Characterization and Evaluation of the Moraxella catarrhalis Oligopeptide Permease A as a Mucosal Vaccine Antigen

Bacterial strains and growth.Moraxella catarrhalis strain ATCC 43617 was obtained from the American Type Culture Collection (Manassas, VA). Isolate 035E was provided by Eric Hansen. Strains 5P34B1, 10P58B2, 12P15B1, 14P25B1, 29P24B1, 33P25B2, 39P29B2, 55P18B3, 63P62B1, and 96P9B1 were sputum isolates obtained from adults with COPD. Strains 135, 238, 555, 2901, 3614, 3584, 5488, 8184, 9483, and 6952 were middle ear fluid isolates obtained via tympanocentesis. Pulsed-field gel electrophoresis of genomic DNA cut with SmaI showed that the strains are genetically diverse. M. catarrhalis strains were grown on brain heart infusion (BHI) plates at 37°C with 5% CO₂ or in BHI broth with shaking at 37°C.

Chemically competent Escherichia coli strains Top10 and BL21(DE3) were obtained from Invitrogen (Carlsbad, CA) and were grown at 37°C on Luria-Bertani (LB) plates or in LB broth.

Nucleotide sequence analysis.M. catarrhalis genes encoding putative surface proteins, including lipoproteins and outer membrane proteins with characteristic signal sequences, were identified based on the genome sequence of strain ATCC 43617 using a genome mining approach as described previously (44). The oppA gene was defined by homolog analysis using a BLAST search at the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences of oppA genes amplified from 10 clinical isolates of M. catarrhalis and the ATCC 43617 strain were determined at the Roswell Park Cancer Institute (RPCI) DNA sequencing facility with sequencing primers P1, P5, and P6 listed in Table 1. These sequences were assembled with the Sequencher program and aligned with the ClustalW alignment function of the MacVector program.

Genomic DNA and RNA purification.Genomic DNA of M. catarrhalis strains was purified with the Wizard genomic DNA purification kit (Promega, Madison, WI) according to the manufacturer's instructions. The total RNA of M. catarrhalis 035E was isolated with the Qiagen RNeasy minikit (Qiagen, Valencia, CA) followed by DNase I treatment by using RQ1 RNase-free DNase I from Promega and finally cleaned up with the Qiagen RNeasy minikit according to the protocols provided with the kits.

RT-PCR.Reverse transcriptase PCR (RT-PCR) was performed using the Superscript II reverse transcriptase from Invitrogen (Carlsbad, CA). The first-strand cDNA was synthesized with 2 pmol of the reverse primer P3 (Table 1) and 1.5 μg of total RNA extracted from the M. catarrhalis 035E strain. A 10% cDNA reaction mixture was then used in the second-step PCR with both forward (P2) and reverse (P3) primers (Table 1) and Hotmaster mix (5 Prime Inc., Gaithersburg, MD) to amplify a 223-bp fragment of the oppA gene. A parallel reaction without reverse transcriptase was performed as a negative control to exclude amplification from contaminating DNA. One hundred nanograms of genomic DNA extracted from the M. catarrhalis 035E strain was used in PCR as a positive control.

