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The Journal of Immunology, 2004, 173: 4598-4606.
Copyright © 2004 by The American Association of Immunologists

The Emerging Pathogen Moraxella catarrhalis Interacts with Complement Inhibitor C4b Binding Protein through Ubiquitous Surface Proteins A1 and A21

Therése Nordström*, Anna M. Blom{dagger}, Arne Forsgren* and Kristian Riesbeck2,*

Departments of * Medical Microbiology and {dagger} Clinical Chemistry, Lund University, Malmö University Hospital, Malmö, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Moraxella catarrhalis ubiquitous surface protein A2 (UspA2) mediates resistance to the bactericidal activity of normal human serum. In this study, an interaction between the complement fluid phase regulator of the classical pathway, C4b binding protein (C4BP), and M. catarrhalis mutants lacking UspA1 and/or UspA2 was analyzed by flow cytometry and a RIA. Two clinical isolates of M. catarrhalis expressed UspA2 at a higher density than UspA1. The UspA1 mutants showed a decreased C4BP binding (37.6% reduction), whereas the UspA2-deficient Moraxella mutants displayed a strongly reduced (94.6%) C4BP binding compared with the wild type. In addition, experiments with recombinantly expressed UspA150–770 and UspA230–539 showed that C4BP (range, 1–1000 nM) bound to the two proteins in a dose-dependent manner. The equilibrium constants (KD) for the UspA150–770 and UspA230–539 interactions with a single subunit of C4BP were 13 µM and 1.1 µM, respectively. The main isoform of C4BP contains seven identical {alpha}-chains and one {beta}-chain linked together with disulfide bridges, and the {alpha}-chains contain eight complement control protein (CCP) modules. The UspA1 and A2 bound to the {alpha}-chain of C4BP, and experiments with C4BP lacking CCP2, CCP5, or CCP7 showed that these three CCPs were important for the Usp binding. Importantly, C4BP bound to the surface of M. catarrhalis retained its cofactor activity as determined by analysis of C4b degradation. Taken together, M. catarrhalis interferes with the classical complement activation pathway by binding C4BP to UspA1 and UspA2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Moraxella catarrhalis was earlier considered to be a harmless commensal in the respiratory tract, but is now acknowledged as an important mucosal pathogen. It is the third leading bacterial cause of acute otitis medium in children after Streptococcus pneumoniae and Haemophilus influenzae (1, 2, 3). M. catarrhalis is also a common cause of sinusitis and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. In recent years, focus has been on both its outer membrane protein composition and on its interactions with the human host (4). The outer membrane proteins of M. catarrhalis that are suggested as virulence determinants include, among others, Moraxella IgD binding protein (MID)3 (3), catarrhalis outer membrane protein B (CopB), protein CD, M. catarrhalis adherence protein, and ubiquitous surface protein (Usp) (for reviews, see Refs.3 and 4). The MID, also designated Hag (for hemagglutinin), has recently been demonstrated to function as an adhesin (5, 6, 7, 8, 9, 10, 11). Moreover, mice immunized with MID764–913 cleared M. catarrhalis much more efficiently as compared with mice immunized with BSA (12). The conserved 81-kDa CopB plays a role in iron acquisition, and the heat-modifiable CD protein functions as an adhesin to nasal and inner ear mucins (13, 14, 15). In addition to MID, immunization with CopB or CD proteins has shown to induce protective Abs in a mouse pulmonary clearance model (16, 17). M. catarrhalis adherence protein was recently discovered and shown to exhibit both lipolytic and adhesive properties (18).

The UspA family has been studied in detail and consists of UspA1, UspA2, and the hybrid protein, UspA2H (19, 20). UspA1 and UspA2 have molecular masses of 88 and 62 kDa, respectively. However, both proteins migrate as high molecular mass complexes in SDS-PAGE. The amino acid sequences of UspA1 and UspA2 are only 43% identical, but both proteins bear a common epitope of 140 aa residues with 93% identity. Abs against the common epitope have been found to be protective against M. catarrhalis infections in a mouse pulmonary clearance model (21). Both UspA1 and UspA2H are responsible for adhesion of M. catarrhalis to epithelial cells in vitro (10, 20).

The majority of clinical M. catarrhalis isolates are serum resistant (22, 23). Interestingly, Moraxella mutants deficient in UspA2 or both UspA1 and UspA2 are much more susceptible to killing by human serum as compared with the wild-type counterpart or a UspA1 mutant. Therefore, it has been suggested that M. catarrhalis UspA2 is an important outer membrane protein associated with serum resistance due to interaction with vitronectin (24, 25).

The complement system is the first line of innate defense against pathogenic microorganisms, and activation of this system leads to a cascade of protein deposition on the bacterial surface, resulting in formation of the membrane attack complex and opsonization of the pathogen, followed by phagocytosis. C4b-binding protein (C4BP) is a fluid phase regulator of the classical pathway of complement activation. C4BP inhibits the formation and accelerates the decay of the C3 convertase (C4bC2a), and it also serves as a cofactor to factor I in the proteolytic degradation of C4b (26, 27, 28). C4BP is a large glycoprotein that is present in the plasma in several forms. The major form is composed of seven identical {alpha}-chains (70-kDa subunits) and one {beta}-chain (45 kDa) (Fig. 1A) (29). The {alpha}- and {beta}-chains consist of repeating domains of ~60 aa designated complement control protein (CCP) domains (30, 31). The unique {beta}-chain of the major isoform of C4BP binds to the vitamin K-dependent anticoagulant protein S. Consequently, in the majority of serum, C4BP circulates in a 1:1 high affinity noncovalent complex with protein S (29).



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  		FIGURE 1. Schematic illustration of plasma C4BP and recombinant C4BPSTOP8. A, The main C4BP isoform in serum consists of seven identical {alpha}-chains and one {beta}-chain. The {alpha}-chains have eight CCP domains, whereas the {beta}-chain has three CCP domains. All chains are held together by disulfide bridges involving the nonrepeat carboxy-terminal regions. B, rC4BPSTOP8 has a single {alpha}-chain with a stop codon introduced after CCP8 and is devoid of the carboxy-terminal. rC4BPSTOP8 was manufactured in kidney cells 293; ATCC CRL-1573.

