Binding of Vitronectin by the Moraxella catarrhalis UspA2 Protein Interferes with Late Stages of the Complement Cascade(基础医学)

Binding of Vitronectin by the Moraxella catarrhalis UspA2 Protein Interferes with Late Stages of the Complement Cascade

http://www.100md.com 感染与免疫杂志 2006年第3期

     Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390

    Section of Infectious Diseases, Evans Biomedical Research Center, Boston University Medical Center, Boston, Massachusetts 02118

    Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts 06105

    ABSTRACT

    Many Moraxella catarrhalis strains are resistant to the bactericidal activity of normal human serum (NHS). The UspA2 protein of the serum-resistant strain O35E has previously been shown to be directly involved in conferring serum resistance on this strain. Testing of 11 additional serum-resistant M. catarrhalis wild-type isolates and their uspA1 and uspA2 mutants showed that the uspA1 mutants of all 11 strains were consistently serum resistant and that the uspA2 mutants of these same 11 strains were always serum sensitive. Analysis of complement deposition on four different serum-resistant M. catarrhalis strains and their serum-sensitive uspA2 mutants showed that, for three of these four strain sets, the wild-type and mutant strains bound similar amounts of early complement components. In contrast, there was a significant reduction in the amount of the polymerized C9 on the wild-type strains relative to that on the uspA2 mutants. These same three wild-type strains bound more vitronectin than did their uspA2 mutants. UspA2 proteins from these three strains, when expressed in Haemophilus influenzae, bound vitronectin and conferred serum resistance on this organism. Furthermore, vitronectin-depleted NHS exhibited bactericidal activity against these same three serum-resistant wild-type strains; addition of purified vitronectin to this serum restored serum resistance. In contrast, binding of the complement regulator C4b-binding protein by the M. catarrhalis strains used in this study was found to be highly variable and did not appear to correlate with the serum-resistant phenotype. These results indicate that binding of vitronectin by UspA2 is involved in the serum resistance of M. catarrhalis; this represents the first example of vitronectin-mediated serum resistance on a microbe.

    INTRODUCTION

    Moraxella catarrhalis is a gram-negative, unencapsulated bacterium that frequently colonizes the human nasopharynx (29, 59). This organism is the third-most-common cause of acute otitis media in infants and very young children (14) and can also cause more serious disease in the respiratory tract of adults, likely being responsible for 2 to 4 million exacerbations of chronic obstructive pulmonary disease each year in the United States (40).

    Among the various phenotypic attributes of M. catarrhalis that could be involved in virulence expression by this organism, its ability to evade complement-mediated killing by normal human serum (NHS) was recognized many years ago (for reviews, see references 12 and 55). van Dijk and colleagues (23) first proposed that serum resistance might be a virulence factor for M. catarrhalis because serum-resistant strains were isolated more frequently from adult patients than from healthy individuals (23, 24, 28). The lack of an appropriate animal model for M. catarrhalis disease precludes direct testing of this hypothesis in vivo, although more recent studies indicate that the majority of M. catarrhalis isolates are serum resistant (36, 62).

    A number of different gene products of M. catarrhalis have been linked to the serum-resistant phenotype. Mutations in four different genes encoding proteins exposed on the surface of the outer membrane including UspA2 (2), CopB (20), OMP CD (25), and OMP E (41) have been shown to have deleterious effects on serum resistance. At least three genes encoding products involved in lipooligosaccharide biosynthesis, including galE (63), kdsA (33), and kdtA (45), have been shown to be necessary for normal expression of serum resistance by M. catarrhalis. To date, however, only the UspA2 protein has been shown to be directly involved in the expression of serum resistance (6).

    UspA2 or the very similar UspA2H protein (30) is expressed by almost every strain of M. catarrhalis studied in this laboratory to date. In a recent survey of nasopharyngeal isolates from a pediatric population in Europe, nearly every strain contained either a uspA2 or uspA2H gene (36). UspA2 is present on the M. catarrhalis cell surface as a dense layer of short projections (22, 44), and purified or recombinant UspA2 proteins have been reported to bind vitronectin (Vn; also known as S protein) (34), C4b-binding protein (C4BP) (42), and fibronectin (56). The physiological relevance of these binding activities remains to be fully elucidated, but binding of the complement regulators vitronectin or C4BP could affect serum resistance. Over a decade ago, another laboratory reported that serum-resistant but not serum-sensitive M. catarrhalis isolates bound vitronectin [C. M. Verduin, M. Jansze, J. Verhoef, A. Fleer, and H. van Dijk, Complement resistance in Moraxella (Branhamella) catarrhalis is mediated by a vitronectin-binding surface protein, abstr. 143, Clin. Exp. Immunol. 97(Suppl. 2):50, 1994]. More recently, binding of C4BP by M. catarrhalis was proposed to interfere with activation of the classical complement pathway (42).

    In the present report, analysis of the interaction of four serum-resistant M. catarrhalis wild-type strains and their serum-sensitive uspA2 mutants with complement components revealed that, for three of these four strains, UspA2 likely interferes with polymerization of C9, thereby interfering with proper formation of the membrane attack complex (MAC) in bacterial outer membranes. The binding of vitronectin to the UspA2 proteins of these three strains was shown to be responsible for this effect. In addition, analysis of mutants showed that C4BP binding was apparently not related to the serum-resistant phenotype of these same M. catarrhalis strains.

    MATERIALS AND METHODS

    Bacterial strains, plasmids, and culture conditions. Bacterial strains and plasmids used in this study are listed in Table 1. M. catarrhalis strains were grown at 37°C in brain heart infusion (BHI) broth (Difco/Becton Dickinson, Sparks, Md.) or on BHI agar plates. When appropriate, BHI was supplemented with kanamycin (15 μg/ml) or spectinomycin (15 μg/ml). Neisseria gonorrhoeae strains were grown in N. gonorrhoeae liquid medium (35) or on chocolate agar plates. Haemophilus influenzae strains were grown at 37°C in BHI broth supplemented with 10% (vol/vol) Levinthal base (4) or on BHI plates with 5% (vol/vol) Levinthal base; when appropriate, ampicillin was added to a final concentration of 10 μg/ml. All agar plates were incubated in an atmosphere containing 95% air-5% CO2.

