The Moraxella catarrhalis outer membrane protein CD contains two
distinct domains specifying adherence to human lung cells
Christine Akimana1 & Eric R. Lafontaine2
1
Department of Medical Microbiology and Immunology, University of Toledo Health Sciences Campus, Toledo, OH, USA; and 2Department of Infectious
Diseases, University of Georgia Athens College of Veterinary Medicine, Athens, GA, USA
Correspondence: Eric R. Lafontaine,
Department of Infectious Diseases, University
of Georgia Athens College of Veterinary
Medicine, 220 Riverbend Road, South
Building Room 146, Athens, GA 30602, USA.
Tel.: 1706 542 2863; fax: 1706 542 5771;
e-mail: [email protected]
Received 4 December 2006; revised 1 February
2007; accepted 5 February 2007.
First published online 28 March 2007.
DOI:10.1111/j.1574-6968.2007.00693.x
Editor: Stephen Smith
Keywords
OMPCD; McaP display system; cell binding
domains; thrombospondin type 3 repeats.
Abstract
Most Moraxella catarrhalis isolates express a highly-conserved outer membrane
protein of 453 residues designated OMPCD, which has been previously shown to
mediate binding to A549 human lung cells. Here, it is reported that two distinct
domains of the M. catarrhalis strain O35E OMPCD protein specify adherence.
Truncated proteins were expressed in Escherichia coli to demonstrate that OMPCD
residues 1–240 as well as 241–400 are important for attachment to A549 cells, and
database searches indicated that amino acids 285–299 resemble an adhesive motif
found in eukaryotic proteins termed thrombospondin-type 3 repeat (TT3R).
Cellular enzyme-linked immunosorbent assay using His-tagged proteins demonstrated that residues 236–300 of OMPCD, containing the TT3R motif, specify
adhesive properties. Furthermore, these assays revealed that a purified protein
encompassing residues 16–236 binds to A549 cells. The two cell-binding domains of
OMPCD were further defined to amino acids 16–150 and 261–300 by utilizing a
surface-display system, which was constructed from the M. catarrhalis autotransporter protein McaP, to express foreign peptides on the surface of recombinant bacteria.
Introduction
Moraxella catarrhalis is a Gram-negative organism causing
20% of bacterial otitis media in children (Cripps et al.,
2005) and 10% of respiratory infections in adults with
chronic obstructive pulmonary diseases (COPD) (Murphy
et al., 2005). Of significance, 80% of infants in developed
countries will experience otitis media (Cripps et al.,
2005) and COPD is a leading cause of death worldwide
(www.nhlbi.nih.gov/resources/docs/04_chtbk.pdf). Thus, developing a vaccine against M. catarrhalis would have a
beneficial impact on the health of children and the elderly.
The outer membrane (OM) protein OMPCD exhibits
characteristics of a promising vaccine candidate. It is highly
conserved among M. catarrhalis strains (Murphy et al., 1993;
Hsiao et al., 1995), is expressed by most isolates and
contains surface-exposed epitopes (Sarwar et al., 1992).
Immunization of mice with OMPCD elicits the production
of antibodies binding to the bacterial surface (Murphy et al.,
1999), and COPD patients recovering from M. catarrhalis
infections produce antibodies against the protein (Murphy
et al., 2003). Furthermore, OMPCD likely plays a key role in
pathogenesis due to its ability to bind to middle ear mucin
(Reddy et al., 1997) and to A549 human lung cells (Holm
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c
et al., 2004). Targeting functionally relevant regions of
OMPCD in a vaccine may thus have the advantage of
interfering with the establishment of M. catarrhalis in
susceptible individuals. Other adhesins have been proven to
be effective vaccine antigens. For instance, vaccines licensed
for use in the US against Bordetella pertussis contain the
adhesin FHA (http://www.cdc.gov/nip/publications/pink/
pert.pdf). Binding domains of adhesins also have potential
as therapeutic agents to block adherence to host surfaces.
One example is a peptide corresponding to the binding
domain of the adhesin SAI/II, which was used to inhibit
adherence of Streptococcus mutans to salivary receptors and
prevent colonization of the teeth (Kelly et al., 1999). The
present study identifies regions of M. catarrhalis OMPCD
important for adherence to A549 cells, and thus represent
potential vaccine components.
