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Research Article
7 October 2014

Role of the Oligopeptide Permease ABC Transporter of Moraxella catarrhalis in Nutrient Acquisition and Persistence in the Respiratory Tract


Moraxella catarrhalis is a strict human pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, resulting in significant worldwide morbidity and mortality. M. catarrhalis has a growth requirement for arginine; thus, acquiring arginine is important for fitness and survival. M. catarrhalis has a putative oligopeptide permease ABC transport operon (opp) consisting of five genes (oppB, oppC, oppD, oppF, and oppA), encoding two permeases, two ATPases, and a substrate binding protein. Thermal shift assays showed that the purified recombinant substrate binding protein OppA binds to peptides 3 to 16 amino acid residues in length regardless of the amino acid composition. A mutant in which the oppBCDFA gene cluster is knocked out showed impaired growth in minimal medium where the only source of arginine came from a peptide 5 to 10 amino acid residues in length. Whether methylated arginine supports growth of M. catarrhalis is important in understanding fitness in the respiratory tract because methylated arginine is abundant in host tissues. No growth of wild-type M. catarrhalis was observed in minimal medium in which arginine was present only in methylated form, indicating that the bacterium requires l-arginine. An oppA knockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the Opp system mediates both uptake of peptides and fitness in the respiratory tract.


Moraxella catarrhalis, a Gram-negative diplococcus, is a commensal of the upper respiratory tract, as well as a human-specific pathogen responsible for 10 to 20% of otitis media in children and approximately 10% of exacerbations of chronic obstructive pulmonary disease (COPD) in adults in the United States (16). Otitis media is the most common reason for pediatric office visits and the prescribing of antibiotics to children, typically by the age of 3 years (712). COPD is the third leading cause of death in the United States, affecting at least 24 million people, and costs $50 billion in health care expenses each year (1316). COPD is considered a major unmet medical need that is increasing in prevalence throughout the world (17, 18). Antibiotic resistance is an additional concern, with nearly 100% of clinical isolates of M. catarrhalis displaying resistance to β-lactam drugs with the ability to protect other β-lactam-sensitive bacteria in the surrounding environment (3, 9, 1923).
In the last 2 decades, M. catarrhalis has transitioned from being considered an emerging to an established pathogen. Since this bacterium was previously considered to be a commensal, the literature on mechanisms that allow M. catarrhalis to thrive in the harsh environment of the human respiratory tract is sparse, and the associated virulence mechanisms are not well studied (1, 2, 2429). There is a need to understand bacterial physiology and metabolism during infection as these pathways and traits could be exploited for development of new treatments (30). For example, M. catarrhalis is an arginine auxotroph, yet it is not known whether the bacterium can utilize methylated arginine, the predominant form of arginine available in the airways (3136). The annotated oligopeptide permease (opp) gene cluster in the M. catarrhalis genome has homology with the peptide import ATP-binding cassette (ABC) transport systems composed of permease, ATPase, and substrate binding proteins (37, 38). ABC transporters are potential targets for development of antimicrobial agents and vaccines (3942). Little is known about the role and function of ABC transport systems in M. catarrhalis, including the Opp system.
The goal of this study was to characterize the function of the Opp system in M. catarrhalis and its impact as a nutritional virulence factor both in vitro and in vivo. Understanding the mechanism of such systems will provide further insight into mechanisms by which M. catarrhalis survives in the harsh environment of the respiratory tract.


Bacterial strains, plasmids, and growth.

M. catarrhalis strain 035E, provided by Eric Hansen, is a prototype otitis media strain that was isolated from the middle ear fluid of a child with otitis media in Dallas, TX. The plasmid pUCK18K (43) was a gift from Anthony Campagnari. Plasmid pWW115 was a gift of Wei Wang and Eric Hansen (44). Bacteria were grown on brain heart infusion (BHI) agar plates overnight at 35°C with 5% CO2 or in BHI broth at 37°C with shaking at 225 rpm unless otherwise specified. Chemically defined medium (CDM) is a minimal nutrient medium containing the essential elements M. catarrhalis requires to survive (36, 45).

Construction of mutants and complemented oppA strain.