    Construction of uspA1 and uspA2 mutants of M. catarrhalis strains. Twelve serum-resistant M. catarrhalis strains were subjected to transformation with the suicide plasmid pUSPA1KAN (2) to inactivate their uspA1 genes. Similarly, these same strains were transformed with the suicide plasmid pELU2P44SPEC (30) to inactivate their uspA2 genes. M. catarrhalis strains 7169, FIN2344, and O12E were also transformed with the suicide plasmid pAA2 (6) to construct the uspA2 deletion mutants used in the complement deposition experiments.

    Human sera. NHS was prepared as previously described (6). Inactivation of complement was achieved by heating NHS at 56°C for 30 min to obtain heat-inactivated serum (HIS). To prepare zymosan-activated NHS (ZAS) (used as a positive control for detection of polymerized C9), 80 mg of zymosan A (Sigma, St. Louis, Mo.) was boiled in 2 ml of normal saline for 2 h, and then a 10-μl portion of this suspension was added to 90 μl of NHS and incubated for 1 h at 37°C. Following centrifugation at 10,000 x g for 15 min at 4°C, the supernatant fluid was collected and designated ZAS. Factor B-depleted human serum (lacking alternative complement pathway activity) was purchased from Quidel (San Diego, Calif.).

    Serum bactericidal assay. The ability of bacterial strains to resist the bactericidal activity of NHS was measured by a described previously assay (6). To assess the relative roles of the classical and alternative complement pathways in killing of serum-sensitive M. catarrhalis mutants, factor B-depleted serum was used to selectively block the alternative pathway, and NHS containing 10 mM MgCl2 and 10 mM EGTA was used to selectively block the classical pathway (6).

    Western blot-based detection of complement deposition on N. gonorrhoeae and M. catarrhalis strains. Bacterial strains were grown to mid-logarithmic phase in broth and then resuspended in Veronal-buffered saline (VBS) containing 5 mM MgCl2 and 1.5 mM CaCl2 (VBS++) to a final optical density at 600 nm of 1.0. Portions (500 μl) of these bacterial suspensions were mixed with 400 μl of VBS++ and 100 μl of either NHS or HIS contained in microcentrifuge tubes on ice. Next, the tubes were incubated at 37°C for 20 min. To stop complement activation, the tubes were transferred to crushed ice and incubated for 5 min. The bacterial cells were then pelleted by centrifugation at 16,000 x g for 5 min at 4°C. The cells were washed three times using 1 ml of ice-cold VBS containing 0.1% (wt/vol) gelatin (GVBS). The final pellets were resuspended in 100 μl of phosphate-buffered saline (PBS) and digested by being boiled in 50 μl of 3x digestion buffer (16) to prepare whole-cell lysates for Western blot analysis. To detect SC5b-9 deposition, proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 7.5% (wt/vol) polyacrylamide gels under nonreducing conditions and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, Mass.). For detection of C1q, C4, and C3, proteins were resolved by SDS-PAGE using 12.5% (wt/vol) polyacrylamide gels under reducing conditions (i.e., 5% [vol/vol] 2-mercaptoethanol in digestion buffer) and transferred to nitrocellulose membranes (Schleicher & Schuell BioScience, Keene, N.H.). To detect C7, samples were analyzed as for C1q, C4, and C3 except that the samples were subjected to SDS-PAGE under nonreducing conditions. After incubation of membranes with the appropriate primary antibodies against SC5b-9, C1q, C3, C4, and C7 (Quidel), horseradish peroxidase-conjugated secondary antibodies were added, and antigen-antibody complexes were visualized by chemiluminescence using Western Lightning Chemiluminescence Reagent Plus (New England Nuclear, Boston, Mass.). Antibodies to the M. catarrhalis CopB protein (19) or Hag protein (44) were used to probe membrane-bound samples to verify equivalent loading of samples. To standardize loading for the N. gonorrhoeae samples, membranes were probed using monoclonal antibody (MAb) 2C3, which recognizes the N. gonorrhoeae H.8 lipoprotein (5). This antigen ranges in size from 23.5 kDa to 28.5 kDa among different N. gonorrhoeae strains (21).

    Binding of human serum-derived vitronectin to bacterial cells. Whole-cell lysates were prepared from the four wild-type strains and their respective uspA2 mutants as described above and were used to detect binding of vitronectin present in NHS or HIS to these bacteria. The samples were subjected to SDS-PAGE in 12.5% (wt/vol) polyacrylamide gels under reducing conditions, and the separated proteins were transferred to nitrocellulose membranes and probed with a MAb against human vitronectin (Quidel). For recombinant H. influenzae strains, bacterial cells grown to mid-logarithmic phase were incubated with 10% NHS for 20 min at 37°C, and whole-cell lysates were prepared and analyzed as described above. Samples containing equivalent amounts of recombinant UspA2 were used for these analyses.