Materials and methods
Strains, plasmids and culture conditions
Strains and plasmids are described in Table 1. Recombinant Escherichia coli was cultured using Luria–Bertani
(LB) medium (Fisher Bioreagentss) supplemented with
FEMS Microbiol Lett 271 (2007) 12–19
13
Moraxella catarrhalis OMPCD adherence epitopes
Table 1. Plasmids and strains
Strain or plasmid
M. catarrhalis
O35E
O35E.CD1
E. coli
EPI300
TUNER
Plasmids
pCC1
pCC1.3
pMHCD1.2
pCACD240
pCACD400
pIFmcap17
pCAmcapD53–336
pCAmcaP.CD16–150
pCAmcaP.CD150–236
pCAmcaP.CD236–300
pCAmcaP.CD261–330
pETcoco-1
pCAHISCD16–453
pCAHISCD16–236
pCAHISCD200–330
pCAHISCD236–300
pCAHISCD266–366
pCAHISCD300–400
pGEX6p-1.Hia.541–714.F681E
Description
Source or references
Wild-type isolate
Isogenic ompCD mutant of strain O35E
Holm et al. (2004)
Holm et al. (2004)
Cloning strain
Expression strain
Epicentre
Novagen
Cloning vector
pCC1-based adherence negative control
O35E-ompCD ORF cloned into pCC1
pMHCD1.2 in which codon specifying aa 240 was changed to a stop codon
pMHCD1.2 in which codon specifying aa 400 was changed to a stop codon
O35E-mcaP ORF cloned into pCC1
pIFmcap17 in which the DNA specifying aa 53–336 of McaP was deleted and
replaced with unique NsiI/PacI sites; adherence negative
pCAmcapD53–336 into which an amplicon encoding aa 16–150 of O35E
ompCD was cloned
pCAmcapD53–336 into which an amplicon encoding aa 150–236 of O35E
ompCD was cloned
pCAmcapD53–336 into which an amplicon encoding aa 236–300 of O35E
ompCD was cloned
pCAmcapD53–336 into which an amplicon encoding aa 261–330 of O35E
ompCD was cloned
Protein expression vector, specifies six N-terminal histidines
pETcoco-1 encoding O35E-OMPCD aa 16–453
pETcoco-1 encoding O35E-OMPCD aa 16–236
pETcoco-1 encoding O35E-OMPCD aa 200–330
pETcoco-1 encoding O35E-OMPCD aa 236–300
pETcoco-1 encoding O35E-OMPCD aa 266–366
pETcoco-1 encoding O35E-OMPCD aa 300–400
Plasmid encoding a mutated GST-Hia fusion protein that does not bind to
epithelial cells
Epicentre
Holm et al. (2004)
Holm et al. (2004)
This study
This study
Lipski et al. (2007)
This study
15 mg mL 1 chloramphenicol. Where indicated, the Epicentre Copy Control Induction Solution was added to LB
medium as previously reported (Holm et al., 2004). A549
human lung cells were cultured as described elsewhere
(Holm et al., 2004).
Recombinant DNA techniques
Standard molecular biology methods were performed as
outlined elsewhere (Sambrook & Russell, 2001). The codons
specifying aa 240 or 400 of the O35E-ompCD ORF (contained in pMHCD1.2) were changed to a stop codon by the
use of the Stratagene QuickChanges II XL site-directed
Mutagenesis Kit. The DNA region of the O35E-mcaP gene
(contained in pIFmcaP17) specifying 53–336 aa of McaP
was deleted and replaced with unique NsiI and PacI sites by
the use of site-directed mutagenesis. Plasmids were sequenced to confirm that only the desired mutations were
introduced.
FEMS Microbiol Lett 271 (2007) 12–19
This study
This study
This study
This study
Novagen
This study
This study
This study
This study
This study
This study
Joseph St-Geme
Construction of McaP-OMPCD hybrids
PCR products encoding various regions of OMPCD were
amplified from M. catarrhalis O35E with Platinum Pfx DNA
Polymerase (Invitrogen) using primers containing PacI
(forward) or NsiI (reverse) sites, and cloned into the cognate
sites of pCAmcaPD53–336. The resulting ‘pCAmcaP.CD’
plasmids were tested for their ability to specify expression
of McaP-OMPCD chimeric proteins by western blot using
antibodies against McaP (Fig. 5a) or OMPCD (data not
shown). Plasmids were sequenced to verify that mutations
were not introduced in ompCD amplicons.