Overlap extension PCR.Overlap extension PCR is a PCR-based strategy to create chimeric genes by fusion of independent fragments in the absence of restriction sites (50). We utilized overlap extension PCR to generate the linear double-stranded DNA for oppA mutant construction via homologous recombination. The transforming DNA for an oppA mutant was composed of three fragments: F1/oppAUSG (oppA upstream gene sequence), F2/aphA-3 gene (kanamycin resistance gene), and F3/oppADSG (oppA downstream gene sequence). The locations of these three fragments in relation to the oppA gene are depicted in Fig. 1A. Primers (P9 through P14) (Table 1) for each fragment were designed to include a 10-nucleotide (nt) 5′ extension with sequence complementary to the end of the adjacent fragment. The resulting F1, F2, and F3 fragments would have 20-nt complementary overlapping regions at adjacent ends, which are necessary for the fusion. The first-step PCR was carried out to amplify the three fragments individually with a high-fidelity DNA polymerase, Pfu (Stratagene, Cedar Creek, TX). Genomic DNA extracted from M. catarrhalis strain 035E was used as template for F1 and F3 PCR. Plasmid pUC18K containing the nonpolar kanamycin resistance cassette (29) was used as template for F2 PCR. F1, F2, and F3 were then purified by the Qiaquick PCR purification kit (Qiagen, Valencia, CA). Ten nanomoles of each purified fragment was mixed in the absence of additional primers in a second-step PCR with Pfu. The PCR program consisted of 10 repetitive cycles with a denaturing step at 94°C for 30 s, an annealing step at 50°C for 1 min, and an elongation step at 72°C for 5 min. The fusion product was subsequently amplified by Pfu in the third PCR with the forward primer P9 and the reverse primer P14 (Table 1). This amplicon consisted of 816 bp of upstream sequence of oppA and 930 bp of downstream sequence of oppA flanking the kanamycin cassette. The amplicon was purified from a 1% agarose gel with the Qiaquick gel extraction kit (Qiagen, Valencia, CA) and sent for sequencing to the RPCI DNA sequencing facility.

• Open in new tab
• Download powerpoint

FIG. 1.

Characterization of the oppA gene. (A) Schematic illustration of oppA with the other oligopeptide transporter genes in the genomes of M. catarrhalis ATCC 43617 and S. pyogenes. Numbers indicate the initial and terminal nucleotide positions of each gene within the GenBank sequence. (B) Ethidium bromide-stained agarose gel showing amplicons of the oppA gene amplified from 20 clinical isolates of M. catarrhalis and the ATCC 43617 strain. Lanes 1 and 23, DNA standards; lanes 2 to 11, amplicons from middle ear fluid isolates 135, 238, 555, 2901, 3584, 3614, 5488, 6952, 8184, and 9483, respectively; lane 12, amplicon from ATCC 43617; lanes 13 to 22, amplicons from sputum isolates 5P34B1, 10P58B2, 12P15B1, 14P25B1, 29P24B1, 33P25B2, 39P29B2, 55P18B3, 63P62B1, and 96P9B1, respectively. The sizes of the DNA standards (in kilobases) are indicated on the right. (C) Results of reverse transcriptase PCR (RT-PCR) with RNA extracted from M. catarrhalis 035E to detect oppA transcript. Lane 1, DNA standards; lane 2, RT-PCR product; lane 3, RT-PCR in the absence of reverse transcriptase; lane 4, PCR product from genomic DNA template. DNA standards are noted in base pairs on the left.

Mutant construction.The isogenic oppA mutant strain was constructed by transformation of M. catarrhalis strain 035E (28) with the overlap extension PCR product. Briefly, strain 035E was grown to an optical density at 600 nm (OD₆₀₀) of 0.2. A volume of 100 μl of the bacterial culture was then spread on a BHI agar plate and air dried. Two circles (2-cm diameter) were marked on the plate. Either 30 μl of distilled water or 30 μl of water containing 100 ng of DNA was dropped onto these circles, followed by incubation at 37°C with 5% CO₂ for 5 h. Bacteria within each circle were harvested with a cotton swab and spread on BHI plates containing 50 μg/ml kanamycin. After 24 h of incubation at 37°C with 5% CO₂, numerous colonies were present on the selection plate following DNA transformation but no visible colony was present on the water control plate. The mutant colonies were examined by PCR and sequencing as described in Results.

Cloning of the oppA gene.The pCATCH plasmid is an expression vector engineered to express recombinant lipoprotein and has been used previously to express M. catarrhalis lipoproteins (3, 11). The 1,967-bp oppA gene encoding the mature OppA protein was amplified from M. catarrhalis strain ATCC 43617 with primers P7 and P8 (Table 1) and ligated into pCATCH between restriction sites NcoI and BamHI. The ligation mixture was transformed into chemically competent E. coli strain Top10 and grown on a kanamycin (50-μg/ml) selection plate. The OppA expression plasmid was named pCATCH/OppA.

Southern blot assay.Southern blot assays were performed with genomic DNA that was restricted with PvuII and NdeI using a Hoefer TransVac vacuum blotting unit according to the manufacturer's instructions (Hoefer, San Francisco, CA). Probes were biotinylated with an NEBlot Phototope-Star detection kit (New England Biolabs) according to the manufacturer's instructions.