 
Several bacterial pathogens bind C4BP, and hence avoid deleterious effects of the complement system. C4BP binding has been demonstrated in many isolates of Streptococcus pyogenes, and the amino-terminal hypervariable regions of several members of the M family proteins are responsible for the binding (32). All clinical isolates of Bordetella pertussis expressing filamentous hemagglutinin, and also Eschericha coli K1 expressing the outer membrane protein A, bind C4BP (33, 34). Furthermore, Neisseria gonorrhoeae has been reported to bind C4BP through porin protein 1A and 1B (Por 1A and Por 1B) (35) and type IV pili (36).

In this study, we demonstrate that M. catarrhalis binds C4BP, and that UspA1 and UspA2 are responsible for the binding. M. catarrhalis mutants lacking these two proteins separately or in combination showed that UspA2 is expressed on the bacterial surface at a higher density than UspA1. Experiments with recombinant proteins demonstrated that both UspA1 and UspA2 bind C4BP in a dose-dependent manner. Furthermore, the binding site for UspA1/A2 is localized within the CCP2, CCP5, and CCP7 domains of the C4BP {alpha}-chain. By investigating the degradation of C4b, C4BP bound to the surface of M. catarrhalis was found to have preserved cofactor activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and culture conditions

The clinical M. catarrhalis isolates BBH18, RH4, and Bc5 have recently been described in detail (5, 8). The M. catarrhalis strains were routinely cultured in brain heart infusion (BHI) liquid broth or on BHI agar plates at 37°C. The UspA1-deficient mutants were cultured in BHI supplemented with 1.5 µg/ml chloramphenicol (Sigma-Aldrich, St. Louis, MO), and UspA2-deficient mutants were incubated with 7 µg/ml zeocin (Invitrogen Life Technologies, Carlsbad, CA). Both chloramphenicol and zeocin were used for growth of the double mutants.

Antibodies

Rabbits were immunized i.m. with 200 µg of recombinant full-length UspA1 emulsified in CFA (Difco; Becton Dickinson, Heidelberg, Germany), and boosted on days 18 and 36 with the same dose of protein in IFA (9). Blood was drawn 3 wk later. To increase the specificity, the anti-UspA1 antiserum was affinity-purified with Sepharose-conjugated recombinant UspA1. To ensure that the antiserum reacted with the same affinity to both recombinant UspA150–770 and UspA230–539, the antiserum was examined with ELISA. UspA150–770 or UspA230–539 (20 nM) were immobilized in microtiter plates and incubated with increasing concentrations of the antiserum, followed by HRP-conjugated goat anti-rabbit antiserum diluted 1/1000 (Dakopatts, Glostrup, Denmark). The binding of anti-UspA1 antiserum to both UspA1 and UspA2 was also confirmed by Western blots. Hence, the antiserum was designated anti-UspA1/A2 polyclonal Ab (pAb). The pAbs, mAb104 and mAb67, against C4BP were kindly provided by Dr. B. Dahlbäck (Department of Clinical Chemistry, Lund University, Malmö, Sweden). mAb104 was raised against CCP1, whereas mAb67 was against CCP4 as described by Blom et al. (37). The rabbit anti-C3b pAb was purchased from Dakopatts. The anti-C4c and anti-C4d mAbs were from Quidel (San Diego, CA).

Construction and characterization of UspA1/A2-deficient M. catarrhalis

The UspA-coding genes were amplified as two cassettes using DyNAzyme II DNA Polymerase (Finnzymes, Espoo, Finland), introducing the restriction enzyme sites, BamHI and HindIII, at the ends of the first cassette, and HindIII and XhoI at the ends of the second cassette. Resulting PCR fragments were digested with appropriate restriction enzymes and cloned into the pET26b(+) vector (Novagen, Madison, WI). A chloramphenicol resistance gene cassette from pLysS (Novagen) was amplified by PCR, using specific primers introducing the restriction enzyme site for HindIII. After digestion, the PCR product was ligated into the uspA1 gene. A zeocin resistance gene cassette was amplified from the plasmid pEM7/Zeo (Invitrogen Life Technologies) with specific primers introducing HindIII, and the resulting PCR product was digested and ligated into the uspA2 gene. M. catarrhalis strains RH4 and BBH18 were transformed by electroporation using a Genepulser apparatus (Bio-Rad, Hercules, CA) and the settings 2.5 kV, 25 µF, and 200 {Omega}. After transformation, bacteria were cultured in BHI broth without antibiotics for 6 h, and thereafter grown on BHI solid medium supplemented with chloramphenicol and/or zeocin. Resulting mutants were screened by PCR and the protein expression was analyzed by Western blot and flow cytometry.

Serum bactericidal assay

Normal human serum (NHS) was obtained from five healthy volunteers. The blood was clotted for 30 min at room temperature (RT), and thereafter incubated on ice for 60 min. After centrifugation, the sera were pooled, aliquoted, and stored at –70°C. Serum that was inactivated at 56°C for 30 min was used as a control. The M. catarrhalis strains and mutants were diluted in DGVB2+ (2.5 mM Veronal buffer, pH 7.3, containing 0.1% (w/v) gelatin, 1 mM MgCl2, and 0.15 mM CaCl2). Bacteria (104 CFU) were incubated together with 10% of NHS or heat-inactivated NHS in a final volume of 100 µl. This mixture was incubated at 37°C, and at time 0, 5, 10, 15, 20, and 30 min, 10-µl aliquots were removed and spread onto BHI agar plates.