Construction and purification of recombinant
proteins
PCR fragments specifying various portions of OMPCD were
amplified from M. catarrhalis O35E using primers containing AscI (forward) or PacI (reverse) sites, and cloned into the
cognate sites of pETcoco-1 (Novagen). The resulting ‘pCAHISCD’ plasmids were sequenced to confirm that they
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
14
specify the intended portions of OMPCD fused to six
N-terminal histidine residues. These plasmids were introduced into E. coli TUNER for overexpression of recombinant HIS-OMPCD proteins, which were extracted from
TUNER cells and purified from soluble fractions/inclusion
bodies as recently described for His-tagged McaP proteins
(Lipski et al., 2007). Purified proteins were shown by
western blot to react with antibodies against both the
histidine tag and OMPCD (data not shown). The GST-Hia
protein was purified from TUNER cells harboring the
plasmid pGEX6p-1.Hia.541-714.F681E using Pierce’s BPER GST Spin Purification Kit. Protein concentrations were
determined with the BCATM Protein Assay Kit (Pierce).
Antibodies
Female BALB/c mice were immunized with the purified
recombinant protein specified by plasmid pCAHISCD.16–453 as previously reported (Lipski et al., 2007).
These antibodies were shown to specifically bind to
OMPCD by Western blot using cell lysates of M. catarrhalis
O35E and its ompCD mutant O35E.CD1 (data not shown).
The OMPCD-specific monoclonal antibody 3.9 H has been
described previously (Murphy et al., 1999). Anti-GST and
anti-His antibodies were purchased from Novagen. The
McaP-specific antibodies against the peptide PEP1 (Bullard
et al., 2005) or against aa 51–650 (Lipski et al., 2007) were
described elsewhere.
Adherence assays
Duplicate visual adherence assays were performed as previously reported (Holm et al., 2004; Melillo et al., 2006). For
each well, five randomly selected microscopic fields were
photographed; bound bacteria were enumerated for each
field and divided by the number of epithelial cells present.
Results are expressed as the mean number of bound bacteria
( SE) per epithelial cell; assays were repeated on at least
four separate occasions. The constructs depicted in Figs 2
and 5 were also tested using a viable cell count attachment
assay (Holm et al., 2004). The results of these experiments
were consistent with the data obtained with visual assays
(data not shown).
Cellular enzyme-linked immunosorbent assay
(ELISA)
Cellular binding assays were performed using A549 cells
fixed with paraformaldehyde as outlined by others (Laarmann et al., 2002). Briefly, fixed monolayers were blocked
for 1 h, incubated 3 h with purified proteins, washed,
incubated for 1 h with mouse antibodies against GST or
HISCD.16–453, washed, and incubated with a goat antimouse horse radish peroxidase (HRP)-conjugated antibody.
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
C. Akimana & E.R. Lafontaine
Protein binding was measured by adding 1-step Turbo TMB
substrate solution (Pierce) and measuring absorbance at
450 nm. A GST-fusion protein containing residues 541–714
of the Haemophilus influenzae adhesin Hia was used as a
negative control in the experiments. This particular protein
also contains a mutation abolishing the adhesive properties
of Hia (Joseph St-Geme, Duke University Medical Center,
pers. commun.). For all cellular ELISA, no protein controls
[i.e. monolayers incubated with phosphate-buffered saline
(PBS)] were included and probed with HISCD.16–453 or
GST antibodies; the absorbance values at 450 nm for these
controls were near or below those of the GST-Hia-negative
control depicted in Fig. 3b (data not shown). In addition,
duplicate wells of 96-well plates were coated with purified
proteins to verify antibody reactivity (data not shown).
Flow cytometry, protein preparations and
Western blot analysis
Flow cytometry was performed as described elsewhere
(Lipski et al., 2007). Antibodies against aa 51–650 of McaP
were used to demonstrate that McaP-OMPCD hybrid proteins are expressed on the surface of recombinant E. coli
bacteria (data not shown). The methods used to prepare cell
lysates, extract sarkosyl-insoluble OM proteins and perform
Western blot experiments were described by others (Bullard
et al., 2005; Lipski et al., 2007). Equivalent loads of protein
were analyzed by immunoblotting.
Nucleotide sequence analysis and statistical
methods
Plasmids were sequenced at the University of Michigan
Biomedical Research Core Facilities. Chromatograms were
analyzed with ChromaTool (BioTools, Inc.). GraphPad
Prism 4 was used for statistical analyses.
