INFECTION AND IMMUNITY, Apr. 2004, p. 1906–1913 Vol. 72, No. 40019-9567/04/$08.00�0 DOI: 10.1128/IAI.72.4.1906–1913.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The Moraxella catarrhalis Porin-Like Outer Membrane Protein CD Isan Adhesin for Human Lung Cells

Melissa M. Holm, Serena L. Vanlerberg, Ian M. Foley, Darren D. Sledjeski,and Eric R. Lafontaine*

Department of Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio 43614-5806

Received 4 August 2003/Returned for modification 4 November 2003/Accepted 27 December 2003

The outer membrane protein CD (OMPCD) of Moraxella catarrhalis is an outer membrane protein withseveral attributes of a potential vaccine antigen. We isolated four transposon mutants of strain O35E on thebasis of their reduced binding to A549 human lung cells in microcolony formation assays, and we determinedthat they contain a transposon in ompCD. We also found that these transposon insertions had pleiotropiceffects: mutants grew slower, became serum sensitive, bound �10-fold less to A549 cells, and appearedtransparent when grown on solid medium. We confirmed that these various phenotypes could be attributedsolely to disruption of ompCD by constructing the isogenic strain O35E.CD1. O35E-ompCD was cloned, andrecombinant Escherichia coli bacteria expressing the gene product exhibited a 10-fold increase in adherence toA549 cells. This is the first report of M. catarrhalis ompCD mutants, and our findings demonstrate that thisgene product is an adhesin for human lung cells.

The gram-negative bacterium Moraxella catarrhalis is apathogen of the human respiratory tract that causes otitis me-dia in young children (17, 20, 33, 44) and lower respiratorytract infections in adults with chronic obstructive pulmonarydisease (44, 62, 63). Patients with underlying conditions appearto be particularly susceptible, as illustrated by the increasingnumber of cases of M. catarrhalis-caused wound infections,bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia,meningitis, pericarditis, and endocarditis (8, 14, 17, 33, 44, 51,65, 68, 70, 71).

Little is known about pathogenesis by M. catarrhalis. Mostresearch has thus far focused on the identification and char-acterization of a few outer membrane proteins for vaccinedevelopment purposes. These include the adhesins UspA1 (30,36, 37, 40, 41), UspA2H (36, 41), Hag (21, 22, 31, 54), McaP(69), and MID (23, 24, 26, 43, 52), the serum resistance factorUspA2 (3, 4, 15, 18, 36, 40, 41), the iron acquisition proteinsCopB (2, 5, 11, 28, 29, 64), LbpA/LbpB (11, 13, 19, 74), TbpA/TbpB (13, 16, 50, 74), and OmpB1 (12, 13, 38, 39), and thehighly conserved proteins outer membrane protein E (OMPE)(9, 10, 45) and OMPCD (32, 46, 47, 61). Although no specificbiological function has been attributed to OMPCD, this mol-ecule is predicted to be structurally similar to bacterial porins(47) and binds middle ear mucin (59). Thus, OMPCD may beinvolved in nutrient acquisition and/or adherence to mucosalsurfaces.

The present study describes the isolation and characteriza-tion of ompCD mutants of the M. catarrhalis wild-type strainO35E, and the data demonstrate that OMPCD is an adhesinfor A549 human lung epithelial cells.

MATERIALS AND METHODS

Strains, plasmids, tissue culture cell lines, and growth conditions. Strains andplasmids are described in Table 1. M. catarrhalis strains were grown at 37°C inTodd Hewitt (TH) broth (Difco) or on TH agar plates in an atmosphere of92.5% air–7.5% CO2. M. catarrhalis transposon mutants were selected with 20 �gof kanamycin (KAN)/ml. Escherichia coli strains were grown in Luria-Bertani(LB) broth (Difco) or on LB agar plates. For the selection of recombinant E. coliclones, the LB medium was supplemented with either 100 �g of ampicillin/ml, 50�g of KAN/ml, or 15 �g of chloramphenicol/ml. For adherence and serumbactericidal assays with recombinant E. coli cells, 5-ml cultures were grownovernight at 37°C with shaking (200 rpm). These overnight cultures were dilutedinto 20 ml of fresh broth supplemented with 0.25 ml of 1000X CopyControlinduction solution (Epicentre) and grown at 37°C for 2 h with vigorous shaking(300 rpm). Chang (conjunctival epithelium; ATCC CCL20.2), A549 (type IIalveolar lung epithelium; ATCC CCL85), and human middle ear epithelial cells(HMEE) were cultured as described elsewhere (31).