    Depletion of vitronectin from NHS. Immunoglobulin G (IgG) antibody was purified from rabbit polyclonal vitronectin antiserum (Advanced Research Technologies, San Diego, Calif.) by using Gamma Bind Plus beads (Amersham Biosciences, Piscataway, N.J.). Approximately 5 mg of this IgG antibody was coupled to 2.5 ml of Affi-Gel Hz Hydrazide Gel using an Affi-Gel Hz Immunoaffinity kit (Bio-Rad, Hercules, Calif.) at room temperature (RT) overnight. To deplete NHS of vitronectin, a 500-μl portion of NHS was incubated with the gel described above in a small chromatography column for 1 h at 4°C with gentle mixing. The liquid was then allowed to drain from the gel. Low-speed centrifugation was used to collect residual liquid from the gel, and all liquid portions were pooled and stored at –70°C until used. As a control, a 500-μl portion of NHS was incubated with a gel that had been coupled to IgG antibody purified from normal rabbit serum under the same conditions described above; the resultant liquid was designated as mock-treated serum. The extent of vitronectin depletion was >90%, as assessed by both Western blot analysis and the use of an enzyme-linked immunosorbent assay for vitronectin (Innovative Research, Southfield, Mich.). When a Western blot control assay was used to determine whether this absorption method affected the complement in the serum, there was little difference in the amounts of C1q in the mock-treated serum and the vitronectin-depleted serum.

    Evaluation of the effect of vitronectin depletion on the bactericidal activity of NHS. Both the vitronectin-depleted serum and the mock-treated serum were used in bactericidal assays at a final concentration of 30% (vol/vol) in VBS++ in a 50-μl reaction volume. In some of these assays, 2.5 μg of purified human monomeric vitronectin (Innovative Research) was added to the vitronectin-depleted serum. In an additional control assay, 2.5 μg of this purified vitronectin was added to 30% HIS, which was then tested for its bactericidal activity.

    Construction of recombinant H. influenzae strains expressing M. catarrhalis UspA2 proteins. The uspA2 open reading frames from M. catarrhalis strains O35E, O12E, FIN2344, and 7169 were PCR amplified using the oligonucleotide primer pair AA52 (5'-GAAAACCATGAAACTTCTCCC-3') and AA54 (5'-TGCCTAGGAAAGCTTTTTGCCTAGGAAAGCTTTT-3'; the AvrII site is underlined). Each of these PCR amplicons was ligated by using PCR sewing (32) to another PCR amplicon that contained the promoter region of the H. influenzae ompP2 gene (39) that had been amplified using the oligonucleotide primers P2-pro-5'-SphI (5'-ACATGCATGCAGATTTATGGATAGCCTTAG-3'; the SphI site is underlined) and P2-pro-3'U2-5' (5'-GTTTCATGGTTTTCATAATTTGTATTCCTTATGGTTG-3'). The final PCR products were digested with both SphI and AvrII, ligated to the SphI/AvrII-digested plasmid pGJB103M (6), and used to electroporate H. influenzae strain DB117 (54). Ampicillin-resistant clones were screened for UspA2 expression by Western blot analysis of whole-cell lysates (6). One UspA2-positive clone derived from each M. catarrhalis strain was verified by nucleotide sequence analysis; these plasmids were designated pAAO35EU2-P2, pAAO12EU2-P2, pAAFIN2344U2-P2, and pAA7169U2-P2, respectively. As a negative control, the ompP2 promoter was cloned into pGJB103M after being amplified with P2-pro-5'-SphI and P2-pro-3'-AvrII (5'-GTCCTAGGAATTTGTATTCCTTATGGTTG-3'; the AvrII site is underlined); the resultant plasmid was designated pAA-P2-pro. These recombinant H. influenzae strains expressed different amounts of the UspA2 proteins, and densitometric analysis with Kodak 1D software, version 3.5.3 (Scientific Imaging Systems, New Haven, CT) was used to quantitate the differences among these strains. To standardize loading for Western blot detection of UspA2, membranes were stained with Ponceau S dye, and a prominent protein band of approximately 40 kDa was used as the internal control.

    Use of flow cytometry to analyze binding of complement components and regulators to bacteria. Binding of purified C4BP to M. catarrhalis and N. gonorrhoeae strains was assessed by using flow cytometry as previously described (42). Briefly, 2 x 108 to 3 x 108 CFU was incubated with 2.5 μg of purified C4BP (Advanced Research Technologies) in PBS containing 3% (wt/vol) fish gelatin (Sigma) (PBS-FG) in a 100-μl reaction volume at 37°C for 1 h. The bacteria were then washed three times with 500 μl PBS-FG, followed by incubation with 1 μg of a MAb to C4BP (Quidel) in a 100-μl final volume at RT for 30 min. After three washes with 500 μl PBS-FG, the bacteria were incubated with 1 μg of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody (Abcam, Cambridge, Mass.) for 30 min at RT. Finally, the bacteria were washed three more times with PBS-FG, and the final pellet was suspended in 1 ml of PBS and analyzed by flow cytometry using a FACScan flow cytometer (Becton-Dickinson). As a negative (isotype) control, all strains were incubated with both the primary and the secondary antibodies but without prior incubation with C4BP.

    To measure binding of C3 derived from NHS, bacteria were incubated with 10% NHS in VBS++ for 20 min at 37°C, put on ice for 5 min, and then washed three times with ice-cold GVBS. The washed cells were then incubated with polyclonal goat antiserum to C3 (Quidel; 1:200 dilution in a 100-μl reaction volume) for 20 min at RT, washed three times, incubated with FITC-conjugated rabbit anti-goat antibody (Abcam), and analyzed by flow cytometry. To measure binding of NHS-derived C4BP, C4c, and C4d, bacteria were incubated with 20% NHS in VBS++ for 30 min at 37°C, put on ice for 5 min, and then washed three times with ice-cold GVBS. The washed cells were incubated with MAbs (1 μg each in a 100-μl reaction volume) specific for C4BP, C4c, or C4d (Quidel) for 20 min at RT, washed three times, incubated with FITC-conjugated goat anti-mouse antibody (Abcam), and analyzed by flow cytometry. As a negative (isotype) control, all strains were incubated with both the primary and the secondary antibodies but without prior incubation with NHS.