Results
Selected features of the M. catarrhalis O35EompCD gene product
Analysis of OMPCD using tools provided by the Expasy
Proteomics server identified three domains of interest
(Fig. 1). PSIPRED identified eight b-strands within the first
236 aa of the protein, each connected by loops of 4–39
residues, which likely form a b-barrel domain anchoring
OMPCD in the OM of M. catarrhalis. This putative anchor
was followed by a region of 87 aa (residues 236–323), rich in
alanine (12%) and proline (20%), that links the b-barrel
portion of OMPCD to a C-terminal domain (323–453 aa)
likely located in the periplasm. BLASTP searches using NCBI
indicated that this latter part of OMPCD contains OMPA
FEMS Microbiol Lett 271 (2007) 12–19
15
Moraxella catarrhalis OMPCD adherence epitopes
(a)
C-terminal
β barrel
(aa 330–437 = OMPA/MotB domains)
Linker
236
323
(b)
OMPCD-
GGRLAPAVPVAPVAEPVAEPVVAPAPVILPKPEPEPVIEEAPAVIEDIVVDSDGDGVPDHLDACPGTPVNTVVDPRGCPVQVNLVEE
TT3R-QEDSDNDGVGDACDN
OprF- CSDSDNDGVCDNVDK
Fig. 1. Structural features of OMPCD. (a) depicts different regions of the O35E ompCD gene product with the position of residues defining key
structural as well as functional features. (b) compares the aa sequences of the OMPCD linker region, the consensus sequence of the thrombospondin
type III repeat (TT3R) and aa 200–214 of Pseudomonas aeruginosa OprF. Bold and underlined aa are perfectly conserved between OMPCD and TT3R or
between OprF and TT3R.
FEMS Microbiol Lett 271 (2007) 12–19
(a)
64
50
OMPCD-reactive
antibodies
36
22
(b)
Bacteria/epithelial cell
*
4.5
*
3.0
*
Adherence to
A549 cells
1.5
40
0
AC
D2
40
pC
AC
D
D1
pC
pM
HC
C1
.3
.2
0.0
pC
(Pfam domain# PF00691, 7e-18) and MotB (COG1360, 3e16) motifs, previously shown to interact with peptidoglycan
(Koebnik, 1995; Grizot & Buchanan, 2004; Parsons et al.,
2006). The structural features predicted for OMPCD are
characteristically found in members of the OmpA-like
family of proteins (Koebnik et al., 2000), which include the
adhesins OmpA of E. coli (Shin et al., 2005) and OprF of
Pseudomonas aeruginosa (Azghani et al., 2002). Our observations are consistent with previously published findings
by Murphy et al. (1993) while studying OMPCD of
M. catarrhalis strain ATCC25240.
The O35E-OMPCD linker region was found to contain a
motif resembling a thrombospondin type III repeat (TT3R)
between residues 285 and 299 (Fig. 1b; Pfam domain #
PF02412). Thrombospondins are multifunctional eukaryotic proteins that contain several TT3R motifs binding to
both integrins and calcium (Adams, 2001). Interestingly,
P. aeruginosa OprF, also an adhesin for A549 cells (Azghani
et al., 2002), contains a potential TT3R motif between
aa 200–214 (see Fig. 1b). On the basis of these observations,
it was hypothesized that this TT3R motif specifies adhesive
properties in OMPCD. To test this hypothesis, truncated
proteins were expressed in E. coli and their ability to confer
adherence to A549 cells was measured. The first construct
tested (pCACD240) specifies aa 1–240 of OMPCD and does
not contain the TT3R motif. The second construct
(pCACD400) produces residues 1–400 of OMPCD, encompassing the TT3R motif (see Fig. 1). As shown in Fig. 2a, the
truncated proteins migrated with lower MW of 50 kDa
(pCACD400) and 29 kDa (pCACD240). Figure 2b demonstrates that the adherence of E. coli expressing full-length
OMPCD (pMHCD1.2) or the protein truncated at residue
400 (pCACD400) is fivefold greater than the adherence-
Fig. 2. Western blot analysis (a) and adherence (b) of Escherichia coli
expressing OMPCD proteins. (a) Immunoblotting was performed with
whole cell preparations using the OMPCD-specific antibody 3.9 H. MW
markers are shown to the left in kilodaltons. (b) For adherence assays,
E. coli strains were grown in LB broth supplemented with Copy Control
Induction Solution for 5 h and incubated with A549 cells for 30 min
before washing off unbound bacteria and staining with Giemsa. The
asterisks indicate that the increase in adherence, compared with the
negative control pCC1.3, is statistically significant (P o 0.05 using a
Mann–Whitney test).