Construction of the oppA mutant strain was previously described (41). A second mutant strain was constructed in which the entire oligopeptide permease gene cluster was knocked out using overlap extension PCR and homologous recombination as previously described (41, 46) (Fig. 1). Briefly, the transforming DNA for the oppBCDFA mutant was composed of three overlapping fragments that included 1 kb upstream of oppB (fragment 1), a nonpolar kanamycin resistance cassette amplified from plasmid pUCK18K (fragment 2) (43), and 1 kb downstream of oppA (fragment 3) using the oligonucleotide primers listed in Table 1. The insert and surrounding sequences of the mutant were confirmed by sequence analysis.
FIG 1 Diagram of the opp gene cluster in the wild-type strain and the deleted segments in the oppA and oppBCDFA knockout mutants (not to scale). Black arrows denote open reading frames; gray arrows indicate kanamycin resistance cassette.
TABLE 1 Oligonucleotide primer sequences
F, forward; R, reverse. Bold text indicates restriction enzyme sites.
The oppA mutant strain was complemented using the pWW115 plasmid as previously described (44). Briefly, plasmid pWW115 was purified from an overnight culture in BHI broth of 035E-pWW115 supplemented with 100 μg/ml spectinomycin using a QIAprep Spin Miniprep kit (Qiagen). A 2,400-bp fragment (139spec) spanning from 250 bp upstream of the oppA start site to 118 bp downstream of the stop site of oppA was amplified by PCR using primers that included a BamHI site and a SacI site (Table 1). The 139spec fragment and pWW115 were digested with BamHI and SacI (Invitrogen). Ligation was performed using T4 DNA ligase and 30 ng of pWW115 and 20 ng of 139spec. The oppA mutant strain was transformed with the ligation mixture by spotting 2.5 μl of the ligation mixture onto a BHI agar plate inoculated with 100 μl of 035E at an OD600 of 0.2 and incubated for 5 h at 37°C. Spots were then spread onto BHI agar plates that contained 100 μg of spectinomycin and incubated overnight. Resulting transformed colonies (complemented oppA strain, designated oppA C′) were confirmed with PCR and immunoblotting (Fig. 2L).
FIG 2 Characterization of purified proteins, antisera, and mutants. (A) Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel of the N terminus of the purified recombinant protein OppB. (B) Immunoblot assay of recombinant protein OppB probed with immunopurified rabbit antiserum raised to a purified recombinant amino-terminal fragment of protein OppB (1:100,000). (C and D) Immunoblot assay of whole bacterial lysates of the wild-type 035E strain (C) and the oppBCDFA knockout mutant (D) probed with OppB antiserum (1:10,000). The asterisk indicates OppB. A cross-reactive band of ∼34 kDa is seen in both lysates. (E) Coomassie blue-stained SDS-PAGE gel of purified recombinant protein OppF. (F) Immunoblot assay of purified recombinant protein OppF probed with adsorbed rabbit antiserum raised to purified recombinant protein OppF (1:10,000). (G and H) Immunoblot assay of whole bacterial lysates of the wild-type 035E strain (G) and the oppBCDFA knockout mutant (H) probed with OppF antiserum (1:10,000). (I to L) Immunoblot assay of whole bacterial lysates of the wild-type 035E strain (I), the oppBCDFA knockout mutant (J), the oppA knockout mutant (K), and the oppA complemented strain (L) probed with OppA antiserum (1:100,000). Molecular mass makers are noted on the left in kilodaltons (kDa).

Cloning of the N-terminal region of the oppB gene and of the oppF gene.

A 678-bp fragment of the N-terminal region of the gene encoding the mature OppB protein and the 969-bp region of the gene encoding the entire OppF protein were amplified by PCR from 035E genomic DNA using the primers noted in Table 1 and ligated into plasmid pET 100 D-TOPO (Invitrogen). The ligation mixtures were transformed into the chemically competent Escherichia coli strain Top10 and grown on Luria broth (LB) plates containing 100 μg/ml carbenicillin. The expression plasmids were named pNOppB and pOppF, respectively.

Expression and purification of recombinant proteins.

Plasmids pNOppB and pOppF were transformed into E. coli strain BL21(DE3) (Invitrogen) to express N-terminal OppB and OppF with a six-histidine amino-terminal tag. A volume of 100 ml of LB broth containing 300 μg/ml carbenicillin was inoculated with 5 ml of an overnight culture of bacteria containing the expression vector. Following growth to an optical density of 0.7 at 600 nm (OD600), protein expression was induced with 4 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 4 h at 37°C. Bacteria were harvested by centrifugation at 4,000 × g for 15 min at 4°C. Pellets were resuspended in 10 ml of lysis buffer (20 mM sodium phosphate, 500 mM NaCl, 1 mg/ml lysozyme, 1× Protease Arrest [G Biosciences], pH 7.4) and mixed by nutation for 30 min at room temperature. The suspensions were then sonicated with a Branson Sonifier 450 at setting 5, using an 80% pulsed cycle of four 30-s bursts with 2-min pauses. The sonicated bacterial lysates were centrifuged at 10,000 × g for 20 min at 4°C. The pellet containing the N-terminal region of OppB was suspended in a guanidinium-binding buffer (30 mM NaH2PO4, 150 mM NaCl, 6 M guanidinium chloride, pH 7.4). The pellet containing OppF was suspended in a urea-binding buffer (30 mM NaH2PO4, 150 mM NaCl, 6 M urea, pH 7.4).
Recombinant proteins were purified with Talon metal affinity resin (BD Biosciences, Palo Alto, CA) according to the manufacturer's instructions. Briefly, 2 ml of a 50% Talon resin suspension was centrifuged at 2,400 × g for 5 min, and the storage buffer was removed. The beads were equilibrated with binding buffer, guanidine or urea, and incubated with the bacterial lysate suspension for 20 min at room temperature with nutation. The resin with bound protein was centrifuged as described above and washed two times with 2 volumes of binding buffer. Proteins were eluted by incubating the resin with 1.5 ml of elution buffer (150 mM imidazole, 50 mM NaH2PO4, 270 mM NaCl, and 6 M guanidine chloride [N-terminal OppB] or 6 M urea [OppF], pH 7.4) for 10 min at room temperature with nutation, followed by centrifugation. The elution step was repeated, and the concentration of the pooled eluates was determined using the Lowry assay (Sigma). Recombinant OppA was purified as previously described (41).