    Binding of radiolabeled C4BP to bacteria. Purified C4BP was labeled with [125I]iodine using the chloramine-T method to a specific activity of 805 kcpm/μg. Plate-grown M. catarrhalis or N. gonorrhoeae cells were suspended in PBS supplemented with 2% (wt/vol) bovine serum albumin (BSA). Portions of these suspensions containing 2 x 108 to 3 x 108 CFU were incubated with 300 ng of 125I-labeled C4BP for 1 h at 37°C in a 200-μl volume of PBS-BSA. The bacteria were then washed three times using 500 μl of PBS-BSA, and the radioactivity associated with the bacterial pellet was measured using a gamma counter.

    Statistical analysis. Statistical analysis was carried out by applying Student's t test using Excel 2003 software (Microsoft Corporation, Redmond, WA). P values of <0.05 were considered significant.

    RESULTS

    Effect of uspA1 and uspA2 mutations on serum resistance. The UspA2 protein of M. catarrhalis strain O35E has been shown to be directly involved in the serum-resistant phenotype of this strain (6). Recently, a uspA1 mutant of a different M. catarrhalis strain (BBH18) was reported to have significantly reduced resistance to serum killing (42). To determine whether other strains of this pathogen might be dependent on UspA1 for serum resistance, uspA1 and uspA2 mutants of 11 additional M. catarrhalis strains were tested in a serum bactericidal assay using 10% NHS (Fig. 1). These strains were isolated in five different geographic areas: Texas, New York, North Carolina, Tennessee, and Finland. In every set of strains tested, the uspA1 mutant either was as serum resistant as its parent strain or had only slightly reduced serum resistance, whereas each uspA2 mutant was exquisitely serum sensitive (Fig. 1).

    uspA2 mutants of M. catarrhalis are killed via the classical complement pathway. The preceding results reaffirmed the importance of the UspA2 protein in serum resistance. To investigate further how these strains evaded complement-dependent killing by NHS, four strains of M. catarrhalis (O35E, O12E, FIN2344, and 7169) were selected for detailed examination. These strains were found to differ in the extent and means by which they interacted with the complement regulator C4BP (discussed below). Newly constructed uspA2 deletion mutants of strains O12E, FIN2344, and 7169 were used together with the previously described O35E uspA2 deletion mutant O35E2 (6) in a series of bactericidal activity assays. All four wild-type strains and their uspA2 mutants survived equally in heat-inactivated NHS (Fig. 2, black bars). As expected from previous studies (2, 6), the four wild-type strains resisted killing by NHS, while their uspA2 mutants were exquisitely serum sensitive (Fig. 2, white bars). To investigate the role of the alternative pathway in this bactericidal activity, factor B-depleted serum (which has a nonfunctional alternative pathway) was tested; it was found that the wild-type strains survived, whereas the uspA2 mutants were readily killed (Fig. 2, gray bars). This result indicated that the classical pathway alone is sufficient for the killing of these uspA2 mutants. To selectively block the classical pathway while retaining alternative pathway function, these wild-type strains and mutants were incubated with NHS in the presence of MgCl2 and EGTA (Fig. 2, striped bars). With all four strain sets, both the wild-type strains and the uspA2 mutants survived at equivalent levels, indicating that the classical pathway initiates and sustains killing of these uspA2 mutants by NHS. In a control experiment, NHS containing MgCl2 and EGTA was tested in a hemolytic assay with rabbit erythrocytes and shown to be sufficient in alternative pathway activity (data not shown). It should be noted that, in a previous study (6), killing of the serum-sensitive uspA2 mutant O35E2 by NHS was shown to be IgG dependent.

    Deposition of the early components of the complement cascade on M. catarrhalis. To determine the stage of the complement cascade that was blocked by these four serum-resistant wild-type M. catarrhalis strains, these strains and their uspA2 mutants were incubated with either HIS or NHS, lysed, and probed by Western blotting to detect deposited complement components. To obtain points of reference, these same procedures were performed with the well-characterized N. gonorrhoeae strains FA19 (serum resistant) and UU1 (serum sensitive) (51) as positive and negative controls, respectively, for classical pathway-mediated activity. Strain FA19 is serum resistant by virtue of its ability to bind C4BP (51).

    C1q. The first complement component is C1q, which binds to the antibody-antigen complex and triggers the activation of the classical complement pathway (53). No C1q deposition on the N. gonorrhoeae strains was observed (Fig. 3A). In contrast, C1q deposition on M. catarrhalis was readily detectable. Equivalent amounts of C1q appeared to be deposited on the wild-type parent strain and the respective uspA2 mutants of O35E, O12E, and FIN2344. The uspA2 mutant of 7169 bound little or no detectable C1q for reasons that are not clear at this time. As expected, no C1q deposition was observed with either the wild-type strains or the uspA2 mutants incubated with HIS (Fig. 3A).

    C4. The subsequent step in classical pathway activation is C4 binding. C4 is composed of three polypeptide chains, the -chain (93 kDa), -chain (75 kDa), and the -chain (33 kDa), linked by disulfide bonds. Upon activation, a small fragment (C4a) is released from the -chain, allowing the C4b fragment to attach covalently to the target through an internal thioester bond in its -chain. The polyclonal antiserum to C4 used in the present study reacted primarily with the 75 kDa -chain of C4/C4b (Fig. 3B). With the N. gonorrhoeae control strains, there was more C4b detected on strain UU1 than on FA19 (Fig. 3B). There were no appreciable differences in the amount of C4b deposited on the four wild-type M. catarrhalis strains compared to their uspA2 mutants (Fig. 3B), suggesting that UspA2 did not affect the complement system prior to this step. These data also indicate that the extent of C4b processing on the wild type and uspA2 mutants was similar because the C4b -chain is associated only with unprocessed C4b. As expected, C4b was not detected on bacteria incubated with HIS (Fig. 3B).