DNA cloning and protein expression

Genomic DNA was extracted from M. catarrhalis Bc5 using a DNeasy tissue kit (Qiagen, Hilden, Germany). The UspA-coding genes were amplified using DyNAzyme II DNA Polymerase with specific primers introducing BamHI and HindIII restriction enzyme sites. The UspA1 and UspA2 are considered to be autotransporters (38). Thus, the signal peptides and the C-terminal sequences are most likely not involved in the function of the proteins. Therefore, to avoid presumptive toxicity and to increase solubility, the signal peptides and the hydrophobic C-terminal regions were not included. The resulting PCR products corresponded to the truncated proteins designated UspA150–770 and UspA230–539. The PCR products were cloned into pET26b(+) vector and the resulting plasmids were transformed into the host E. coli DH5{alpha}. Thereafter, the plasmids encoding for the Usp-proteins were transformed into the expressing host BL21(DE3) (Novagen). All constructs were sequenced using the BigDye Terminator Cycle Sequencing version 3.1 Ready reaction sequencing kit (Applied Biosystems, Foster City, CA). The expression and purification of the recombinant proteins was done as previously described (7).

Outer membrane protein preparations

M. catarrhalis forms vesicles and secretes outer membrane components into the surrounding medium (39). EDTA and heat induce vesicle formation, and this has proved to be a convenient and reliable method for extraction of M. catarrhalis outer membrane proteins. To analyze the outer membrane proteins of the UspA1/A2-deficient M. catarrhalis mutants and the wild-type strains, vesicle formation was induced using 0.05 M Na2HPO4, 0.15 M NaCl, 0.01 M EDTA (pH 7.4) at 56°C (39). After centrifugation, the supernatants were concentrated using Vivaspin columns (Vivascience, Hannover, Germany). Finally, vesicles were analyzed on SDS-PAGE and Western blot.

SDS-PAGE and detection of proteins on membranes (Western blots)

SDS-PAGE was run as described before (5). Gels were stained with Coomassie brilliant blue R-250 (Bio-Rad). Electrophoretical transfer of protein bands from the gel to an Immobilon-P membrane (Millipore, Bedford, MA) was done at 20 V overnight to transfer the high molecular mass complexes. After transfer, the Immobilon-P membrane was blocked in PBS with 0.1% Tween 20 (PBS-Tween) containing 5% milk powder. After several washings in PBS-Tween, the membrane was incubated with rabbit anti-UspA1/A2 antiserum diluted 1/500 in PBS-Tween, including 2% milk powder, for 1 h at RT. HRP-conjugated goat anti-rabbit antiserum diluted 1/1000 was added after washings in PBS-Tween. After incubation for 1 h at RT and additional washings in PBS-Tween, development was performed with ECL Western blotting detection reagents (Amersham Pharmacia Biotech, Uppsala, Sweden).

Complement proteins

Human C4BP was purified from human plasma (40). Recombinant wild-type C4BP (rC4BP) was expressed in human kidney cells 293 (ATCC CRL-1573; American Type Culture Collection, Manassas, VA), and purified using affinity chromatography with mAbs against the {alpha}-chains of C4BP (41). rC4BPSTOP8, containing only one {alpha}-chain (Fig. 1B), was prepared in the same way, with the exception of a deletion of the nonrepeat carboxy-terminal region involved in the polymerization of the chains. rC4BP lacking single CCP domains (C4BP mutants) were constructed by overlapping extension PCR, and were expressed in kidney cells 293, followed by purification with affinity chromatography (37). All the mutants were extensively characterized to show that no folding problem was introduced by mutagenesis (37) and were previously used to define binding site for several ligands (34, 35, 36, 42, 43). C4b, C3b, and factor I were from Advanced Research Technologies (San Diego, CA).

Flow cytometry analysis

The UspA1/A2-protein expression and the capacity for M. catarrhalis to bind C4BP were analyzed by flow cytometry. The M. catarrhalis wild-type strains and UspA1/A2-deficient mutants were grown on solid medium overnight and washed twice in PBS containing 3% fish gelatin (Sigma-Aldrich) (PBS-gelatin). To analyze UspA1/A2 expression, the bacteria (108) were incubated with the anti-UspA1/A2 antiserum in 100 µl of PBS-gelatin for 1 h at 37°C. The bacteria were washed and incubated for 30 min at RT with FITC-conjugated goat anti-rabbit pAb (Dakopatts) diluted according to the manufacturer’s instructions. After three additional washes, the bacteria were analyzed in a flow cytometer (EPICS, XL-MCL; Coulter, Hialeah, FL). C4BP binding to whole bacteria was analyzed by incubation of the bacteria (108) with 2.5 µg of C4BP in PBS-gelatin for 1 h at 37°C. After washings, the bacteria were incubated with 0.5 µg of anti-C4BP pAb per milliliter for 30 min at RT. Thereafter, the bacteria were washed and incubated for 30 min at RT with FITC-conjugated goat anti-rabbit pAb. After three additional washes, bacteria were analyzed by flow cytometry. All incubations were kept in a final volume of 100 µl of PBS-gelatin, and the washings were done with the same buffer. The anti-C4BP pAb and FITC-conjugated anti-rabbit pAb were added separately as a negative control for each strain analyzed.

To analyze C4BP cofactor activity, M. catarrhalis BBH18 wild-type (108) was incubated with 5 µg of C4BP in PBS-gelatin for 1 h at 37°C, followed by washings and addition of 10% of C4BP depleted normal human serum. After 30 min at 37°C, the bacteria were washed and 1 µg/ml anti-C4c or anti-C4d mAbs (Quidel) were added. After another 30 min, the bacteria were washed and FITC-conjugated goat anti-mouse pAb (Dakopatts) was added. In another set of experiments, the bacteria were incubated with anti-C3d pAb (Dakopatts) (1/20) after incubation with serum, followed by a FITC-conjugated anti-rabbit pAb. The bacteria were washed and analyzed by flow cytometry. M. catarrhalis BBH18 wild type without preincubation with C4BP was used as negative control.