Development of antisera to recombinant proteins.

Purified recombinant N-terminal OppB and full-length OppF were sent to Covance (Denver, PA) for antibody production in New Zealand White rabbits using a 77-day protocol (OppB) and a 59-day protocol (OppF). Briefly, 250 μg of purified protein was emulsified 1:1 in complete Freund's adjuvant for initial subcutaneous immunization. Subsequent immunization followed a 3-week cycle of boosts with 125 μg of protein emulsified 1:1 in incomplete Freund's adjuvant. Serum was collected 2 weeks after the second (day 59) or third (day 77) boost.
To remove background antibodies, the OppF antiserum was adsorbed with the oppBCDFA mutant as follows. A 50-ml late-logarithmic-phase culture of the oppBCDFA mutant was harvested by centrifugation, washed in phosphate-buffered saline (PBS), and suspended in a 1-ml 1:100 dilution of serum. The suspension was incubated at 4°C for 30 min, and bacterial cells were removed by centrifugation at 4,000 × g for 15 min at 4°C. The adsorbed serum was then filter sterilized using a 0.45-μm-pore-size filter. Background antibodies in the OppB antiserum were removed by elution from a Sepharose column. As per instructions, CNBr-Sepharose 4B beads (1 g) (GE Healthcare Bio-Sciences, Pittsburgh, PA) were swelled with 1 mM HCl (50 m) and washed five times with 1 mM HCl (30 ml). Beads were coupled with the recombinant N-terminal OppB protein (1 mg/ml) in a tube previously difiltrated with five volumes (5 ml) of coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, 0.05% Zwittergent, pH 8.3) and incubated at 4°C overnight. Beads were blocked with 0.2 M glycine (10 ml) for 2 h at room temperature with nutation and then washed five times alternately with 30 ml of high- and low-pH buffers (coupling buffer and 0.1 M sodium acetate [NaOAc], 0.5 M NaCl, pH 4). Finally, beads were washed with 100 ml of coupling buffer with no Zwittergent, followed by 30 ml of PBS, pH 7.8, and stored at 4°C. To elute OppB-specific antibodies, columns were washed with 100 ml of PBS, followed by incubation with a 5-ml 1:5 dilution of the antiserum in PBS for 30 min at room temperature with nutation. The column was washed with 150 ml of PBS, pH 5. Antibodies were eluted with 3 ml of 0.1 M glycine, pH 2.5, followed by 10 ml of PBS, pH 4. The eluted antibodies were buffered with 1 ml of 1 M Tris, pH 8, and concentrated to ∼ 2 ml using Amicon-Ultra 4 tubes (Millipore, Billerica, MA).

Thermal shift assay.

Thermal shift assays were performed using a Stratagene Mx3005P real-time PCR instrument (Stratagene, La Jolla, CA) as previously described (47). Purified OppA in buffer was studied at a concentration of 5 μg in a 30-μl volume to which select amino acids or peptides were added to a final concentration of 1 mM. SYPRO orange was added as a fluorescence reporter at a 1,000-fold dilution from its stock solution. The change in fluorescence was monitored using a Cy3 filter, with excitation and emission wavelengths of 545 nM and 568 nM, respectively. The temperature was raised from 25°C to 98°C in 0.5°C intervals for 45 min, with fluorescence readings taken at each interval. The fluorescence data were plotted and normalized. The first derivative of the curve was calculated to provide the melting temperatures (Tms) using the software program GraphPad Prism, version 5.0, as previously described (47, 48). Results were reported as the averages with standard deviations of at least three values, which were obtained from three independent experiments performed in duplicate. A temperature shift of >2°C is considered significant in this assay (47, 49). Statistical significance was further determined by performing a two-tailed t test. A P value of ≤0.05 was considered significant. Protein stability was assessed by SDS-PAGE. A second independent protein preparation yielded similar results.

Assessment of bacterial growth.