negative control. Removing the OMPCD linker region
(pCACD240) reduced binding to lung cells by 50%, which
is consistent with the TT3R motif specifying adhesive
2007 Federation of European Microbiological Societies
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16
C. Akimana & E.R. Lafontaine
β barrel
(a)
16
Linker
236
C-terminal
323
453
HISCD.16 –453
HISCD.16 – 236
HISCD.200 – 330
HISCD.266 – 366
HISCD.300 – 400
(b) 0.7
*
*
HISCD.236 – 300
0.6
*
Absorbance450 nm
0.5
*
*
0.4
*
*
0.3
*
0.2
*
0.1
0.0
16–453
16–236 200–330 266–366 300–400 236–300 Negative
Fig. 3. Binding of recombinant His-OMPCD proteins to A549 cells. (a) Schematic representation of His-tagged proteins encompassing various portions
of OMPCD, which were used in cellular ELISA. (b) Binding of purified recombinant proteins to A549 cells was measured by cellular ELISA. Monolayers
were incubated with 0 (white), 5 (light grey), 50 (dark grey) or 150 (black) nmoles of the indicated proteins. Results are expressed as the mean SE of
absorbance at a wavelength of 450 nm. Duplicate experiments were performed on at least three separate occasions. The asterisks indicate that the
difference in the absorbance at 450 nm compared with the negative control (at the same protein amount) is statistically significant (P o 0.05 using a
Mann–Whitney test).
properties. Interestingly, the adherence of E. coli harboring
pCACD240 is 2.5-fold greater than that of the negative
control, suggesting that the b-barrel region of OMPCD also
contains a cell-binding domain.
Direct binding of purified recombinant HisOMPCD proteins to A549 cells
To delineate the adherence domain(s) of OMPCD, the
histidine-tagged proteins encompassing different portions
of the adhesin depicted in Fig. 3a were overexpressed,
purified, and tested for their ability to bind to A549
pneumocytes in cellular ELISA. As shown in Fig. 3b, purified
proteins specifying aa 200–330 or aa 236–300 bound to
A549 cells whereas polypeptides corresponding to residues
266–366 or 300–400 did not bind to monolayers. These
results indicate that residues 236–300 of OMPCD, which
contain the TT3R motif, specify adhesive properties. Cellular ELISA also revealed that a fusion protein corresponding
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c
to the b-barrel region of OMPCD (HISCD.16–236) binds to
A549 cells at levels equivalent to the positive control
HISCD.16–453 (Fig. 3b). These data are consistent with
our analysis of recombinant bacteria expressing truncated
OMPCD proteins (Fig. 2) and support the hypothesis that
the adhesin contains two distinct cell-binding domains: one
located in the b-barrel region and a second one in the linker
region between residues 236 and 300.
Display of OMPCD polypeptides on the surface
of recombinant bacteria
To conclusively demonstrate that OMPCD harbors two
distinct binding domains, the M. catarrhalis OM protein
McaP was used to display portions of OMPCD on the
surface of E. coli. These recombinant bacteria were then
tested for their ability to attach to A549 cells. McaP was
recently shown (Lipski et al., 2007) to be an autotransporter
protein containing an OM anchor region of 12 b-strands
FEMS Microbiol Lett 271 (2007) 12–19
17
Moraxella catarrhalis OMPCD adherence epitopes
350
(a)
M1
ATA50
375
linker
(α-helix)
Q51
F650
383
53 (PacI)
(b)
M1
ATA50
Q51
53 (PacI)
(c)
M1
336 (Nsi I)
ATA50
Q51
Signal
sequence
336 (Nsi I)
OMPCD peptide
Surface-exposed
passenger domain
Transporter domain
(OM membrane anchor)
Fig. 4. Construction of an in-frame deletion in the O35E McaP protein for the display of OMPCD peptides on the surface of bacterial cells. (a) Depicts
different regions of the O35E mcaP gene product with the position of residues defining key structural and functional features. (b) Shows the truncated
McaP protein encoded by the plasmid pCAmcaPD53–336. (c) Illustrates chimeric OMPCD-McaP proteins.