Recombinant DNA techniques. Standard molecular biology methods wereperformed as described previously (60). M. catarrhalis genomic DNA was pre-pared with the Invitrogen Easy-DNA kit. Plasmid DNA was purified with theQIAprep Spin Miniprep system (Qiagen). The North2South chemiluminescentnucleic acid hybridization and detection system (Pierce) was used to performSouthern blotting experiments. A 1.2-kb DNA fragment containing a kanr car-tridge was obtained from the plasmid pUC4K and used as a probe in some ofthese experiments. The 1.2-kb ompCD-specific DNA probe was obtained by PCRusing the oligonucleotide primers P1 and P2 (see below).

PCR and cloning. Amplification of DNA fragments was performed with thePlatinum Pfx DNA polymerase (Invitrogen) unless indicated otherwise. TheompCD-specific oligonucleotide primers P1 (5�-GTGACAGTCAGCCCACTA-3�) and P2 (5�-TTGCTACCAGTGATTACTGC-3�) were used to amplify a1.2-kb DNA fragment from strain O35E that corresponds to a truncated openreading frame (ORF). The primers P3 (5�-GGATCGCTATGCTAAAATAGGTGC-3�) and P4 (5�-TCAAAAGCTAAGAAAACCGCT-3�) were used to gen-erate a 1.6-kb amplicon from O35E containing the complete ompCD ORF andwhich was utilized as template in sequencing reactions as well as in cloningexperiments with the Epicentre CopyControl PCR cloning system. Taq DNApolymerase (Invitrogen) was used in other PCR-based experiments. The plasmidpCC1.3 corresponds to the Epicentre CopyControl vector pCC1, into which themanufacturer’s control DNA insert was cloned.

Transposon mutagenesis and adherence assays. M. catarrhalis O35E transpo-son mutants were obtained using the EZ::TN �KAN-2� transposome (Epicen-tre), and mutants were screened in microcolony formation assays to identifythose substantially reduced in their adherence to A549 cells, as we have previ-ously reported (31). The method used to quantitatively measure the adherenceof M. catarrhalis to human tissue culture cell lines has been described elsewhereand involves a 3-h incubation prior to washing unbound bacteria (31). Adherence

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, Medical College of Ohio, Health EducationBuilding, 3055 Arlington Ave., Toledo, Ohio 43614-5806. Phone: (419)383-6626. Fax: (419) 383-3002. E-mail: elafontaine@mco.edu.

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assays with E. coli recombinant cells involved a 5-min incubation prior to washingunbound bacteria.

Construction of isogenic mutants. An amplicon of 1.2 kb containing a trun-cated ompCD ORF from strain O35E was generated with the primers P1 and P2(see above) and was ligated into the vector pUC19, yielding the recombinantplasmid pELCD. The latter was linearized with DraIII, treated with Pfu DNApolymerase (Stratagene) to render the restricted ends blunt, and ligated with a1.2-kb SmaI DNA fragment containing the kanr cassette from the plasmidpUC4K. This ligation mixture was introduced into E. coli TOP10, and transfor-mants were selected for resistance to KAN, thereby yielding the plasmidpELCDKAN. A 2.4-kb amplicon, which corresponds to a truncated O35E-ompCD gene interrupted by the kanr cartridge in the middle of the ORF, wasgenerated from pELCDKAN using the primers P1 and P2. This PCR productwas then electroporated into M. catarrhalis strain O35E. The resulting kanr

colonies were screened by PCR with primers P1 and P2 to identify potentialisogenic ompCD mutants (data not shown). Southern blotting experiments wereperformed to confirm that proper allelic exchange had occurred in the isogenicmutant O35E.CD1 (data not shown).