Growth curves were performed using a Bioscreen C automated growth curve analysis system (Oy Growth Curves AB Ltd., Helsinki, Finland). M. catarrhalis strains were grown in BHI broth overnight with shaking at 37°C and 225 rpm. Growth curves were performed with a 200-μl inoculum of the overnight cultures diluted 1:1,000 in BHI broth or 1:250 in CDM. In some experiments, overnight cultures were used to inoculate new cultures, which were then grown to an OD600 of 0.5 (mid-log phase) and then diluted 1:250 in CDM. Each growth condition was performed as five replicate wells in each experiment, with optical density measurements taken at 600 nm in 1-h intervals at 37°C with constant shaking (machine settings: fast speed, high amplitude). All peptides were added to CDM to a final concentration of 0.25 mg/ml. Each experiment was repeated at least twice. Visualization was aided by Daniel's XL Toolbox add-in for Excel, version XYZ, by Daniel Kraus, Wurzburg, Germany.

Pulmonary clearance model.

The mouse pulmonary clearance model has been described previously (50, 51). All procedures were approved by the University at Buffalo Institutional Animal Care and Use Committee. BALB/c mice were challenged simultaneously with the wild type and the oppA mutant, and clearance of the strains was assessed. Briefly, overnight cultures of M. catarrhalis 035E wild type and the oppA mutant were used to inoculate 50 ml of BHI broth cultures, which were then grown to log phase (OD600 of ∼0.3 to 0.4 or ∼108 CFU/ml). Bacteria were harvested by centrifugation, and each sample was resuspended in 5 ml of PBS with gelatin, calcium, and magnesium (PBSG) (137 mM NaCl, 2.7 mM KCl, 4.3 mM NaHPO4, 1.4 mM KH2PO4, 0.125 mM CaCl2, 0.5 mM MgCl2, and 0.1% gelatin, pH 7.3). Aliquots of the suspensions were diluted and plated to confirm the starting number of bacteria (∼109 CFU). A volume of 5 ml of each culture suspension (total, ∼109 CFU) was placed in the nebulizer of an inhalation exposure system (model 099C A4212; Glas-Col, Terre Haute, IN). The equipment settings were as follows: 10 min of preheating, 40 min of nebulization, 30 min of cloud decay, 10 min of decontamination, vacuum flow meter at 60 ft3/h, and compressed airflow meter at 10 ft3/h. BALB/c mice (n = 10 per group) were placed in the chamber during this time.
At 3 h postchallenge, the mice were euthanized by inhalation of isoflurane. Lungs were harvested and homogenized on ice in 5 ml of PBSG using a tissue homogenizer. Aliquots (50 μl) of each lung homogenate were plated on chocolate agar, and a second aliquot was plated on chocolate agar containing 15 μg/ml of ribostamycin and incubated at 35°C with 5% CO2 overnight; each experiment was performed in duplicate. Colonies were counted the following day to determine the concentration of bacteria in the lungs at 3 h after aerosol challenge. The number of colonies on the ribostamycin plates was used to calculate the concentration of the oppA mutant. The number of colonies on the ribostamycin plate was subtracted from the number of colonies on plates with no antibiotic to calculate the concentration of wild-type bacteria in lungs. Statistical significance was determined by performing a two-tailed t test. A P value of ≤0.05 was considered significant. This experiment was repeated with similar results.


The oligopeptide permease (opp) gene cluster.

A genome-mining approach has previously identified oppA of the oligopeptide permease (opp) gene cluster as a conserved gene in M. catarrhalis that was characterized as a mucosal vaccine antigen (41, 52). As previously described, the opp gene cluster contains five genes (Fig. 1) with greatest similarity (68.5% to 80%) to the opp genes of Streptococcus pyogenes (41). Genes oppB and oppC have been annotated as encoding permease proteins, oppD and oppF have been annotated as encoding ATPase proteins, and oppA has been annotated as the substrate binding protein in M. catarrhalis located at position 1298180 to 1303459 on the BBH18 strain in GenBank (accession number NC_014147.1) (38). PCR was used to assess 20 strains of M. catarrhalis isolated from patients with otitis media or COPD from different geographic locations for the presence of oppB, oppC, oppD, and oppF. A PCR product of identical size was amplified from each strain, indicating that these genes are present in all strains tested (data not shown).

Characterization of recombinant proteins and antisera.