Expression and purification of His-OppA protein.pCATCH/OppA was transformed into E. coli strain BL21(DE3) to express OppA as a lipoprotein with a thrombin-cleavable C-terminal hexahistidine tag. A volume of 500 ml LB broth containing 50 μg/ml kanamycin was inoculated with 20 ml overnight culture of bacteria harboring the expression vector. Following growth to an OD₆₀₀ of 0.6, OppA expression was induced with 3 mM IPTG (isopropyl-β-d-thiogalactopyranoside) for 4 h at 37°C. The bacteria were then harvested by centrifugation at 4,000 × g for 15 min at 4°C. The pellet was suspended in 10 ml of lysis buffer (20 mM sodium phosphate, 500 mM NaCl, 1 mg/ml lysozyme, 100 μg/ml Pefabloc [Roche, Indianapolis, IN], pH 7.4) and mixed with a nutator for 30 min at 4°C. The suspension was then sonicated with a Branson Sonifier 450 at setting 6, using an 80% pulsed cycle of 4 30-s bursts with 2-min pauses. The sonicated bacterial lysate was centrifuged at 10,000 × g for 20 min at 4°C. The supernatant containing His-OppA protein was saved for protein purification.

His-OppA was purified with Talon metal affinity resin (BD Biosciences, Palo Alto, CA) according to the manufacturer's instructions. Two milliliters of a 50% suspension of the Talon resin was loaded onto a 20-ml chromatography column. The resin storage buffer was drained, and the remaining 1-ml beads were equilibrated with 2 × 10 ml binding buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.4). After equilibration, the Talon beads were incubated with 10 ml of bacterial supernatant in the column with both ends capped for 20 min at room temperature with rocking. The unbound protein was drained, and the bound protein was washed two times with 20 ml wash buffer (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, 10 mM beta-mercaptoethanol, pH 7.4) and eluted with 5 ml elution buffer (20 mM sodium phosphate, 500 mM NaCl, 150 mM imidazole, pH 7.4) by gravity flow. The eluates were collected every 500 μl in each tube. Tubes containing concentrated protein fractions were pooled and subjected to buffer exchange into phosphate-buffered saline (PBS) by using a Centricon YM-10 filter device (Millipore Corporation, Danvers, MA). The concentration of the purified protein was determined by bicinchoninic acid assay (Pierce, Rockford, IL). The quality of the purified protein was examined by SDS-PAGE and Coomassie blue staining.

Development of antisera to OppA and whole bacteria.Purified recombinant His-OppA protein was sent to Covance (Denver, PA) for antibody production in New Zealand White rabbits using a 118-day protocol. Briefly, 250 μg purified OppA was emulsified 1:1 in complete Freund's adjuvant for initial subcutaneous injection. Subsequent immunizations followed a 3-week cycle of boosts with 125 μg OppA emulsified 1:1 in incomplete Freund's adjuvant. Test bleeds were taken approximately 10 days after the boosts. Final bleeds were taken 2 weeks after the 5th boosts.

Rabbit anti-035E antisera were raised by Covance according to a protocol described previously (18). New Zealand White rabbits were injected subcutaneously and intramuscularly twice with a 4-week interval with 10⁹ CFU of M. catarrhalis 035E emulsified with incomplete Freund's adjuvant (1:1). Blood samples were collected 2 weeks after the final injection.

Rabbit antiserum to OMP CD was produced in our laboratory according to a protocol described by Adlowitz et al. (2).

SDS-PAGE and immunoblot assay.Whole-cell extracts of M. catarrhalis clinical strains, 035E, and the 035E/oppA mutant were prepared by suspending a loop of bacterial colonies grown on the BHI agar plates in 100 μl PBS. The bacterial suspension was then mixed with 100 μl 2× SDS-PAGE sample buffer (Laemmli buffer) and boiled for 10 min. Twenty microliters of each whole-cell extract was separated on a 10% SDS-PAGE gel and transferred to nitrocellulose membrane.