    C3. C3 represents the most abundant complement protein and plays a critical role in both the classical and alternative pathways (38). It is composed of a 117-kDa -chain and a 75-kDa -chain held together by disulfide bonds. Akin to C4, activation of C3 results in covalent binding of C3b via a labile thioester directly to bacterial targets or to the activating C3-convertases (C4bC2a or C3bBb) already assembled on the bacterial surface, the latter leading to the formation of the C5 convertases that are required for further activation of the terminal pathway (38). More C3b was deposited on N. gonorrhoeae UU1 (serum sensitive and does not bind C4BP) than on N. gonorrhoeae FA19 (serum resistant and binds C4BP) (Fig. 3C). Differences in the amounts of C3 deposited on the wild-type M. catarrhalis strains and their uspA2 mutants appeared to be modest at best. To measure this more quantitatively, flow cytometry was used to measure C3 binding. The serum-sensitive N. gonorrhoeae strain UU1 bound more C3 than did the serum-resistant FA19 strain (Fig. 3D), confirming the difference seen by Western blot analysis (Fig. 3C). Of the four pairs of M. catarrhalis wild-type strains and uspA2 mutants, only the FIN2344 wild-type strain bound significantly less C3 than its uspA2 mutant (Fig. 3D). As expected, C3 was not detected on bacteria incubated with HIS (Fig. 3C).

    Deposition of late components of the complement cascade on M. catarrhalis strains. The late stages of complement activation include the formation of the MAC, which starts with the activation of C5, leading to the generation of C5b and the sequential binding of C6, C7, and C8. The last component to participate in this complex is C9, which undergoes a conformational change from a globular to an elongated form and binds to the C5b-8 complex, which traverses the bacterial outer membrane. Finally, more C9 is recruited to form a C9 polymer, leading to the formation of pores in the membrane and eventually the death of the cell (38). The deposition of two components (i.e., C7 and polymerized C9) of the MAC on bacterial cells was analyzed in this study.

    C7. Consistent with the results obtained with C4 and C3, there was a reduction in the amount of C7 deposited on N. gonorrhoeae strain FA19 relative to that deposited on strain UU1 (Fig. 4A). The uspA2 mutants of strains O35E, O12E, and FIN2344 appeared to bind slightly more C7 than their respective wild-type parent strains. As expected, C7 was not detected on bacteria incubated with HIS (Fig. 4A).

    Polymerized C9. The MAb used to detect polymerized C9 recognizes a neoepitope that is formed when C9 polymerizes within the MAC. More polymerized C9 was detected on the serum-sensitive N. gonorrhoeae strain UU1 than was bound to the serum-resistant strain FA19 (Fig. 4B). A difference was readily apparent between the wild-type M. catarrhalis strains and their uspA2 mutants, with more polymerized C9 being associated with the mutants (Fig. 4B). Of note, the four strains differed in their "patterns" of C9 polymerization. C9 was distributed equally as trimers and multimers on wild-type FIN2344, and it occurred solely as trimers on wild-type 7169. A faint multimeric C9 band was seen on wild-type O35E, while no C9 was detected on wild-type O12E. In every instance, the uspA2 mutants showed strongly reactive C9 multimers. A decrease of the C9 trimer band with intensification of the C9 multimer band was seen with the FIN2344 uspA2 mutant. Similarly, a shift in C9 reactivity from trimer to multimers was seen with the 7169 uspA2 mutant (Fig. 4B). These differences that occurred at the level of C9 polymerization raised the possibility that the role of the UspA2 protein in serum resistance might involve interference with the final crucial step in activation of the complement cascade.

    M. catarrhalis binds vitronectin from NHS through its UspA2 protein. Several factors have been previously reported to interfere with formation of the MAC. These include vitronectin, clusterin (also known as SP 40,40) and CD59 (also known as protectin or homologous restriction factor) (43, 47, 58). It was also previously reported that purified UspA2 protein from M. catarrhalis strain O35E bound purified vitronectin (34). When the four wild-type M. catarrhalis strains and their respective uspA2 mutants were tested, the wild-type strains O35E, O12E, and 7169 bound more NHS-derived vitronectin than did their uspA2 mutants (Fig. 5). The 7169 uspA2 mutant showed the most dramatic reduction in vitronectin binding relative to its wild-type parent strain. Heat inactivation of the complement system did not eliminate binding of vitronectin to these three wild-type strains (Fig. 5), suggesting that vitronectin binding occurred directly to the bacterium, independently of MAC formation. It should be noted that the wild-type strain FIN2344 bound much smaller amounts of vitronectin than did the other three wild-type strains (Fig. 5).

    Serum-resistant M. catarrhalis strains are killed by vitronectin-depleted NHS. If NHS-derived vitronectin bound to M. catarrhalis inhibited polymerization of C9, then removal of vitronectin from NHS should result in an increased susceptibility of the serum-resistant strains to killing by NHS. To test this hypothesis, vitronectin-depleted NHS (Vn-depleted serum) was used in bactericidal assays with the four serum-resistant M. catarrhalis strains and their uspA2 mutants. The wild-type strains O35E, O12E, and 7169 (which had been shown to bind vitronectin through UspA2) all exhibited a significant increase in serum susceptibility when incubated in Vn-depleted serum (Fig. 6A, B, and D, respectively). Moreover, when purified vitronectin was added to the Vn-depleted serum, these three wild-type strains exhibited their normal serum-resistant phenotype (Fig. 6A, B, and D, respectively). To rule out the presence of bactericidal activity in the purified vitronectin preparation, equal amounts of purified vitronectin were added to HIS and shown to have no effect on the survival of these three wild-type strains (Fig. 6). Interestingly, strain FIN2344, which exhibited a very low level of vitronectin binding (Fig. 5), did not show any increase in susceptibility to killing by Vn-depleted serum (Fig. 6C). The uspA2 mutants of all four strains were exquisitely sensitive to killing by both Vn-depleted serum and mock-treated serum (Fig. 6).