Antiserum to recombinant OppA has been previously described (41). We developed antiserum to an additional two recombinant proteins, one permease and one ATPase protein, OppB and OppF, respectively. The oppB gene encodes a predicted mature protein of 477 amino acids (aa) with a predicted molecular mass of 53.6 kDa. We were unable to express the full-length mature OppB protein, consistent with expression of a permease being toxic for E. coli. Instead, we expressed and purified a truncated 226-amino-acid version of the N-terminal region of this protein from residues 2 through 227, producing a 26-kDa protein with a six-histidine tag on the N terminus. The full-length mature OppF protein consisting of 322 amino acids with a predicted molecular mass of 36.3 kDa was expressed and purified with an N terminus histidine tag. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, subjected to Coomassie blue staining, showed the purified recombinant N-terminal OppB and full-length OppF proteins as single bands of ∼29 kDa and ∼39 kDa, respectively (Fig. 2A and E).
Immunoblot assays with the adsorbed antisera detected bands corresponding to the purified recombinant proteins (Fig. 2B and F), as well the wild-type proteins of OppB at ∼53 kDa and OppF at ∼36 kDa in a whole-cell lysate of the wild-type strain (Fig. 2C and G). These bands were absent from a whole-cell lysate of the oppBCDFA knockout mutant (Fig. 2D and H), confirming that the antisera recognize epitopes on the wild-type OppB and OppF proteins. The antiserum raised to OppB is cross-reactive with another protein approximately 34 kDa in size (Fig. 2C). The size difference between recombinant OppF and wild-type OppF can be attributed to the addition of the histidine tag and associated sequences on the amino terminus of the recombinant protein. Wild-type OppB (Fig. 2C) is significantly larger than the recombinant protein (Fig. 2B) because the antiserum was produced to a recombinant fragment of the N-terminal half of the protein. Antiserum to recombinant OppA recognizes OppA in the wild-type and complemented (oppA C′) strains (Fig. 2I and L); OppA is absent from the oppBCDFA and oppA mutant strains as expected (Fig. 2J and K).

Characterization of mutants.

Overlap extension PCR and homologous recombination were used to replace the entire 6.7-kbp opp gene cluster with a kanamycin resistance cassette (Fig. 1). An oppA mutant has been previously described (41). Mutant strains were confirmed with PCR, sequencing, and immunoblot assays (Fig. 2). Rabbit antiserum to the N-terminal recombinant OppB protein (29 kDa) (Fig. 2A) recognized both the recombinant protein (Fig. 2B) and the wild-type protein in a wild-type whole-cell lysate (53 kDa) (Fig. 2C). No band was detected in the oppBCDFA mutant (Fig. 2D). Rabbit antiserum to recombinant OppF (Fig. 2E) recognized both the recombinant protein of 39 kDa (Fig. 2F) and the wild-type protein of 36 kDa in a wild-type whole-cell lysate (Fig. 2G). No band was detected in the oppBCDFA mutant (Fig. 2H). An immunoblot assay with antiserum to OppA confirmed the absence of OppA in whole-cell lysates of the oppA and oppBCDFA mutants (Fig. 2J and K).
Wild-type, oppBCDFA, oppA, and oppA C′ strains showed similar growth rates in nutrient-rich BHI medium and CDM containing arginine although the mutant strains did not achieve the same density as the wild type in BHI medium (Fig. 3).
FIG 3 Growth curves in brain heart infusion (BHI) broth and chemically defined medium supplemented with arginine (R) of the 035E wild-type (WT, solid black line), oppBCDFA knockout (dashed black line), oppA knockout (gray line), and oppA complemented (oppA C′) (dashed gray line) strains. Each point is the average of five wells, and error bars indicate standard deviations.

OppA binds to peptide substrates.