For the immunoblot assay with the rabbit anti-035E antiserum, the membrane was incubated with a 1:5,000 dilution of the primary antibody in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5) followed by a 1:2,000 dilution of the peroxidase-labeled goat anti-rabbit IgG antibody (KPL, Gaithersburg, MD) in TBST as secondary antibody. Bands were detected by using the SuperSignal West Pico chemiluminescence kit (Thermo Fisher Scientific Inc., Rockford, IL). The image was acquired with an AlphaImager (Alpha Innotech Corporation, San Leandro, CA).

For immunoblotting with the rabbit anti-OppA or anti-CD antibody, the primary antibodies used were a 1:2,000 dilution of test bleed anti-OppA antiserum, a 1:15,000 dilution of final-bleed anti-OppA antiserum, and a 1:2,000 dilution of anti-CD antiserum. A 1:2,000 dilution of the peroxidase-labeled goat anti-rabbit IgG antibody was the secondary antibody followed by colorimetric detection.

For immunoblotting with the mouse antisera pooled following mucosal immunization with recombinant OppA protein, M. catarrhalis 035E, or PBS control, the membranes were incubated with a 1:10,000 dilution of the antisera, probed with a 1:25,000 dilution of peroxidase-conjugated anti-mouse IgG, and developed with chemiluminescence.

Serum from adults with COPD.Serum samples were obtained from adults enrolled in our COPD Study Clinic at the Buffalo Veterans Affairs Medical Center (33, 48). Patients were seen monthly and at times when an exacerbation was suspected. At each visit, serum and expectorated sputum samples were collected and bacteria present in the sputum were identified. Serum samples collected ∼1 month before (preacquisition) and ∼1 month after (postclearance) acquisition and clearance of M. catarrhalis were subjected to whole-cell enzyme-linked immunosorbent assays (ELISAs) to analyze the human systemic immune response to M. catarrhalis infection (33). Nineteen pairs of serum samples with a positive response to homologous infecting strains were used in ELISAs with purified recombinant OppA protein in this study to examine the OppA-specific antibody response. Eight pairs of serum samples collected 2 months apart from patients who never had positive sputum culture for M. catarrhalis during the study were used as negative controls.

ELISA.ELISA was carried out by coating the wells of a 96-well microtiter Immunolon 4 plate (Thermo Labsystems, Franklin, MA) with 500 ng of purified recombinant His-OppA protein overnight in coating buffer (0.1 M Na₂CO₃ and 0.1 M NaHCO₃, pH 9.6). Equal volumes of coating buffer were added to control wells for each well that received OppA. The plate was washed once with PBST (0.5% Tween 20 in PBS buffer) and blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature, after which the plate was washed once again with PBST. Paired COPD patient sera (preacquisition and postclearance) described above were diluted 1:2,000 in diluent buffer (1% BSA in PBST) and added to the sham-coated control wells and His-OppA-coated sample wells in parallel. After incubation for 1 h at room temperature, the plate was washed 4 times with PBST and a 1:6,000 dilution of peroxidase-labeled secondary antibody, goat anti-human IgG (KPL, Gaithersburg, MD), was added. After another 1 h of incubation, the plate was washed 5 times with PBST and developing reagent was added to the wells. The reaction was allowed to proceed in the dark for 10 min and stopped with 2 M sulfuric acid. The absorbance at 450 nm was determined using a Bio-Rad model 3550-UV microplate reader.

The OD value of the sham-coated control wells was subtracted from the OD value of each corresponding sample well to give a normalized OD of each sample. The percent change in the normalized OD between paired samples was calculated with the following formula: [(OD of postclearance sample − OD of preacquisition sample)/OD of preacquisition sample] × 100. ELISAs were repeated in four independent experiments. The percent change in OD of each paired sample was determined as the average of the values derived from the four experiments. The mean and standard deviation (SD) of the percent change of control sera were calculated. The cutoff for a significant percent change of the sample sera was set as (mean + 2 SD) of percent change of controls as previously described (3, 5).