MAbs and characterization of selected protein antigens. The UspA1- andUspA2-specific monoclonal antibody (MAb) 17C7 (4), the UspA1-specific MAb24B5 (18), and the Hag-specific MAb 5D2 (54) have been described elsewhere.The OMPCD MAbs 1D3 and 3.9H have been reported previously (46). Outermembrane vesicles were prepared as described by others (49, 53). Whole-celllysates of M. catarrhalis strains and E. coli recombinant cells were prepared aspreviously reported (18, 53). These preparations were heated at 100°C for 15min, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and either stained with Coomassie blue or electrophoreticallytransferred to polyvinylidene difluoride membranes (Millipore) for Western blotanalysis as previously described (31).

Serum resistance assays. Serum bactericidal assays were performed as re-ported by Aebi et al. (3). Results are expressed as the percentage (� standarddeviation) of bacteria surviving incubation with serum. These assays were per-formed on at least three separate occasions.

RNA purification and QRT-PCR. The methods, primers, and probes used forRNA purification and quantitative real-time PCR (QRT-PCR) in these experi-ments have been described elsewhere (31).

Nucleotide sequence analysis. The nucleotide sequence data were analyzed aspreviously reported (31).

Statistical methods. All statistical analyses were performed using a Mann-Whitney test and GraphPad Prism 2.01 software. P values of �0.05 were con-sidered statistically significant.

Nucleotide sequence accession number. The nucleotide sequence of the M.catarrhalis O35E ompCD gene has been deposited in GenBank under the acces-sion number AY493741.

RESULTS

ompCD mutations have pleiotropic effects. We recently de-scribed a mutagenesis and screening approach which identifiednine adherence mutants in M. catarrhalis strain O35E (31).These mutants showed substantially lower binding to A549human lung cells in microcolony formation assays, and allcontained a transposon in the hag gene. This screening ap-proach also yielded four adherence mutants that were notdescribed in our original report because their growth was sig-nificantly impaired. Figure 1 illustrates this defect for the rep-resentative mutant O35E.TN52. We also found that these fourslower-growing mutants expressed wild-type levels of Hag,UspA1, and UspA2 (Fig. 2). Since Hag appears to be the mainadhesin for A549 and HMEE cells (23, 31), we reasoned thatthe apparent adherence defect exhibited by the four slow-growing mutants in microcolony assays might simply resultfrom their poor growth.

Even though Southern blotting experiments suggested thatthese four isolates are not siblings (data not shown), theircolonies all appeared transparent in contrast to the opaquemorphology of the parent strain O35E (Fig. 3). We also ob-served that all four isolates lacked expression of a major OMPof 55 kDa (Fig. 4A). Several larger antigens were also absent inthe mutants (Fig. 4A).

TABLE 1. Plasmids and strains

Strain or plasmid Description Source or reference

M. catarrhalis strainsO35E Wild-type isolate 4O35E.1 uspA1 isogenic mutant of strain O35E; adherence negative for

Chang cells3

O35E.2 uspA2 isogenic mutant of strain O35E; serum sensitive 3, 4O35E.TN2 hag transposon mutant of strain O35E; adherence negative for

A549 and HMEE cells31

O35E.TN52 ompCD transposon mutant of strain O35E This studyO35E.TN313 ompCD transposon mutant of strain O35E This studyO35E.TN593 ompCD transposon mutant of strain O35E This studyO35E.TN649 ompCD transposon mutant of strain O35E This studyO35E.CD1 Isogenic ompCD mutant of strain O35E This study

E. coli strainEPI300 Cloning strain Epicentre

PlasmidspCC1 Cloning vector EpicentrepUC19 Cloning vector New England BiolabspUC4K Source of the kanr cassette AmershamBiosciencespCC1.3 pCC1 into which the control insert provided by the manufacturer

was cloned; adherence negative, serum sensitiveThis study

pELCD Truncated O35E-ompCD gene cloned into pUC19 This studypELCDKAN pELCD containing the kanr cassette of pUC4K in the middle of