OppA is predicted to bind peptides, but its binding specificity has not been tested directly (38). The substrate binding affinity of OppA to peptides was examined with thermal shift assays (38, 47, 49). The melting temperature of a protein bound to its substrate will be higher than that of the protein alone because the protein-substrate complex is more stable and requires more energy to denature. An unstable or partially folded protein will not yield a consistent, reproducible melt curve. Purified recombinant OppA displayed a melting temperature (Tm) of 50.6 ± 0.7°C (Table 2 and Fig. 4). OppA was incubated with peptide substrates ranging in size from a single arginine molecule to a peptide composed of 16 amino acid (aa) residues, and the shift in melting temperature was recorded. Peptides composed of 3 amino acids up to at least 16 residues in length resulted in a significant upward shift of ≥5.9°C in the Tm of OppA, indicating binding of these peptides to OppA (Table 2). In contrast, peptides of two residues in length or single amino acids did not cause a significant shift in the Tm of OppA, indicating an absence of binding (Table 2).
TABLE 2 Results of thermal shift assays with recombinant purified OppA
Amino acid or peptideNo. of residuesMean Tm ± SD (°C)b
Nonea050.6 ± 0.7
R150.4 ± 0.6
VR252.5 ± 0.3
AR252.6 ± 0.4
FRA356.5 ± 0.1*
ARA357.4 ± 0.2*
FRAP457.2 ± 0.0*
ARAA457.9 ± 0.2*
VANAP559.1 ± 0.3*
VANRP559.4 ± 0.2*
ARAAA558.0 ± 0.2*
RPPGFSPFR957.9 ± 0.1*
ARAAAAARAA1061.9 ± 0.4*
ARAAAARAARAA1262.5 ± 0.3*
ARAAAARAAARAA1363.4 ± 0.7*
None, OppA alone.
*, significant shift in Tm compared to recombinant OppA alone (P ≤ 0.004).
FIG 4 Results of thermal shift assays of purified recombinant OppA alone and with the addition of peptides of 5 aa residues in length as noted. Each curve represents the average of three values. The melting temperature (Tm) is indicated by the arrows approximating the midpoint of the curves, which were determined by analyzing the first derivative of these curves.
Since M. catarrhalis is an arginine auxotroph, we hypothesized that the presence of an arginine molecule in the peptides was required for OppA to bind to the peptide substrate. Two peptides composed of 5 aa residues were tested, where only one of the two peptides contained an arginine molecule. The peptide VANRP shifted the Tm of OppA upward 8.8°C to 59.4 ± 0.2°C (Fig. 4 and Table 2). This was not different from that of the peptide with no arginine, VANAP, which resulted in a Tm of 59.1 ± 0.3°C (Fig. 4 and Table 2). Thus, OppA does not have a specific affinity for peptides containing arginine. This observation was further confirmed by testing additional peptides composed of 3 or 4 aa residues with and without arginine. The addition of each of these peptides resulted in a significantly positive temperature shift compared to OppA alone, whether arginine is present or absent in the peptide observed (Table 2). We conclude that OppA binds peptides of ≥3 aa and up to at least 16 aa, regardless of the presence of arginine in the peptide.

Peptide utilization mediated by the Opp system.

Given that OppA binds a wide range of peptide substrates (Table 2), we performed experiments to assess whether utilization of peptides is mediated by the Opp system. Growth rates of the wild type, oppA and oppBCDFA knockout mutants, and the oppA C′ strains were compared. CDM containing no arginine was used as the base medium for all experiments, and arginine was added to the medium as a free molecule or in the form of a peptide (Table 2). In all growth experiments, the oppA mutant showed the same growth pattern as the oppBCDFA mutant, and the growth of the complemented oppA C′ strain was similar to that of the wild type. For simplicity, only the oppBCDFA mutant is shown unless otherwise noted (Fig. 5). The peptide 5 aa residues in length (VANRP) supported growth of the wild type but not the oppBCDFA or oppA mutant, while the complemented oppA C′ strain restored growth to wild-type levels (Fig. 5). When CDM was supplemented with free arginine or peptides of 4 residues or less containing an arginine molecule, there was no difference in growth between the wild type and oppBCDFA mutant (Fig. 6A; only the 3-residue peptide shown). A 10-aa residue peptide supported growth of the wild type, and after approximately 20 h, the oppBCDFA mutant also began growing (Fig. 6B). A 16-aa residue peptide supported the growth of both the wild-type and mutant strains (Fig. 6C). We conclude that the Opp system mediates import of peptides of ≥5 aa residues and <10 aa residues in length.
FIG 5 Growth curves in chemically defined medium of the 035E wild-type (WT) (solid black line), oppBCDFA knockout (dashed black line), oppA knockout (solid gray line), and oppA complemented (oppA C′) (dashed gray line) strains supplemented with the peptide VASRP as the only source of arginine. Each point is the average of five wells, and error bars indicate standard deviations.
FIG 6 Growth curves in chemically defined medium of the 035E wild-type (WT) and the oppBCDFA knockout strains supplemented with peptides as the only source of arginine as indicated. Each point is the average of five wells, and error bars indicate standard deviations.

Growth with methylated arginine.

Both prokaryotes and eukaryotes utilize protein arginine methylation, and the degradation of these proteins releases methylated arginine molecules (31, 53). An abundance of methylated arginine is present in the respiratory tract, but whether M. catarrhalis will grow with methylated arginine has not been tested previously (26, 29, 31, 53). Arginine has three different methylation states, asymmetric methylation (NMMA), symmetric dimethylation (SDMA), and asymmetric dimethylation (ADMA) (Fig. 7) (26, 31, 53). Inflammation stimulates the production of nitric oxide (NO) from the l-arginine precursor and is inhibited by asymmetrically methylated arginine. Growth of M. catarrhalis in the presence of each variation of methylated arginine was examined. CDM containing the minimal nutritional requirements for M. catarrhalis but lacking arginine was supplemented with arginine (0.8 mM) in its three different methylation states. None of the methylated arginines supported growth of M. catarrhalis, whereas nonmethylated l-arginine supported growth (Fig. 8). We conclude that M. catarrhalis requires l-arginine, which may be a limiting nutrient in the respiratory tract, for growth (31, 33, 54).
FIG 7 Diagram showing the structure of arginine and the three possible methylation sites: asymmetric methylation (NMMA), symmetric dimethylation (SDMA), and asymmetric dimethylation (ADMA).
FIG 8 Growth curve in chemically defined medium of the 035E wild-type strain supplemented with 0.8 mM l-arginine (Arg), monomethylated arginine (NMMA), symmetrically dimethylated arginine (SDMA), and asymmetrically dimethylated arginine (ADMA) as the only sources of arginine. Each point is the average of five wells, and error bars indicate standard deviation,.