Whole-cell ELISA.M. catarrhalis strain 035E and the 035E/oppA mutant strain were grown in BHI broth to an OD₆₀₀ of 0.2, harvested by centrifugation, and resuspended in PBS. A volume of 100 μl of the suspension was added to each well of the 96-well microtiter Immunolon 4 plate (Thermo Labsystems, Franklin, MA). Wells with PBS alone were included as controls. The rest of the experiment was performed according to the protocol described above for ELISA with OppA. Primary antibodies, either rabbit anti-OppA antibody, rabbit anti-CD antibody (positive control), or rabbit anti-protein 140 (protein 140 is a non-surface-exposed protein) (negative control), diluted in 2-fold serial dilutions from 1:10,000 to 1:40,000, were added to the wells. The secondary antibody was a 1:4,000 dilution of peroxidase-labeled goat anti-rabbit IgG (KPL, Gaithersburg, MD).

Flow cytometry.M. catarrhalis was grown to mid-logarithmic phase, and 100 μl of culture was centrifuged at 4,000 × g for 5 min. The bacterial pellet was resuspended in an appropriate dilution of heat-inactivated (56°C for 30 min) rabbit serum in PBS. Appropriate dilutions were determined by performing initial experiments at multiple 2-fold dilutions to identify the dilution of serum that yielded the greatest signal-to-noise ratio. Two negative controls were included: (i) bacteria incubated in PBS alone and (ii) bacteria incubated with rabbit antiserum directed at a nonsurface protein, protein 140. Bacteria incubated with polyclonal antiserum from rabbits immunized with whole bacterial cells were used as a positive control. Bacteria were incubated with antisera for 1 h at 37°C. The bacteria were washed with PBS and resuspended in a 1:10 dilution of antibody conjugated to fluorescein isothiocyanate (KPL). After 30 min of incubation at 37°C, the sample was diluted with 900 μl PBS and fluorescence was detected with a FACSCalibur flow cytometer using Cell Quest 3.1 software (BD Biosciences, San Jose, CA).

Mucosal immunization of mice.Groups of six BALB/c mice were immunized intranasally with either 50 μg purified recombinant OppA plus 1 μg cholera toxin, formalin-killed M. catarrhalis 035E plus 1 μg cholera toxin, or 1 μg cholera toxin alone in PBS buffer as a sham immunization control. Intranasal immunization was performed on days 0, 10, and 20 by placing a pipette tip with 5 μl immunogen at each nostril of a conscious mouse for it to sniff. Immunized mice were challenged on day 28 as described below.

Mouse pulmonary clearance model.The mouse pulmonary clearance model was used as described previously (43). An overnight culture of M. catarrhalis 035E was inoculated into 100 ml BHI broth with an OD₆₀₀ of 0.05 and grown to an OD₆₀₀ of 0.3. Bacteria were collected by centrifugation and resuspended in 10 ml PCGM buffer (4.3 mM NaHPO₄, 1.4 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, 5 mM CaCl₂, 0.5 mM MgCl₂, 0.1% gelatin, pH 7.3). An aliquot of suspension was diluted and plated to determine the starting concentration of bacteria, which was approximately 1 × 10⁹ CFU per ml in each experiment. Ten milliliters of the bacterial suspension was placed in the nebulizer of a Glas-Col inhalational exposure system, model 099C A4212 (Glas-Col, Terre Haute, IN). Immunized mice were challenged using this inhalation system with the following settings: 10 min of preheating, 40 min of nebulization, 30 min of cloud decay, 10 min of decontamination, vacuum flow meter at 60 cubic feet/h, and compressed airflow meter at 10 cubic feet/h.

Three hours postchallenge, the mice were anesthetized by inhalation of isoflurane and bled by retro-orbital puncture. Serum was heat inactivated at 56°C for 30 min and stored at −20°C for later use. After serum was collected, the anesthetized mouse was euthanized by administration of additional isoflurane. Lungs were then harvested and homogenized on ice in 5 ml PCGM buffer using a tissue homogenizer. Aliquots (20 μl) of undiluted and 1:10 diluted lung homogenate were plated in duplicate and incubated at 35°C with 5% CO₂ overnight. Colonies were counted the following day. The statistical significance of colony counts between groups of immunized and sham-immunized mice was determined by two-tailed t tests. A P value of ≤0.05 was considered significant.