    Recombinant M. catarrhalis UspA2 proteins expressed in H. influenzae confer serum resistance and bind vitronectin. The uspA2 genes from these four M. catarrhalis strains were cloned and expressed in H. influenzae strain DB117 under the control of the H. influenzae ompP2 promoter on a multicopy plasmid. These recombinant H. influenzae strains expressed various levels of UspA2 protein, with the 7169 UspA2 protein being expressed at a level approximately three to fivefold lower than the others, as determined by densitometry (Fig. 7A). However, all four recombinant H. influenzae strains expressing UspA2 proteins were fully resistant to the bactericidal activity of 10% NHS, whereas the H. influenzae strain carrying the vector plasmid with the ompP2 promoter was fully serum sensitive (Fig. 7B). Cells of these four recombinant H. influenzae strains were also tested for their ability to bind vitronectin from NHS. When cell lysates containing equivalent amounts of UspA2 proteins from these recombinant strains were probed with the vitronectin MAb, it was found that the recombinants expressing the O35E, O12E, and 7169 UspA2 proteins all bound readily detectable amounts of vitronectin (Fig. 7C). No binding of vitronectin was observed with the vector-only strain, and barely any binding was observed with the strain expressing the FIN2344 UspA2 protein (Fig. 7C). When the amount of recombinant FIN2344 UspA2 protein was increased in this assay, vitronectin binding was readily detectable (data not shown). These data confirmed that M. catarrhalis strains O35E, O12E, and 7169 bound NHS-derived vitronectin through their UspA2 protein while the FIN2344 UspA2 protein exhibited much less vitronectin binding.

    M. catarrhalis strains differ in their binding of purified C4BP. It was recently reported that two serum-resistant M. catarrhalis strains (i.e., RH4 and BBH18) bound the complement regulator C4BP mainly through their UspA2 protein (42). However, the preceding experiments indicated that UspA2-mediated vitronectin binding was most likely responsible for the serum-resistant phenotype of M. catarrhalis strains O35E, O12E, and 7169. To determine whether C4BP binding was involved with the serum-resistant phenotype of these three strains, we tested these three strains and their uspA1 and uspA2 mutants for their ability to bind purified C4BP. In addition, we tested the other nine wild-type, serum-resistant M. catarrhalis strains and their mutants (Fig. 1) for this binding activity. Flow cytometric analysis revealed that these 12 strains differed markedly in both the amount of C4BP bound and the binding moiety; these differences are summarized in Table 2. N. gonorrhoeae strains FA19 and UU1 (51) were used as positive and negative controls, respectively, for C4BP binding in these flow cytometric experiments (Fig. 8A and B).

    Data from four M. catarrhalis strains representing the different C4BP binding phenotypes (Table 2) are presented in Fig. 8. The wild-type O35E strain (Fig. 8C) bound low levels of purified C4BP; a further reduction in C4BP binding was observed with its uspA1 mutant (Fig. 8D), whereas its uspA2 mutant (Fig. 8E) bound marginally more C4BP than the wild type. The wild-type O12E strain (Fig. 8F) appeared to bind very little C4BP, and the uspA1 (Fig. 8G) and uspA2 (Fig. 8H) mutations had little effect on this minimal binding. Strain FIN2344 (Fig. 8I) bound a relatively large amount of C4BP, mainly via its UspA1 protein, because its uspA1 mutant (Fig. 8J) bound almost no C4BP, whereas its uspA2 mutant (Fig. 8K) bound levels of C4BP only slightly lower than those bound by the wild-type strain. Finally, the wild-type 7169 strain (Fig. 8L) and its uspA1 mutant (Fig. 8M) both bound large amounts of C4BP, comparable to C4BP binding by the positive control strain N. gonorrhoeae FA19 (Fig. 8A), whereas there was a very large reduction in binding observed with the 7169 uspA2 mutant (Fig. 8N).

    Binding of radiolabeled C4BP to M. catarrhalis strains. Measurement of the binding of radioiodinated C4BP to these strains was used to confirm the flow cytometry-derived data described above. N. gonorrhoeae FA19 and UU1 (Fig. 9A) were used as positive and negative controls, respectively, for C4BP binding. M. catarrhalis strain O35E (Fig. 9B) bound 10-fold-less C4BP (i.e., 3,000 cpm) than N. gonorrhoeae FA19. The O35E uspA1 and uspA2 mutants (Fig. 9B) bound less C4BP than O35E. Once again, the O12E wild type and its respective uspA1 and uspA2 mutants (Fig. 9C) bound barely detectable amounts (i.e., 400 to 800 cpm) of C4BP. M. catarrhalis FIN2344 (Fig. 9D), and its uspA2 mutant again exhibited high-level binding of C4BP (i.e., 45,000 to 49,000 cpm), whereas its uspA1 mutant bound barely detectable levels of C4BP. Both the wild-type 7169 strain (Fig. 9E) and its uspA1 mutant bound very high levels of C4BP (i.e., 60,000 cpm), whereas the 7169 uspA2 mutant bound barely detectable levels. Collectively, these results confirm those obtained by flow cytometry.

    Measurement of the cofactor activity of C4BP bound to M. catarrhalis cells. C4BP can function as a cofactor for factor I to inactivate surface-bound C4b by cleaving it to release the C4c fragment while the C4d fragment remains attached to the bacterial surface. The C4c MAb used in this study recognizes both C4b and C4c but not C4d (51). In contrast, the C4d MAb used in this study binds both C4b and the C4d fragment (51). Therefore, C4d binding is a reflection of the number of C4b molecules deposited on the bacterium and is not influenced by C4b processing. In contrast, C4BP cofactor activity will result in the release of C4c into solution and a corresponding decrease in binding of the C4c MAb. A higher C4d/C4c ratio indicates more C4BP cofactor function on the bacterial surface (51).