Role of oppA gene in the murine respiratory tract.

To assess the role of OppA in fitness and persistence in the respiratory tract in vivo, a competitive challenge was performed with the wild type and the oppA knockout mutant. Groups of BALB/c mice (n = 10) were subjected to aerosol challenge with an equal inoculum of wild-type and the oppA mutant strains simultaneously. An ∼2-fold reduction in the concentration of the oppA mutant in the lungs was observed 3 h after challenge compared to the wild-type strain (P < 0.025, two-tailed t test) (Fig. 9). Thus, the oppA mutant was impaired in its capacity to persist in the murine respiratory tract compared to wild-type M. catarrhalis. We conclude that OppA plays a role in fitness and persistence in the murine respiratory tract.
FIG 9 Results of pulmonary clearance in mice following simultaneous aerosol challenge by equal numbers of M. catarrhalis bacteria of the 035E wild-type (WT) strain and the oppA knockout mutant strain (109 CFU). The numbers of CFU/ml in homogenized lung tissues were determined at 3 h following challenge. Results are the averages of 10 animals per group; error bars show standard deviations. The numbers of CFU in lung tissue of the oppA mutant were significantly lower than wild-type values (Student's t test, P < 0.03).


M. catarrhalis contributes significantly to worldwide morbidity and mortality. Little is known about how this organism survives and causes disease in the human respiratory tract, which is a hostile environment for bacteria (12, 24, 25). l-Arginine, an essential nutrient that M. catarrhalis must acquire to survive, is especially limiting during periods of inflammation that produce nitric oxide, such as an exacerbation of COPD (17, 18, 55). The present study advances our understanding of M. catarrhalis pathogenesis with the following novel observations on nutrient acquisition and survival in the respiratory tract. (i) The substrate binding protein, OppA, binds peptides and is stabilized by a peptide substrate. (ii) The Opp system mediates the utilization of peptides between 5 and 10 amino acid residues in length. (iii) M. catarrhalis requires l-arginine and cannot utilize methylated arginine, which is found in abundance in the respiratory tract. (iv) The oppA mutant strain is cleared more rapidly than the wild-type strain in a mouse pulmonary challenge model, indicating a role for OppA in persistence in the respiratory tract.
ABC transporters are composed of permease proteins, ATPase proteins, and substrate binding proteins and play essential roles in fitness and survival, making them attractive targets for development of new therapies and vaccines (3740, 56, 57). Similar to other bacteria, the M. catarrhalis opp gene cluster has two putative permease genes (oppB and oppC), two putative ATPases (oppC and oppD), and the oppA gene encoding the substrate binding protein. OppA is a promising vaccine antigen candidate, but the function of the Opp system in M. catarrhalis has not been investigated (41). We show through a thermal shift assay that OppA functions as a substrate binding protein with peptide substrates ranging in size from 3 aa to a minimum of 16 aa residues in length (Fig. 4 and Table 2). Binding is determined by the size of the peptide rather than composition since peptides of the same size but different compositions show similar binding. We also show that OppA does not require the presence of an arginine molecule to bind, which is similar to OppA homologues that bind to a wide range of peptide substrates (56).
To investigate the role of the Opp system in peptide import, a series of growth curves comparing M. catarrhalis wild-type and the opp mutant strains were performed in minimal medium lacking free arginine and supplemented with a range of arginine-containing peptides. There may be size restriction on what can be utilized by the Opp system (56). Although OppA binds a range of peptide substrates, the Opp system mediated uptake of a limited size range of peptides compared to those bound by OppA (Table 2). The Opp system of M. catarrhalis is essential for mediating uptake of peptides between 5 and 10 aa residues in length, yet peptides of smaller or larger size were not dependent on the Opp system for uptake. We speculate that smaller and larger peptides are being imported by other mechanisms, such as the general porin complex in the case of small peptides or through an uptake system that has not yet been characterized (58). An alternative explanation for the larger peptides is that they may undergo proteolytic degradation in the periplasm by any number of annotated peptidase enzymes present in the genome of M. catarrhalis that have not been characterized (38, 59, 60). The resulting smaller peptide fragments are then taken up to support growth. From these data we conclude that the Opp system mediates uptake of peptides between 5 aa residues and 10 aa residues in length.
Acquiring arginine from the surrounding environment is essential for M. catarrhalis survival, yet utilization of methylated arginine (Fig. 7) has not been investigated. Methylated arginine is abundant in the respiratory tract released through the proteolysis of host proteins (31, 33, 54). Additionally, l-arginine is the precursor molecule to nitric oxide (NO) production, which is inhibited by methylated arginine (26, 3133, 54, 55, 61). The balance of methylated arginine and l-arginine in the lungs has been described as the “arginine paradox,” where l-arginine is limited due to sequestration in cellular compartments, inhibition of additional damage pathways, and competition for arginase activity, as well as for the production of NO (26, 62). Conditions where inflammation is common, such as COPD, result in increased levels of methylated arginine as well as stimulation of the NO pathway, further limiting the availability of free l-arginine for uptake by M. catarrhalis (17, 18, 26, 31, 3335, 55, 63). Thus, we were interested to determine if M. catarrhalis could utilize methylated arginine.
Wild-type M. catarrhalis was unable to grow with any form of methylated arginine, monomethylarginine, symmetric dimethylarginine, or asymmetric dimethylarginine. Therefore, acquiring nonmethylated arginine from the environment of the respiratory tract is critical for survival in vivo. Thus, the Opp system may play an essential role in fitness by mediating the uptake of peptides containing l-arginine that has not been methylated.
M. catarrhalis is a strict human pathogen, and thus animal models that mimic human disease are limited (24, 64). We used the mouse pulmonary clearance model, which is the most widely used model (41), to investigate the role of the Opp system in vivo. In a competition assay, the wild-type strain persisted longer in the murine respiratory tract than the oppA mutant (Fig. 9). These results indicated that the Opp system is contributing to fitness and survival in the respiratory tract.
In summary, this work advances our understanding of the mechanisms of virulence of M. catarrhalis through the following novel observations. (i) The substrate binding protein OppA binds peptides of 3 amino acids to at least 16 amino acids in length independent of amino acid content. (ii) The Opp system facilitates uptake of peptides that support the growth of M. catarrhalis. (iii) Methylated arginine does not fulfill the growth requirement of M. catarrhalis for arginine, indicating that l-arginine is required for growth. This observation has important implications in vivo in view of the abundance of methylated arginine in the host respiratory tract, the ecological niche of M. catarrhalis. (iv) The Opp system plays a role in fitness and persistence in the respiratory tract. Based on these observations, we propose that the Opp system functions as a nutritional virulence factor for M. catarrhalis by mediating the acquisition of limiting nutrients, thus facilitating persistence in the harsh environment of the respiratory tract.