the truncated O35E-ompCD geneThis study

pMHCD1.2 Complete O35E-ompCD ORF cloned into pCC1; adheres toA549 cells

This study

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It was previously shown that OMPCD is a heat-modifiableprotein that migrates with a molecular mass of approximately55 kDa (61). We therefore tested by Western blotting whetherthe major OMP missing from the transparent mutants wasOMPCD. Lanes 2 to 5 in Fig. 4B demonstrate that the mutantsno longer detectably expressed OMPCD. Western blotting ex-periments with the OMPCD-specific MAb 3.9H (Fig. 4C) alsosuggested that the higher-molecular-mass antigens missingfrom outer membrane vesicles of the mutants (Fig. 4A) weremultimers of OMPCD. When we analyzed the mutants byPCR, the ompCD-specific primers P1 and P2 yielded an am-plicon of 1.2 kb in the parent strain O35E and one of 2.4 kb inthe transposon mutants (data not shown). These results are

consistent with the 1,221-bp EZ::TN �KAN-2� transposonhaving inserted into the ompCD gene of all four independentlyisolated transparent mutants. This was confirmed by Southernblot analysis with ompCD- and transposon-specific probes(data not shown).

FIG. 1. Growth of M. catarrhalis strains in broth cultures. Symbols:squares, O35E; inverted triangles, O35E.TN52.

FIG. 2. Western blot analysis of M. catarrhalis strains. Proteins present in outer membrane vesicles prepared from the wild-type strain O35E(lane 1), the transposon mutant O35E.TN52 (lane 2), and the isogenic mutant O35E.CD1 (lane 3) were resolved by SDS-PAGE and analyzed byWestern blotting with the UspA1- and UspA2-specific MAb 17C7 (A), the UspA1-specific MAb 24B5 (B), and the Hag-specific MAb 5D2 (C).Note that samples were heated at 100°C for 15 min prior to Western blot analysis. Under these conditions, UspA1 migrates as a 125-kDa proteinand UspA2 migrates as a high-molecular-weight aggregate that is expressed in large amounts (bracket on the right side of panel A). Positions ofmolecular mass markers are shown to the left in kilodaltons. The arrowheads indicate selected antigens.

FIG. 3. Appearance of M. catarrhalis strains on agar plates. Thewild-type strain O35E (bottom left) and the transposon mutantO35E.TN52 (top right) were streaked onto TH agar plates and pho-tographed with lighting from above against a black background. Plateswere incubated for 24 h.

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We constructed the isogenic ompCD strain O35E.CD1 totest whether the various phenotypes of our four transposonmutants could be attributed solely to disruption of the ompCDgene. We found that O35E.CD1 is transparent (data notshown), has an OMP profile indistinguishable from that of thetransposon mutants (Fig. 4A, lane 6), lacks expression ofOMPCD (Fig. 4B and C, lane 6), expresses wild-type levels ofHag, UspA1, and UspA2 (Fig. 2), and exhibits a growth defectsimilar to that of the transposon mutants (data not shown).Furthermore, we observed that O35E.TN52 (4.4% � 8.2%survival) and O35E.CD1 (1.8% � 2.5% survival) were sensitiveto 10% normal human serum, whereas O35E was resistant(117.8% � 15.4% survival). The uspA2 mutant O35E.2 (2.3%� 2.4% survival) was used as a serum-sensitive control (3, 36),and heat inactivation of the serum abolished its ability to killmutants (data not shown).

Taken together, these results demonstrate that ompCD mu-tations affect the growth, colony morphology, and serum resis-tance of M. catarrhalis O35E, but they do not affect expression

of the adhesins UspA1 and Hag or that of the serum resistancefactor UspA2.

The transposon insertion in O35E-ompCD does not signifi-cantly affect expression of the gene located directly down-stream. BLAST searches of the patented M. catarrhalis ge-nome through NCBI databases identified the ompCD ORF(nucleotides 79050 to 77770 of AX067466) as well as the ORFlocated directly downstream (nucleotides 77514 to 75670 ofAX067466). This ORF is predicted to encode a protein with70% identity (83% similarity) to Salmonella enterica serovarTyphimurium GTP-binding elongation factor family proteinBipA (NP_462889.1). To demonstrate that the various pheno-types of ompCD mutants were not due to a polar effect onexpression of the gene downstream of ompCD, we used QRT-PCR to measure the expression of M. catarrhalis bipA. We usedexpression of the unlinked M. catarrhalis gene purH, which werecently reported to be located downstream of the hag gene(31), as a normalization control. No significant change in bipAexpression was observed between the wild-type strain O35E(relative expression � standard deviation, 1.0 � 0.7), theO35E.TN52 transposon mutant (1.73 � 0.3), and theO35E.CD1 isogenic mutant (2.2 � 0.9). Previous work andthese data make it unlikely that the various phenotypes of theompCD mutants are due to changes in transcription of bipA.