C4b-degradation assay

M. catarrhalis RH4 wild type (5 x 108) was incubated with 15 µg of C4BP in 50 mM Tris-HCl (pH 7.4) supplemented with 150 mM NaCl for 1 h at 37°C. After thorough washings in the same buffer, the bacteria were mixed with 250 nM C4b, 60 nM factor I, and trace amounts of 125I-labeled C4b in 50 µl of buffer. As a positive control, 100 nM C4BP was used in fluid phase instead of the preincubated bacteria. As a negative control, the RH4 wild-type Moraxella without preincubation of C4BP was used. The samples were incubated for 2 h at 37°C and the reaction was terminated by the addition of SDS-PAGE sample buffer. The reduced SDS-PAGE was run as described above. Thereafter, the gel was dried and the proteins were visualized with a Personal FX (Bio-Rad) using intensifying screens.

Protein labeling and RIA

Purified recombinant UspA150–770, UspA230–539, or C4BP were labeled with 0.05 mol iodine (Amersham Biosciences, Buckinghamshire, U.K.) per mol protein, using the chloramine-T method (44). The specific activity of 125I-labeled C4BP was 5.7 kcpm/ng. M. catarrhalis strains BBH18 and RH4 wild types and corresponding mutants were grown overnight on solid medium and were washed in PBS with 2% BSA. Bacteria (108) were incubated for 1 h at 37°C with 125I-labeled C4BP (1600 kcpm/sample) in PBS, 2% BSA. After three washings with PBS containing 2% BSA, radiolabeled C4BP bound to bacteria was measured in a gamma counter (Wallac, Espoo, Finland).

Direct ligand binding

Microtiter plates (Nunc-Immuno Module; Nunc, Roskilde, Denmark) were coated with 20 nM of purified recombinant UspA150–770/UspA230–539 in 75 mM sodium carbonate (pH 9.6) at 4°C overnight. Unbound proteins were measured to ensure that UspA150–770 and UspA230–539 bound equally well to the plastic. Plates were washed four times with washing buffer (50 mM Tris-HCl, 0.15 M NaCl, and 0.1% Tween 20, pH 7.5), and blocked for 2 h at RT with washing buffer supplied with 3% fish gelatin (blocking buffer). After four washings, the wells were incubated for 1 h at 37°C with C4BP diluted in 2-fold steps in blocking buffer, with the highest concentration at 1000 nM. Thereafter, the plates were washed and incubated with mAb104 (1/5000) in blocking buffer for 1 h in RT. After additional washings, HRP-conjugated rabbit anti-mouse pAbs (Dakopatts) diluted 1/1000 was added for 1 h at RT. The wells were washed four times and the plates were developed and measured at OD450.

To estimate which CCP domain of C4BP is involved in the binding of UspA1 and UspA2, microtiter plates were immobilized with 40 nM UspA150–770 or UspA230–539. After blocking and washings, the plates were incubated for 1 h at 37°C with triplets of rC4BP mutants (each lacking one CCP) in a concentration of 90 nM, respectively. Thereafter, the wells were washed and incubated with mAb104 and/or mAb67 (1/5000) in blocking buffer for 1 h at RT. After additional washings, HRP-conjugated rabbit anti-mouse pAbs (Dakopatts) diluted 1/1000 was added for 1 h at RT. The wells were washed and developed as above.

Determination of KD

Equilibrium affinity constants were obtained from plots of C4BP binding of UspA150–770 or UspA230–539 as a function of the C4BP concentration (45). The data were fit to the following equation (Equation 1) for a single-site binding isotherm using nonlinear least squares regression analysis: B = Bmax/(1 + (KD/[C4BP])). Bmax was set as the maximum binding of C4BP to UspA150–770 or UspA230–539.

Competition assays

To define saturating conditions of 125I-C4BP, the protein was incubated at increasing concentrations with UspA150–770 or UspA230–539 in microtiter plates. The competition assays were essentially performed as described elsewhere (36). Briefly, microtiter plates were incubated with 20 nM recombinant UspA150–770 or UspA230–539 overnight at 4°C in 75 mM Na2CO3 (pH 9.6). Thereafter, the wells were washed and blocked as described above. After four washings, 125I-labeled C4BP was added (50 kcpm/well), together with various concentrations of unlabeled proteins diluted in blocking buffer, and followed by an overnight incubation at 4°C. After four additional washings, the radioactivity was measured in a gamma counter.

Surface plasmon resonance (Biacore, Uppsala, Sweden)

The interaction between UspA150–770 or UspA230–539 and C4BP was further analyzed using surface plasmon resonance (Biacore 2000; Biacore). Three flow cells on a CM5 sensor chip were activated using 20 µl of 0.2 M 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide with 0.05 M N-hydroxy-sulfosuccinimide at a flow rate of 5 µl/min. The recombinant UspA150–770 and UspA230–539 were then injected at a concentration of 20 µg/ml in 10 mM sodium-acetate buffer (pH 4) over flow cell 2 and 3, respectively, to reach 1700 resonance units. The unreacted groups were blocked with 20 µl of 1 M ethanolamine (pH 8.5). Flow cell 1, which was activated and blocked, was used as a negative control. The association kinetics were studied for a wide range of rC4BPSTOP8 concentrations, diluted in the standard flow buffer (10 mM HEPES-KOH, pH 7.4, 70 mM NaCl, 0.005% Tween 20, 5 mM EDTA). Protein solutions were injected for 100 s to achieve saturation during the association phase at a constant flow rate of 30 µl/min, and the dissociation phase was analyzed for 200 s at the same flow rate. Signals were then normalized by subtracting the nonspecific signal measured in flow cell 1. Between each different rC4BPSTOP8 concentration, the flow cell surfaces were regenerated with a 30-µl injection of 2 M NaCl to remove bound ligand. All sensograms were analyzed using the BiaEvaluation 3.0 software (Biacore) to calculated equilibrium affinity constants.