that is linked to a surfaced-exposed passenger domain by an
a-helical region of 34 residues (Fig. 4a). Site-directed
mutagenesis was used to delete the coding sequence specifying most of the McaP passenger domains (aa 53–336) and to
introduce unique NsiI and PacI sites (Fig. 4b) for the
purpose of cloning PCR fragments that specify foreign
peptides (Fig. 4c). As shown in Fig. 5a, all constructs were
expressed in the OM of recombinant E. coli cells; flow
cytometry experiments using antibodies against McaP also
verified their surface localization (data not shown). Figure
5b demonstrates that the binding of E. coli displaying
OMPCD residues 236–300 or 261–330 on their surface is
fourfold greater than the adherence-negative control
(pCAmcaPD53–336). These results support the hypothesis
that the OMPCD TT3R motif, located between aa 285 and
299, specifies adhesive properties. Adherence assays also
revealed that E. coli expressing OMPCD residues 16–150
binds well to A549 cells. Though the difference between the
adherence of recombinant bacteria displaying OMPCD
aa 150–236 on their surface and the negative control is
statistically significant, the increase in binding was negligible
(Fig. 5b). These data are consistent with aa 16–150 of the
OMPCD b-barrel domain containing an adherence epitope.
FEMS Microbiol Lett 271 (2007) 12–19
Discussion
The aim of this study was to identify the domain(s) of
OMPCD specifying adherence to A549 cells. Of particular
interest is the observation that aa 285–299 of OMPCD
resemble motifs found in thrombospondin molecules designated type III repeats. Thrombospondins are extracellular
glycoproteins of animals with functions in platelet aggregation and regulation of angiogenesis (Adams, 2001). These
molecules are also involved in cell-to-cell and cell-to-matrix
interactions and can bind substrates including integrins,
fibrinogen, fibronectin, laminin and collagen. TT3R motifs
are located in the C-termini of thrombospondins and play
key roles in calcium binding as well as in cell adhesion.
Pseudomonas aeruginosa OprF, also an adhesin for A549
cells, contains a potential TT3R motif between residues 200
and 214 (Fig. 1). Interestingly, the regions of OMPCD
(Murphy et al., 1999) and OprF (Rawling et al., 1995)
encompassing these TT3R motifs have been shown to be
surface-exposed, a location amenable to interacting with a
ligand on epithelial cells. On the basis of these observations,
it was speculated that the TT3R motif of OMPCD specifies a
cell-binding domain for A549 pneumocytes. Of note, the
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18
C. Akimana & E.R. Lafontaine
(a)
98
64
50
McaP-reactive
antibodies
36
*
2.5
Bacteria/epithelial cell
(b)
*
*
2.0
Adherence to
A549 cells
1.5
*
1.0
0.5
0
pC
Am
ca
ca
P.
CD
26
CD
23
P.
133
6
630
0
023
Am
pC
ca
P.
CD
15
-1
5
6
Am
pC
pC
Am
ca
P.
CD
16
-3
3
P∆
53
ca
pC
Am
0
0.0
Fig. 5. Western blot analysis (a) and adherence (b) of Escherichia coli
expressing hybrid McaP-OMPCD proteins. (a) Immunoblotting was
performed with sarkosyl insoluble OM protein preparations and antibodies against the McaP-specific peptide PEP1. MW markers are shown
to the left in kilodaltons. (b) For adherence assays, E. coli strains were
grown in LB broth supplemented with Copy Control Induction Solution
for 1 h and incubated with A549 cells for 3 h before washing off
unbound bacteria and staining with Giemsa. Asterisks indicate that the
increase in adherence (compared with pCAmCAPD53-336) is statistically
significant (P o 0.05 using a Mann–Whitney test).
presence of putative TT3R motifs in OMPCD and OprF has
been reported by others (de Mot & Vanderleyden, 1994).
Database searches using the EMBL-EBI InterPro server also
identified several putative OM proteins that contain putative TT3R motifs (accession number IPR003367); whether
these molecules are adhesins remains to be determined.