OMPCD expression affects the adherence of strain O35E toA549 human lung cells. The apparently reduced binding ofompCD mutants to A549 cells in microcolony formation assaysmight simply reflect their slower growth rate. These assaysentail a 40-h incubation with A549 cells (31). We thereforemeasured the adherence of our mutants after 3 h of incuba-tion, as previously reported (31). Table 2 shows that the trans-parent mutants attached substantially less well to A549 cells, asdid the isogenic ompCD strain O35E.CD1. It should be notedthat the hag mutant O35E.TN2 (31) was used as an adherencenegative control in the assays.

To test whether this was a general defect in adherence, wemeasured the binding of ompCD mutants to Chang monolay-ers and found that they attached at nearly wild-type levels tothese conjunctival cells (Table 2). UspA1 has been reported tobe the major adhesin for Chang cells (3, 36), and the isogenicuspA1 mutant O35E.1 was used as an adherence negative con-trol in our assay. Thus, the ompCD mutants express a func-tional UspA1 adhesin. We also found that the lack of OMPCDexpression did not adversely affect M. catarrhalis binding toHMEE cells (Table 2). We previously reported that Hag is themajor adhesin for HMEE cells (31); thus, the hag mutantO35E.TN2 was used as an adherence negative control in theseexperiments. Taken together, our results demonstrated thatOMPCD expression specifically affects adherence to A549 hu-man lung cells.

Cloning and expression of O35E ompCD by recombinant E.coli cells. To determine whether OMPCD plays a direct role inserum resistance and adherence to A549 cells, we cloned andexpressed this gene in E. coli strain EPI300 using Epicentre’sCopyControl PCR cloning system. This system allowed therecombinant plasmid pMHCD1.2 to be maintained at a verylow copy number. Under these conditions, OMPCD expressionwas not detectable (data not shown). Upon incubation at 37°Cfor 2 h and in the presence the CopyControl inducer solution,which boosts the plasmid copy number to 10 to 200 per cell,

FIG. 4. Western blot analysis of M. catarrhalis strains. Proteinspresent in outer membrane vesicles prepared from the wild-type strainO35E (lane 1) as well as the transposon mutants O35E.TN52 (lane 2),O35E.TN313 (lane3), O35E.TN593 (lane 4), and O35E.TN649 (lane5) and from the isogenic mutant O35E.CD1 (lane 6) were resolved bySDS-PAGE and stained with Coomassie blue (A) or analyzed byWestern blotting with the OMPCD-specific MAbs 1D3 (B) and 3.9H(C). Positions of molecular mass markers are shown to the right inkilodaltons. The arrow as well as arrowheads indicate antigens that aremissing in the transposon as well as the O35E.CD1 mutants.

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however, recombinant bacteria expressed detectable OMPCD(Fig. 5A). When we tested these induced cells in adherenceassays, we found that OMPCD expression increased binding toA549 monolayers by 10-fold after only 5 min of incubation withthese lung cells (Fig. 5B and C). Thus, OMPCD is an adhesinfor A549 cells. OMPCD expression, however, did not conferserum resistance on E. coli (data not shown).

The ompCD gene harbored by the plasmid pMHCD1.2 wassequenced to verify that no mutations were introduced byPCR. This O35E ompCD sequence was also found to be iden-tical to that of ATCC 25240 ompCD (accession numberL10755) and was deposited in the GenBank database.