Dot blot assays

Purified rC4BP variants diluted in 3-fold steps (0.0014–3 µg) in 100 µl of 0.1 M Tris-HCl (pH 9.0) were manually applied to nitrocellulose membranes (Schleicher & Schüll Microscience, Dassel, Germany) using a dot blot device. After saturation, the membranes were incubated for 2 h with PBS-Tween containing 5% milk powder in RT and washed four times with PBS-Tween. Thereafter, 30 kcpm 125I-labeled UspA150–770 or UspA230–539 in PBS-Tween with 2% milk powder was added for 3 h in RT. The bound protein was visualized with a Personal FX (Bio-Rad) using intensifying screens.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of UspA1- and A2-deficient M. catarrhalis mutants

Two clinical isolates (BBH18 and RH4) were mutated by introduction of chloramphenicol and zeocin resistance cassettes in the genes encoding for UspA1 and UspA2, respectively. Resulting mutants were confirmed by PCR. Moreover, absence of UspA1 and/or UspA2 expression was proven by analysis of outer membrane vesicles (i.e., EDTA heat-induced vesicles) in Western blots using an anti-UspA1/A2 antiserum. The BBH18{Delta}uspA1 mutant was deficient in a 115-kDa protein corresponding to UspA1, whereas the BBH18{Delta}uspA2 mutant lacked both a high molecular mass complex >250 kDa and a 100-kDa protein, both corresponding to UspA2 (Fig. 2). The double mutant did not express any proteins that could be recognized by the anti-UspA1/A2 pAb.



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  		FIGURE 2. Western blot analysis of M. catarrhalis BBH18{Delta}uspA1, {Delta}uspA2, and {Delta}uspA1/A2 mutants compared with the wild-type counterpart. To induce vesicle formation, the outer membrane proteins were extracted using an EDTA-containing buffer at 56°C. Resulting proteins were analyzed by Western blots using a rabbit anti-UspA1/A2 antiserum and HRP-conjugated goat anti-rabbit pAb. The BBH18{Delta}uspA1 mutant lacked the 115-kDa band (filled arrowhead), whereas BBH18{Delta}uspA2 expressed neither the high molecular mass protein nor the 100-kDa band (open arrowheads). The BBH18{Delta}uspA1/A2 double mutant was deficient in all three bands.

 
To further examine the M. catarrhalis UspA1 and UspA2 mutants, bacteria were analyzed by flow cytometry using anti-UspA1/A2 pAb. As can be seen in Fig. 3B, only a minor decrease in mean fluorescence intensity (mfi) (from 63.2 to 51.2) was observed with the BBH18{Delta}uspA1 mutant when it was compared with the wild-type counterpart (Fig. 3A). In contrast, M. catarrhalis BBH18{Delta}uspA2 displayed a decreased number of UspA molecules (mfi, 10.3) (Fig. 3C) as compared with the UspA1-deficient mutant. When both UspA1 and UspA2 were knocked out, the resulting mutant was completely deficient in both proteins (Fig. 3D). Similar results were obtained with the M. catarrhalis RH4 wild-type isolate and corresponding mutants (data not shown). Thus, the phenotypes of all the M. catarrhalis mutants were defined and the Western blot results were correlated with the flow cytometry data. In addition, an interesting observation was that our two selected M. catarrhalis isolates (BBH18 and RH4) displayed a higher density of UspA2 as compared with UspA1.



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  		FIGURE 3. Flow cytometry profiles of M. catarrhalis BBH18 wild-type and UspA1/A2-deficient mutants proving a UspA1/A2-dependent C4BP binding. The BBH18 wild-type clinical isolate (A and E), BBH18{Delta}uspA1 (B and F), BBH18{Delta}uspA2 (C and G), or BBH18{Delta}uspA1/A2 (D and H) were incubated with a rabbit anti-UspA1/A2 antiserum (A–D) or C4BP followed by anti-C4BP pAb (E–H). Finally, a FITC-conjugated anti-rabbit antiserum was added. The mfi for each profile is also shown. A typical experiment of three is demonstrated.

 
The M. catarrhalis {Delta}uspA2 mutants are serum sensitive

The wild-type strains and the mutant strains were tested in a serum bactericidal assay. The wild-type strains BBH18 and RH4 were completely resistant to NHS, whereas their derived UspA1 mutants were only partially resistant (Fig. 4). However, the M. catarrhalis {Delta}uspA2 mutants and the double mutants were killed by NHS after 5 min. Both strains and all derived mutants were resistant to heat-inactivated NHS.



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  		FIGURE 4. The M. catarrhalis BBH18{Delta}uspA2 and {Delta}uspA1/A2 mutants were serum sensitive, whereas M. catarrhalis BBH18 wild type was serum resistant and the BBH18{Delta}uspA1 mutant was partially serum resistant. The wild-type ({blacksquare}), {Delta}uspA1 mutant ({blacktriangleup}), {Delta}uspA2 mutant (•), and {Delta}uspA1/A2 mutant ({diamondsuit}) strains were incubated in the presence of 10% NHS. The double mutant was also incubated with 10% heat-inactivated NHS ({diamond}). Numbers of bacteria (CFU) before addition of NHS was defined as 100%. Error bars indicate SD.

 
M. catarrhalis devoid of UspA1 and UspA2 does not bind C4BP

Binding of C4BP to M. catarrhalis was analyzed by flow cytometry using a polyclonal anti-C4BP antiserum. Interestingly, the M. catarrhalis BBH18 isolate strongly bound C4BP (mfi, 99.4) (Fig. 3E). In contrast, BBH18{Delta}uspA1 showed a decreased C4BP binding (mfi, 62.0) compared with the wild-type counterpart (Fig. 3F). Furthermore, BBH18{Delta}uspA2 attracted C4BP to a much lower degree (mfi, 5.4) compared with the BBH18{Delta}uspA1 mutant (Fig. 3G). Consequently, C4BP binding to the BBH18{Delta}uspA1/A2 double mutant was lower (mfi, 2.7) as compared with the single mutants (Fig. 3H). A similar pattern was obtained with the M. catarrhalis RH4 isolate and the corresponding RH4{Delta}uspA1/A2 mutants.