This work was supported by NIH grants DC01220 (T.F.M.), GM094611 (M.G.M.), and 5T32AI007614-12.


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Published In

cover image Infection and Immunity
Infection and Immunity
Volume 82Number 11November 2014
Pages: 4758 - 4766
Editor: B. A. McCormick
PubMed: 25156736


Received: 10 June 2014
Returned for modification: 11 July 2014
Accepted: 18 August 2014
Published online: 7 October 2014


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Megan M. Jones
Department of Microbiology and Immunology, University at Buffalo, The State University of New York, Buffalo, New York, USA
Clinical and Translational Research Center, University at Buffalo, The State University of New York, Buffalo, New York, USA
Antoinette Johnson
Clinical and Translational Research Center, University at Buffalo, The State University of New York, Buffalo, New York, USA
Division of Infectious Diseases, Department of Medicine, University at Buffalo, The State University of New York, Buffalo, New York, USA
Mary Koszelak-Rosenblum
Department of Structural Biology, University at Buffalo, The State University of New York, Buffalo, New York, USA
Hauptman Woodward Medical Research Institute, Buffalo, New York, USA
Charmaine Kirkham
Clinical and Translational Research Center, University at Buffalo, The State University of New York, Buffalo, New York, USA
Division of Infectious Diseases, Department of Medicine, University at Buffalo, The State University of New York, Buffalo, New York, USA
Aimee L. Brauer
Clinical and Translational Research Center, University at Buffalo, The State University of New York, Buffalo, New York, USA
Division of Infectious Diseases, Department of Medicine, University at Buffalo, The State University of New York, Buffalo, New York, USA
Michael G. Malkowski
Department of Structural Biology, University at Buffalo, The State University of New York, Buffalo, New York, USA
Hauptman Woodward Medical Research Institute, Buffalo, New York, USA
Timothy F. Murphy
Department of Microbiology and Immunology, University at Buffalo, The State University of New York, Buffalo, New York, USA
Clinical and Translational Research Center, University at Buffalo, The State University of New York, Buffalo, New York, USA
Division of Infectious Diseases, Department of Medicine, University at Buffalo, The State University of New York, Buffalo, New York, USA


B. A. McCormick


Address correspondence to Timothy F. Murphy, [email protected].

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