DISCUSSION

The M. catarrhalis OMPCD protein exhibits numerous prop-erties of a promising vaccine candidate. This antigen is surfaceexposed and is expressed by virtually all M. catarrhalis isolatestested to date (32, 47, 61). Immunization with recombinantOMPCD confers protective immunity in a mouse pulmonaryclearance test (48), and the protein is an important target ofthe immune response in chronic obstructive pulmonary diseasepatients with M. catarrhalis infections (46). In addition, thepredicted amino acid sequence of OMPCD is highly conservedamong clinical isolates (32, 47), and our results extend thesedata. We found that the nucleotide and predicted amino acidsequences of O35E ompCD were identical to those of ATCC25240 ompCD previously reported by Murphy and colleagues(47). The biological function(s) of OMPCD, however, has notbeen determined. Sequence analysis indicated that it is relatedto porins and that the most closely related gene product is thePseudomonas aeruginosa porin OprF (47). Porins form a largefamily of OMPs that are involved in numerous biological func-tions, including nutrient acquisition (1, 34, 35). Since our datashow that ompCD mutants have slower growth rates, OMPCDmay therefore be involved in passage of a nutrient(s) across theM. catarrhalis outer membrane. Our isogenic ompCD mutantswill facilitate the testing of this hypothesis.

Reddy and coworkers previously reported that OMPCDbinds mucin glycoproteins, suggesting that it may be involvedin adherence to mucosal surfaces (59). We found that ompCD

mutants showed reduced binding to A549 human lung cells,supporting this hypothesis. We also recently showed that theM. catarrhalis O35E Hag protein is an adhesin for A549 cells(23, 31). Thus, one possible explanation for the decreasedadherence of ompCD mutants is that the lack of OMPCD inthe outer membrane affects the proper surface display of Hag,which in turn reduces adherence to A549 cells. Our results,however, argue against this possibility. We have previouslyshown that Hag is a major adhesin for middle ear cells (31).The ompCD mutant O35E.TN52 binds at near-wild-type levelsto HMEE cells, whereas the hag mutant O35E.TN2 no longerattaches (Table 2). The ompCD mutant therefore expresses afunctional Hag adhesin. Furthermore, OMPCD expression byrecombinant E. coli bacteria increased adherence to A549 cellsby 10-fold (Fig. 5). These data provide direct proof thatOMPCD is an adhesin. Interestingly, P. aeruginosa OprF wasrecently shown to be an adhesin for A549 cells (7). Our resultssuggest that both Hag and OMPCD are involved in the bindingof M. catarrhalis to A549 cells. Bacterial adherence is multi-factorial and generally involves several steps, such as initialcontact (from a distance) and close (tight) binding (27, 42, 66,67). Since Pearson et al. demonstrated that Hag forms ex-tended projections that cover M. catarrhalis O35E cells (54),Hag may initially contact A549 cells, whereas OMPCD is nec-essary for a closer interaction. Thus, both Hag and OMPCDmay cooperate in specifically conferring adherence to A549cells. We are currently investigating this hypothesis.

Our data also show that ompCD mutants are serum sensi-tive. This effect, however, might be indirect, since expression ofrecombinant OMPCD does not confer E. coli with the abilityto resist the bactericidal activity of human complement. Thelack of OMPCD expression in the outer membrane may thusaffect proper surface display of a serum resistance factor,which in turn renders bacteria sensitive to complement killing.Candidate serum resistance factors include UspA2 (3, 36),CopB (29), fur-regulated genes (25), OmpE (45), and LOS(75). The transparent appearance of ompCD mutants may alsobe indirectly linked to the lack of OMPCD expression. Forinstance, it has been demonstrated for gram-negative patho-gens such as Haemophilus influenzae (73) and Neisseria men-

TABLE 2. Adherence of M. catarrhalis strains to human cells in vitro

Strain Description% Adherencea

A549b HMEE c Changd

O35E wild type, opaque colony morphology 40.5 � 4.5 27.9 � 4.2 17.4 � 2.7O35E.TN2 Tn mutant, transposon in hag gene, opaque colony morphology 1.3 � 0.3*f 0.7 � 0.1* NDe