To further analyze the interaction between C4BP and M. catarrhalis, 125I-labeled C4BP was added to the two clinical isolates BBH18 and RH4. Both M. catarrhalis strains strongly bound 125I-labeled C4BP (Fig. 5), i.e., 45–55.7% of the added 125I-labeled C4BP bound. However, no significant difference was observed between the M. catarrhalis wild-type strains and the corresponding {Delta}uspA1 mutants. In contrast, the M. catarrhalis {Delta}uspA2 and double mutants did not bind 125I-labeled C4BP above background levels (1.2–5.0% of maximal binding). We also included M. catarrhalis RH4 and BBH18 devoid of the outer membrane protein MID (9) as positive controls. Experiments with these two M. catarrhalis {Delta}mid mutants showed the same C4BP binding as the wild-type counterparts (Fig. 5). Taken together, M. catarrhalis {Delta}uspA1 mutants displayed a 38% decrease in C4BP binding when analyzed by flow cytometry (Fig. 3B), whereas the less sensitive RIA did not demonstrate any significant decrease in 125I-labeled C4BP binding to M. catarrhalis {Delta}uspA1 mutants. Furthermore, M. catarrhalis strongly bound C4BP, and a strict correlation existed between UspA1/A2 expression and C4BP binding.



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  		FIGURE 5. M. catarrhalis UspA2-deficient mutants do not bind C4BP. M. catarrhalis BBH18 and RH4 wild-type isolates were compared with mutants devoid of UspA1, UspA2, or both UspA1 and A2. In addition, E. coli BL21 were included as negative control and two M. catarrhalis {Delta}mid strains as positive controls. Bacteria were incubated with 125I-labeled C4BP, followed by several washes and analysis in a gamma counter. The mean values of three experiments are shown. Error bars indicate SD.

 
C4BP exhibits cofactor activity at the M. catarrhalis cell surface

C4BP serves as a cofactor to Factor I in the degradation of C4b, which results in appearance of the two fragments C4d and C4c. Upon cleavage, it has been demonstrated that C4d remains bound to the surface of the bacteria, but that C4c is released to the surrounding medium resulting in an increased C4d/C4c ratio at the bacterial surface (35). To investigate whether C4BP was active at the M. catarrhalis cell membrane, the BBH18 wild type was coated with C4BP and thereafter incubated with normal human serum depleted of C4BP. Surface-bound C4c and C4d was analyzed by flow cytometry using specific mAbs directed against C4c or C4d. Cofactor activity of C4BP will not alter the amount of C4b measured by mAb against C4d, but will decrease the amount of C4b detected by the mAb against C4c. A higher C4d/C4c ratio was detected at the bacterial surface when M. catarrhalis BBH18 that was preincubated with C4BP (Fig. 6B) was compared with M. catarrhalis that were not preincubated with C4BP (Fig. 6A). Thus, C4BP bound to M. catarrhalis retained its cofactor function because C4b was degraded to C4d. The activity of C4BP was confirmed by analysis of C3b deposition on M. catarrhalis. Because C4BP inhibits C3 convertase, a decreased C3b deposition will be found if C4BP is functionally active. Bacteria were coated with C4BP, followed by incubation with C4BP-deficient serum. C3b deposition was then determined with anti-C3d pAb and FITC-conjugated anti-rabbit pAb. Interestingly, when bacteria were preincubated with C4BP, the C3b deposition significantly decreased, i.e., the mfi were 30–41% lower with C4BP-coated bacteria compared with bacteria only.



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  		FIGURE 6. C4BP retains its cofactor activity when bound to the M. catarrhalis cell surface. A, M. catarrhalis BBH18 without addition of C4BP; or B, bacteria preincubated with C4BP and analysis of bound C4c and C4d. C, Degradation of C4b in the presence of C4BP-coated M. catarrhalis and factor I. A and B, M. catarrhalis BBH18 was preincubated with 5 µg of C4BP, followed by incubation with 10% C4BP-depleted normal human serum for 30 min at 37°C. Thereafter, the bacteria were incubated with anti-C4c or anti-C4d mAbs for detection of the C4b derivates. Finally, FITC-conjugated anti-mouse pAb were added and bacteria were analyzed by flow cytometry. C, The M. catarrhalis RH4 wild type with or without C4BP was incubated with 125I-labeled C4b and factor I, followed by separation of the proteins in SDS-PAGE. C4BP (100 nM) in fluid phase was used as a positive control. The gel was dried and visualized with a phosphor imager. Data are representative for three independent experiments.

 
To further confirm the cofactor activity of C4BP bound to M. catarrhalis, the RH4 wild-type strain with or without addition of C4BP was incubated with factor I and 125I-labeled C4b. When 125I-labeled C4b was incubated in the presence of C4BP-coated M. catarrhalis RH4, the degradation product, C4d, strongly appeared (Fig. 6C). However, no C4b degradation (i.e., C4d was not formed) was seen with bacteria only. Thus, C4BP retained its cofactor capacity to degrade C4b when it was bound to M. catarrhalis.

Recombinant UspA150–770 and UspA230–539 bind C4BP in a dose-dependent manner

To further analyze the interaction between C4BP and the UspA1 and A2, the truncated proteins UspA150–770 and UspA230–539 were recombinantly produced in E. coli. To evaluate whether the C4BP/UspA interaction was dose dependent, microtiter plates coated with UspA150–770 or UspA230–539 were incubated with C4BP at increasing concentrations. Bound C4BP was detected by a specific anti-C4BP mAb. As can be seen in Fig. 7, UspA150–770 and UspA230–539 bound to C4BP in a dose-dependent manner, however, UspA150–770 required higher concentrations of C4BP for binding as compared with UspA230–539. The results from these experiments allowed calculation of apparent KD values of the interactions. KD values were obtained by fitting the data in Fig. 7 to Equation 1. The calculated KD for UspA230–539/C4BP was 26.5 nM, whereas it was 57 nM for UspA150–770/C4BP.