    We used this assay in the context of NHS and measured cofactor activity of C4BP bound on two M. catarrhalis strains, FIN2344 and 7169, which had bound large amounts of purified C4BP (Fig. 8) via their UspA1 or UspA2 proteins, respectively. The C4BP-binding positive control strain N. gonorrhoeae FA19 had a C4d/C4c ratio of 2.9 (Fig. 10A), whereas the N. gonorrhoeae negative control strain UU1 yielded a C4d/C4c ratio of 0.8 (Fig. 10A); this difference was significant (P = 0.0007). When the wild-type M. catarrhalis strains FIN2344 (Fig. 10B) and 7169 (Fig. 10C) were tested by this assay, it was found that the level of cofactor activity was significantly less (P = 0.005 for FIN2344 and P = 0.002 for 7169) than that observed with N. gonorrhoeae FA19 (Fig. 10A). In addition, when these two wild-type M. catarrhalis strains were compared to their respective mutants that did not bind C4BP (i.e., the FIN2344 uspA1 mutant and the 7169 uspA2 mutant), there was no decrease in the C4d/C4c ratios (Fig. 10B and C, respectively).

    These C4BP cofactor activity results suggested either that these two wild-type M. catarrhalis strains, which readily bound purified C4BP at levels equivalent to those bound by N. gonorrhoeae FA19 (Fig. 8), did not bind much C4BP from NHS or that this NHS-derived C4BP was not functional after binding to M. catarrhalis. To address these two possibilities, the amounts of C4BP bound to the same M. catarrhalis cells used in the cofactor activity assays were measured by flow cytometry (Fig. 10F to K). It was noted that N. gonorrhoeae FA19 (Fig. 10D) continued to bind C4BP in the context of NHS very well, while the negative control strain UU1 (Fig. 10E) remained a C4BP nonbinder. In contrast, the two wild-type M. catarrhalis strains (Fig. 10F and I) bound much lower levels of NHS-derived C4BP than did N. gonorrhoeae FA19 (Fig. 10D) and relative to the amounts of purified C4BP bound by these same two strains (Fig. 8 and 9). Under these conditions, the levels of C4BP bound to the wild-type M. catarrhalis strains and their uspA1 and uspA2 mutants (Fig. 10F to K) were low and similar. Collectively, these data provide an explanation for the low and similar C4BP cofactor activity seen among these M. catarrhalis wild-type strains and mutants (Fig. 10B and C).

    DISCUSSION

    The UspA2 protein was previously shown to be directly involved in the serum-resistant phenotype of M. catarrhalis strain O35E (6). In the present study, mutant analysis of an additional 11 wild-type M. catarrhalis isolates proved that expression of UspA2 was essential for serum resistance of these strains (Fig. 1). UspA2, which forms a dense layer of projections on the surface of M. catarrhalis (22, 44), represents an ideal moiety to interact with components or regulators of the complement system. In the present study, we investigated how M. catarrhalis interacts with different components of the complement system in NHS. For this purpose, four serum-resistant strains (O35E, O12E, FIN2344, and 7169) were selected that had already been shown to differ markedly in their binding of purified C4BP (Table 2). Killing of uspA2 mutants of these four strains by NHS was shown to involve the classical complement pathway (Fig. 2). It should be noted that a recent study indicated that M. catarrhalis only weakly activates the mannose-binding lectin pathway of complement activation (17). Analysis of complement deposition on these four M. catarrhalis strains and their uspA2 mutants showed that a readily detectable difference between each pair occurred at the late stages of complement activation, involving the polymerization of C9 and MAC formation (Fig. 4B). These data, based on direct detection of complement components bound to the surface of wild-type and uspA2 mutant pairs of M. catarrhalis, are consistent with an earlier report in which a hemolytic assay was used to measure consumption of complement components by serum-sensitive and serum-resistant isolates of M. catarrhalis (60).

    The finding that UspA2 appeared to interfere with MAC formation, at least in three of the four serum-resistant strains tested in this study, suggested that UspA2 might be either interfering directly with proper MAC formation or binding a regulator that affects this critical step. Examples of both mechanisms are documented in the literature for other serum-resistant microorganisms. For example, the O-polysaccharide of Salmonella enterica serovar Minnesota seemed to physically interfere with MAC insertion into the outer membrane by causing assembly of the MAC away from the bacterial surface (27). Salmonella enterica serovar Typhimurium resisted complement-mediated killing by using its Rck protein to prevent polymerization of C9 (18), whereas Borrelia burgdorferi encodes a CD59-like protein that interferes with MAC formation (43). Other bacteria recruit natural regulators of the complement system that interfere with MAC formation. Rautemaa et al. (52) showed that Escherichia coli can incorporate glycophosphoinositol-anchored protectin (CD59) and acquire resistance to NHS-mediated killing. Other pathogens, especially gram-positive organisms, including both staphylococci (31) and streptococci (13), can bind vitronectin or clusterin or both, although these organisms are not lysed by the MAC.

    Interestingly, M. catarrhalis is one of those pathogens that has been reported to bind the MAC regulator vitronectin. M. catarrhalis clinical isolates were reported to differ in their binding of vitronectin, although the bacterial gene product responsible for this binding activity was not identified [Verduin et al., Clin. Exp. Immunol. 97(Suppl. 2):50, 1994]. McMichael et al. (34) reported that purified UspA2 from M. catarrhalis strain O35E bound purified vitronectin in a dot blot assay. For the first time, a direct correlation was demonstrated between UspA2-mediated binding of vitronectin and the serum-resistant phenotype, at least for three of the four M. catarrhalis strains tested in the present study. This was supported by three findings. First, serum-resistant wild-type strains bound more vitronectin from NHS than did their serum-sensitive uspA2 mutants (Fig. 5). Second, expression of UspA2 from these same three strains in a heterologous genetic background (i.e., H. influenzae) resulted in serum resistance concurrent with vitronectin binding (Fig. 7). Third, vitronectin depletion of NHS resulted in a significant increase in the susceptibility of these three serum-resistant M. catarrhalis strains to bactericidal activity; addition of purified vitronectin to the vitronectin-depleted NHS restored the serum-resistant phenotype of these strains (Fig. 6).