O35E.1 Isogenic uspA1 mutant, opaque colony morphology ND ND 1.5 � 0.4*O35E.TN52 g Tn mutant, transposon in ompCD gene, transparent colony morphology 2.6 � 0.4* 23.9 � 4.4 14.9 � 1.8O35E.TN313 g Tn mutant, transposon in ompCD gene, transparent colony morphology 4.4 � 1.5* ND NDO35E.TN593 g Tn mutant, transposon in ompCD gene, transparent colony morphology 1.3 � 0.2* ND NDO35E.TN649 g Tn mutant, transposon in ompCD gene, transparent colony morphology 0.8 � 0.2* ND NDO35E.CD1 Isogenic ompCD mutant, transparent colony morphology 5.1 � 0.9* ND 16.1 � 2.9

a Adherence is expressed as the mean (� standard error) percentage of bacteria binding to monolayers.b Strains were incubated for 3 h with A549 cells prior to washing unbound bacteria.c Strains were incubated for 15 min with HMEE cells prior to washing unbound bacteria.d Strains were incubated for 15 min with Chang cells prior to washing unbound bacteria.e ND, not determined.f *, the difference in the P value compared to the value for the wild-type strain O35E was found to be statistically significant using a Mann-Whitney test.g Transposon mutants were first identified due to their reduced binding to A549 cells in microcolony formation assays.

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ingitidis (6) that changes in LOS structure and/or levels ofexpression cause differences in colony opacity.

Alternatively, recombinant E. coli bacteria may not expressenough OMPCD to confer serum resistance, or the proteinrequires posttranslational modification that is not achieved inthis heterologous genetic background. It is interesting thatsearches using the NCBI Conserved Domain Search tool indi-cate that OMPCD is related to the OmpA OMP and relatedpeptidoglycan-associated (lipo)proteins family (COG2885; Evalue of 4e-24). E. coli K1 OmpA has been shown to mediateinvasion of human brain microvascular endothelial cells (55,57, 58) and thus is almost certainly involved in physical inter-actions with mammalian cells. Furthermore, E. coli OmpAplays a role in serum resistance. An ompA mutant showedgreater sensitivity to normal human serum (72), and Prasa-darao and colleagues recently demonstrated that OmpA con-tributes to serum resistance by binding to the complement C4bbinding protein (56). Thus, M. catarrhalis OMPCD may verywell play direct roles in resistance to complement killing andcolony opacity.

In summary, this study reports the characterization of M.catarrhalis ompCD mutants isolated by their reduced adher-ence properties. Phenotypic analyses indicate that the lack ofOMPCD expression leads to pleiotropic effects. The availabil-ity of isogenic strains as well as recombinant clones expressingthe protein will facilitate establishing whether OMPCD is di-rectly involved in nutrient acquisition, resistance to comple-ment killing, and/or conferring the opaque appearance of M.catarrhalis wild-type strains. Our finding that OMPCD is anadhesin for human lung cells also suggests that a vaccine con-taining this protein (or portions thereof) may interfere withadherence, which is an important step in bacterial pathogene-sis. Identifying an M. catarrhalis OMPCD epitope(s) involvedin adherence and evaluating its vaccinogenic potential couldsignificantly contribute to the development of a vaccine for thisimportant human pathogen.

ACKNOWLEDGMENTS

This study was supported in part by institutional start-up funds fromthe Medical College of Ohio, a grant from the Thrasher Research

FIG. 5. Western blot analysis of and adherence assays with E. coli recombinant bacteria. (A) Proteins present in whole-cell lysates of EPI300pCC1.3 and EPI300 pMHCD1.2 were resolved by SDS-PAGE and then analyzed by Western blotting with the OMPCD-specific MAb 1D3. Theselysates were prepared from cells grown in the presence of the Epicentre’s CopyControl inducer solution for 2 h. Positions of molecular massmarkers are shown to the right in kilodaltons. (B) Visual adherence assays. Recombinant bacteria were grown under the aforementionedconditions. Unbound bacteria were washed off A549 cells after a 5-min incubation, and the monolayers were stained with Giemsa. (C) Quantitativeadherence assays. Results are expressed as the percentage (� standard error) of recombinant bacteria binding to A549 cells after 5 min ofincubation.

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Fund (award number 02816-6), and a grant from the National Instituteof Allergy and Infectious Diseases, National Institutes of Health(AI051477) to E.R.L.

We thank Tim Murphy at the University of Buffalo and Eric Hansenat the University of Texas Southwestern Medical Center in Dallas forproviding M. catarrhalis strains and antibodies. We also thank ThomasDeMaria at Ohio State University for providing cultures of humanmiddle ear cells. We also thank Tim Murphy, Eric Hansen, RobertBlumenthal, and Mark Wooten for their helpful comments on themanuscript.

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