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  		FIGURE 7. Recombinantly expressed M. catarrhalis UspA150–770 or UspA230–539 binds C4BP in a dose-dependent manner. Recombinant UspA150–770 or UspA230–539 (20 nM) were immobilized in microtiter plates and incubated with increasing concentrations (1–1000 nM) of C4BP followed by detection using an anti-C4BP mAb and HRP-conjugated anti-mouse pAb. The background binding was subtracted from all samples. The mean values of three experiments are shown and error bars indicate SD.

 
The interaction between C4BP and UspA1 or A2 was further confirmed using a competition assay after saturated conditions of UspA150–770 or UspA230–539 and 125I-labeled C4BP were defined (Fig. 8A), were incubated with 125I-labeled C4BP in the presence of increasing C4BP concentrations. Unlabeled C4BP specifically inhibited the binding between 125I-labeled C4BP and UspA150–770 or UspA230–539 (Fig. 8B). A total of 22 nM C4BP was required to block the UspA230–539/125I-labeled C4BP interaction by 50% (IC50), whereas 70 nM was required to block 50% of the UspA150–770 binding to 125I-labeled C4BP.



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  		FIGURE 8. The binding between C4BP and UspA150–770 or UspA230–539 is specific, because C4BP competes with iodine labeled C4BP. A, To define saturating conditions, increasing concentrations of 125I-labeled C4BP was incubated with UspA150–770 or UspA230–539. B, 125I-labeled C4BP (50 kcpm/well) was added together with increasing concentrations of unlabeled C4BP to microtiter plates coated with truncated recombinant UspA1 or A2. Binding at the lowest competitor (i.e., unlabeled C4BP) concentration was defined as 100%. The mean values of three experiments are shown. Error bars correspond to SD.

 
To establish the affinity constant of the UspA/C4BP interaction, binding experiments using surface plasmon resonance (i.e., Biacore) were performed. UspA150–770 and UspA230–539 were immobilized on the surface of a CM5 chip using amino coupling. A single {alpha}-chain of recombinant C4BP (rC4BPSTOP8; Fig. 1B) was injected until saturation was reached (Fig. 9). Due to the low affinity, the association or the dissociation rate affinity could not be calculated. However, the KD (the equilibrium dissociation constant) was calculated from a binding curve showing response at equilibrium plotted against the concentration using steady-state affinity model supplied by BiaEvaluation software (Biacore). The KD for the binding between rC4BPSTOP8 and UspA150–770 or UspA230–539 was 13 µM and 1.1 µM, respectively.



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  		FIGURE 9. Biacore sensogram showing the interaction between UspA230–539 and rC4BPSTOP8. Similar sensogram was obtained with UspA150–770. Various concentrations of purified rC4BPSTOP8 were injected over a CM5-chip with immobilized UspA150–770 and UspA230–539 in separate flow cells. The interaction between UspA150–770 or UspA230–539 and rC4BPSTOP8 at equilibrium was plotted against increasing concentrations of rC4BPSTOP8, which allowed calculation of KD. Identical samples were injected over a control flow cell. Nonspecific binding was subtracted from all samples. rC4BPSTOP8 associated with UspA150–770 or UspA230–539 was measured as arbitrary resonance units.

 
CCP2, -5, and -7 subunits contribute to the binding of rC4BP to UspA1/A2

The main isoform of human C4BP circulating in plasma consists of two types of subunits, i.e., seven identical {alpha}-chains and one {beta}-chain. To identify the subunit that was responsible for the interaction of C4BP with UspA1/A2, we analyzed whether recombinantly produced C4BP containing polymerized {alpha}-chains (but no {beta}-chain) bound to M. catarrhalis. Flow cytometry analyses revealed that rC4BP and plasma-derived C4BP equally bound to M. catarrhalis. This suggested that the {beta}-chain was not involved in the interaction, but that the UspA-dependent binding was localized within the {alpha}-chain.

To evaluate which {alpha}-chain CCP subunit was involved in the C4BP/UspA interaction, eight mutant rC4BP proteins, each lacking one of the eight CCP subunits, were analyzed for binding. Immobilized UspA150–770 or UspA230–539 was incubated with equal amounts of the rC4BP CCP subunit mutants. Interestingly, binding of UspA230–539 to rC4BP lacking either CCP2 or CCP7 strongly decreased (Fig. 10A), and the binding to the {Delta}CCP5 mutant decreased with two-thirds. Similar results were obtained with UspA150–770 (results not shown). The results were also confirmed by dot blots using iodine-labeled UspA150–770 or UspA230–539. Interestingly, we did not observe any inhibition of the interaction between M. catarrhalis and C4BP using NaCl, heparin, or C4b, all of which disrupt bindings based on ionic interactions with CCP1–2 of C4BP.



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  		FIGURE 10. rC4BP deficient in CCP2 or CCP7 does not bind M. catarrhalis UspA230–539. A, Direct binding assay showing UspA230–539 binding to rC4BP mutants. B, Coomassie-stained unreduced SDS-PAGE showing nine purified rC4BP mutants. In A, UspA230–539 was incubated with the different rC4BP mutants in microtiter plates, and thereafter with anti-C4BP mAb104 or mAb67 for rC4BP{Delta}CCP1, followed by HRP-conjugated anti-mouse pAb. The wild type was incubated with both mAbs, but because the affinity is very similar only one is shown. The mean values of three experiments are shown. Error bars correspond to SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrate a novel interaction between the respiratory pathogen M. catarrhalis and the important complement regulator C4BP. Complement resistance is crucial for bacterial virulence. A large number of clinical Moraxella catarrhalis isolates (n = 1350) have been analyzed for serum resistance (23, 46). Interestingly, 89% of the Moraxella isolates from patients with lower respiratory tract infections were strongly or intermediately resistant to complement-mediated killing. In contrast, the percentage of serum-resistant M. catarrhalis harbored by healthy carriers was as low as 41.5%. Binding of complement inhibitors such as C4BP or factor H is a widely spread strategy of serum-resistant pathogens (47).