Three complementary approaches were used to demonstrate that the linker region of OMPCD, containing the
TT3R motif, specifies adhesive properties. Cellular ELISA
identified residues 236–300 as important for cell binding,
and the use of McaP to display portions of OMPCD on the
surface of recombinant bacteria further defined the adherence epitope to aa 261–300. Although it contains the TT3R
motif, the His-tagged protein HISCD.266–366 did not bind
to A549 cells in cellular ELISA (Fig. 3b), suggesting that
residues 261–265 may also be required for the adhesive
properties of the OMPCD linker region. Results of this study
also indicate that OMPCD contains a second cell-binding
epitope located in its b-barrel domain. Recombinant bacteria expressing OMPCD truncated at residue 240 attach to
A549 cells at levels greater than the adherence-negative
control (Fig. 2b), and cellular ELISA demonstrates that a
purified protein encompassing aa 16–236 binds to monolayers (Fig. 3b). The use of the McaP display system further
defined this second cell-binding domain and identified
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residues 16–150 as specifying adhesive properties (Fig. 5b).
Sequence analysis predicts that this portion of OMPCD
contains 5 membrane-embedded b-strands connected by
loops of varying lengths (aa 53–77, aa 89–91, aa 98–125 and
aa 135–142). It is speculated that two of these loops are
surface-exposed (i.e. aa 53–77 and 98–125 aa) and that one
or both of these mediate adherence to A549 cells; the
b-barrel domain of OMPCD is known to contain at least
one surface-exposed epitope (Murphy et al., 1999). Consistent with the hypothesis of this study, deletion of the four
surface-exposed loops in the b-barrel domain of E. coli
OmpA abolishes the ability of this adhesin to bind to human
brain microvascular endothelial cells (Shin et al., 2005).
Furthermore, surface loops located in the N-terminal
b-barrel domain of Neisseria Opa proteins have been shown
to mediate binding to CEACAM ligands (de Jonge et al., 2003).
The results of cellular ELISA suggest that the cell-binding
domain located in the b-barrel domain of OMPCD binds to
A549 pneumocytes better than the adherence epitope located in
the linker region (compare HISCD.16–236 to HISCD.200–330
and HISCD.236–300 in Fig. 3b). We believe, however, that
the difference in binding between HISCD.16–236 and
HISCD.200–330/HISCD.236–300 observed by cellular ELISA
is due to the fact that polyclonal antibodies against OMPCD
residues 16–453 were used to detect the binding of recombinant
proteins to A549 cells. These antibodies may not bind equally
to all three proteins, which encompass different portions of
OMPCD. Whether HISCD.16–236, HISCD.200–330 and
HISCD.236–300 have different binding affinities for A549 cells
remains to be determined and would require the use of a
monoclonal antibody that recognizes all these recombinant
proteins. Though seemingly a good candidate, the His-tag
antibody used in our experiments was not amenable to cellular
ELISA (data not shown).
Preincubation of recombinant bacteria expressing
OMPCD or M. catarrhalis isolates with antibodies against
OMPCD residues 16–453 does not significantly reduce
adherence to A549 cells (data not shown). Murphy and
colleagues elegantly demonstrated that following colonization or infection with M. catarrhalis, a large proportion of
COPD patients develop new antibodies against OMPCD
(Murphy et al., 2003). Using immunoblot assays and GSTOMPCD fusion polypeptides, it was discovered that these
new antibodies preferentially recognize conformational epitopes located in the last 166 residues of the protein (i.e.
287–453 aa of O35E-OMPCD); it is not clear whether the
OMPCD TT3R motif (i.e. aa 285–299) is a specific target of
these antibodies. These observations suggest that OMPCD
contains immunodominant regions which are not relevant
to its biological function as an adhesin. Future studies will
be aimed at identifying key residues responsible for the
adhesive properties of OMPCD and explore the vaccinogenic potential of these cell-binding domains.
FEMS Microbiol Lett 271 (2007) 12–19
19
Moraxella catarrhalis OMPCD adherence epitopes
Acknowledgements
This study was supported by a grant from NIH/NIAID
(AI051477) to E.R.L. Eric Hansen at the University of Texas
Southwestern Medical Center in Dallas and Tim Murphy at
the State University New York at Buffalo are thanked for
providing M. catarrhalis strains and antibodies. Joseph
St-Geme at Duke University Medical Center is also thanked
for providing the GST-Hia fusion protein construct. Thanks
are also due to William Grose, Brian Bullard, Rachel Balder,
Mark Wooten, Robert Blumenthal and Randall Worth for
their helpful comments on the manuscript.
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