However, a definitive conclusion
regarding the function of OMP E awaits further experiments.
The mutants defective in expression of OMP E were more susceptible to
killing by normal human serum compared to their isogenic parent strains
(Fig. 8). A similar observation has been made with mutants of CopB and
UspA2, suggesting that expression of several surface molecules affects
susceptibility to killing of M. catarrhalis by serum
(1, 11).
OMP E, a potential vaccine antigen, is abundantly expressed on the
bacterial surface as indicated by results of immunofluorescence assays
and flow cytometry with MAbs (3). It would be important to
know whether the expression of OMP E is regulated and the extent to
which the protein is expressed in vivo. Sera from adults with COPD and
from some normal adults contain antibodies to OMP E, suggesting that
the protein is expressed in vivo (3). Analysis of PCR
restriction fragment length polymorphisms among clinical isolates and
studies of strain specificity with polyclonal and monoclonal antibodies
indicate that OMP E is conserved among strains of M. catarrhalis (2, 3). These characteristics of abundant expression on the bacterial surface and conservation among strains are
highly desirable for a potential vaccine antigen. Future work should
focus on determining whether immune responses to OMP E are protective
against infection.
The present study contributes to further elucidating the antigenic
structure of OMP E and provides new data indicating that OMP E is
involved in serum resistance and suggesting that OMP E is involved in
growth of the bacterium. The mutants constructed will contribute to
future work to characterize the function of OMP E and further evaluate
OMP E as a vaccine antigen for preventing infections caused by M. catarrhalis.
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ACKNOWLEDGMENTS |
This work was supported by grant AI28304 from the National
Institute of Allergy and Infectious Diseases and the Department of
Veterans Affairs.
Our thanks to Nicole Luke and Anthony Campagnari for advice in
construction of the mutants.
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FOOTNOTES |
*
Corresponding author. Mailing address: VA Western New
York Healthcare System, Medical Research 151, 3495 Bailey Ave.,
Buffalo, NY 14215. Phone: (716) 862-7874. Fax: (716) 862-6526. E-mail: murphyt{at}acsu.buffalo.edu.
Editor:
D. L. Burns
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Infection and Immunity, November 2000, p. 6250-6256, Vol. 68, No. 11
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