Several studies have indicated that complement proteins and regulators are present in the human respiratory tract (48, 49). In addition, complement activity can be detected in the extracellular matrix during inflammation and increased vascular permeability (50). Because C4BP in complex with protein S binds to phospholipid membranes (51, 52), another possibility would be that C4BP promotes adhesion by binding simultaneously to M. catarrhalis and the epithelial cell surface.

Flow cytometry analysis and RIAs of UspA1- and UspA2-deficient mutants revealed that UspA1/A2 was crucial for binding of C4BP. Furthermore, the Western blot and flow cytometry showed that UspA2 was expressed at a much higher density compared with UspA1 (Figs. 2 and 3). Thus, the UspA2-deficient mutants displayed a much stronger decrease in C4BP binding than the UspA1-deficient mutants. This was supported by the results from the serum bactericidal assay (Fig. 4). Interestingly, the BBH18{Delta}uspA1 mutant was only partly serum resistant. This differed from a previous study showing that M. catarrhalis 035E devoid of UspA1 was as resistant to complement-mediated killing as the wild-type counterpart (22).

Recombinantly produced UspA150–770 and UspA230–539 bound C4BP to a similar extent, and both interactions were dose dependent and specific (Figs. 7 and 8). UspA1 and A2 are in part strongly related, but comparison of the amino acid sequences from four M. catarrhalis strains shows only 41.1–46.0% identity and 55.3–63.5% similarity (53). Interestingly, the two outer membrane proteins share a common epitope consisting of 140 aa residues with 93% identity (19), suggesting that the C4BP binding site may be, at least in part, located within this sequence.

In agreement with the direct binding assay (Fig. 7), the UspA150–770/C4BP interaction had a lower binding affinity (KD, 13 µM) as compared with UspA230–539 (KD, 1.1 µM) when analyzed in Biacore. However, both affinity constants were considerably higher than expected. This can be explained by the fact that we used a single rC4BP {alpha}-chain (rC4BPSTOP8) as binding partner in our Biacore analyses. The KD values determined from the interactions where C4BP wild type was used (Fig. 7) were in nanomolar range. Thus, UspA1 and UspA2 most likely interact simultaneously with more than one {alpha}-chain in polymeric C4BP molecule. It has been shown previously for other C4BP ligands that up to four interactions are possible at the same time (31, 54).

The interactions between UspA1 or UspA2 and C4BP appeared to be nonionic because they were not disrupted in presence of high salt concentration or heparin. Moreover, experiments with rC4BP mutants, each deficient in one CCP module, revealed that CCP2 and CCP7 on the {alpha}-chain are the main binding sites for both recombinant UspA230–539 (Fig. 10A) and UspA150–770. Furthermore, the {Delta}CCP5 mutant had lost two-thirds of its binding capacity. Several pathogens interact with CCP2, whereas M. catarrhalis is the first bacterium described that uses CCP7 as a recognition site for C4BP binding. Streptococcus pyogenes M proteins bind C4BP in a nonionic manner and recognize CCP1 and CCP2, and their binding sites overlap to some extent with the binding site for C4b (32, 41). Filamentous hemagglutinin from Bordetella pertussis interacts with C4BP in a very similar way as C4b; an ionic binding with a cluster of charged amino acids on the interface of CCP1 and CCP2 (33, 43). In parallel, the interaction between porins Por1A and Por1B from N. gonorrhoeae and C4BP also requires CCP1. The Por1A-C4BP and Por1B-C4BP interactions are based on hydrophobic and ionic bindings, respectively (35). Furthermore, isolated type IV pili from N. gonorrhoeae display an ionic binding to C4BP that involves CCP1 and CCP2 (36). Finally, outer membrane protein A from E. coli K1 interacts with CCP3 on C4BP, and the binding is based on a hydrophobic interaction (34). Thus, with the exception of M. catarrhalis, CCP1–3 are the major modules of the C4BP {alpha}-chain that are used by several bacterial species.

The ability of M. catarrhalis to bind C4BP suggests that the species uses C4BP’s capacity to inhibit the complement-mediated attack in two ways. Firstly, C4BP bound to the surface of M. catarrhalis maintains its activity to degrade C4b to C4c and C4d (Fig. 6). Consequently, such degradation prevents C4b from participating in the opsonization of the pathogen. Secondly, because surface-bound C4BP binds C4b, the formation of C3 convertase (C4bC2a) will most likely be inhibited and its decay accelerated. This may help M. catarrhalis to avoid membrane attack complex-mediated lysis.

Taken together, we have presented several lines of evidence on M. catarrhalis UspA1 and UspA2 binding to C4BP, a factor that inhibits the classical pathway of the complement system. Because UspA2 is expressed at a higher density as compared with UspA1, UspA2 most likely has a stronger impact on M. catarrhalis binding to C4BP and, therefore, contributes to M. catarrhalis serum resistance and, consequently, virulence.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Alfred Österlund Foundation, the Anna and Edwin Berger Foundation, the Crafoord Foundation, the Greta and Johan Kock Foundation, the Swedish Medical Research Council, the Swedish Society of Medicine, and the Cancer Foundation at the University Hospital in Malmö. Back

2 Address correspondence and reprint requests to Dr. Kristian Riesbeck, Department of Medical Microbiology, Malmö University Hospital, Lund University, SE-205 02 Malmö, Sweden. E-mail address: kristian.riesbeck{at}mikrobiol.mas.lu.se Back

3 Abbreviations used in this paper: MID, Moraxella IgD binding protein; CopB, Catarrhalis outer membrane protein B; Usp, ubiquitous surface protein; C4BP, C4b-binding protein; CCP, complement control protein; BHI, brain heart infusion; NHS, normal human serum; pAb, polyclonal Ab; PBS-Tween, PBS with 0.1% Tween 20; RT, room temperature; PBS-gelatin, PBS containing 3% fish gelatin; mfi, mean fluorescence intensity. Back

Received for publication March 23, 2004. Accepted for publication July 22, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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