    Vitronectin has been shown to regulate complement at the level of MAC assembly at two stages. (i) It binds to the metastable membrane-binding site of C5b-7 (46, 49). (ii) It binds to the assembling C5b-9 complex and prevents tubular polymerized C9 formation (47). The heterogeneity of C9 complexes seen on the different M. catarrhalis strains used in this study may be explained by regulation of MAC formation at two levels. Blockade at the level of C5b-7 would lead to the absence of C9 trimer, as seen with the wild-type strain O12E (Fig. 4B) and might explain the slight increase in C7 bound by the O35E and O12E uspA2 mutants (Fig. 4A). Blockade at the level of C5b-9 may permit formation of C9 trimers but not polymers, as seen with the wild-type strain 7169 (Fig. 4B). The increased amount of C9 polymerization on the FIN2344 uspA2 mutant compared to the wild-type strain, both of which bound vitronectin very poorly, may suggest that UspA2 itself could block further C9 polymerization. It is also possible that UspA2 may be involved directly, interfering with insertion of fully formed C5b-9 into the bacterial outer membrane. Vitronectin is a multifunctional protein; another important function it subserves is cell attachment by binding to the v3 and v5 integrins via its Arg-Gly-Asp (RGD) domain (50). It is possible that vitronectin binding to M. catarrhalis may facilitate bacterial attachment to human cells and merits further study.

    Nordstrm et al. (42) recently proposed that M. catarrhalis interferes with activation of the classical complement pathway by binding the complement regulator C4BP to its UspA1 and UspA2 proteins. C4BP interferes with the classical complement pathway by both acting as a cofactor with factor I in the cleavage of C4b and inhibiting the formation and accelerating the decay of the C3 convertase (10). In fact, within the past decade, there have been numerous reports describing binding of C4BP to different pathogens and the association of this binding with either serum resistance or increased virulence. These pathogens that bind C4BP include Streptococcus pyogenes (57), N. gonorrhoeae (51), Escherichia coli K1 (48), and Neisseria meningitidis (26). A number of these pathogens interact with the second complement control protein domain (CCP2) of the eight CCP domains on the -chain of C4BP (42). M. catarrhalis is the first bacterium described that uses CCP7 as a recognition site for C4BP binding; in addition, it can also bind to CCP2 and (to a lesser extent) CCP5 (42). With the exception of M. catarrhalis, CCP1 to CCP3 are the major domains of the C4BP -chain that are used for binding by several bacterial pathogens (7-9, 48, 57). No specific feature of C4BP binding has yet been identified that, together with factor I, maximizes processing of C4b, which will yield bound C4d in excess of C4c, but we speculate that the specificity of CCP binding and its nature (ionic versus hydrophobic) may vary among M. catarrhalis isolates, thereby producing some that are more susceptible to C4BP processing than others.

    When 12 serum-resistant M. catarrhalis strains were tested for their binding of C4BP in the present study, it was found that these strains differed in both the amount of C4BP bound and in whether this binding involved UspA1 or UspA2 (Table 2). It was expected that differences in total C4BP binding by these wild-type strains might be reflected by differences in serum resistance. However, even those wild-type strains that bound very small amounts of C4BP (i.e., strain O12E) (Fig. 8) were as resistant to killing by 10% NHS as strains that bound very large amounts of C4BP (i.e., strain 7169) (Fig. 8). Moreover, it was observed that all the uspA1 mutants were essentially as serum resistant as their wild-type parent strains and that the uspA2 mutants were consistently serum sensitive (Fig. 1), regardless of their ability to bind C4BP. A similar phenomenon was previously reported for Bordetella pertussis, whose filamentous hemagglutinin (designated FHA) protein binds C4BP; a B. pertussis fha mutant that did not bind C4BP was found to be serum resistant (15). Similarly, C4BP has been shown to bind the gonococcal type IV pilus (9), but complement regulation and serum resistance as a result of this interaction were not demonstrated.

    Among the strains tested in this study, those that bound the largest amounts of purified C4BP were FIN2344 and 7169, which used their UspA1 or UspA2 proteins, respectively, for this purpose (Fig. 8 and 9). It must be noted, however, that in the serum bactericidal assay (and likely in vivo), M. catarrhalis encounters C4BP in the context of other serum components. When these two wild-type strains and their uspA1 and uspA2 mutants were incubated with NHS and tested for C4BP cofactor activity (Fig. 10), there were no significant differences between the wild-type strains and their respective mutants which did not bind purified C4BP. Additional experiments measuring the binding of C4BP from NHS to these M. catarrhalis strains (Fig. 10) established that the lack of cofactor activity could be attributed to a marked decrease in C4BP binding to the wild-type strains in the presence of serum. Nordstrm et al. (42) reported that purified C4BP bound to wild-type M. catarrhalis strains retained its cofactor activity; however, a comparative analysis of cofactor function on mutants that did not bind C4BP was not included in that study. We speculate that binding of C4BP to M. catarrhalis is blocked or severely impeded by a serum component.

    It is important to note that resistance of the wild-type M. catarrhalis strains O35E, O12E, and 7169 to killing by NHS was not completely eliminated by vitronectin depletion (Fig. 6), a result which suggests that vitronectin binding is not the only mechanism protecting these strains. However, at least for these three strains that bound vitronectin to avoid serum killing, binding of C4BP appeared not to be involved in serum resistance.

    The likely existence of another UspA2-mediated serum resistance mechanism that does not involve vitronectin is reinforced by the results obtained with the FIN2344 strain and its uspA2 mutant. Increased binding of C3 by the FIN2344 uspA2 mutant (Fig. 3D) suggests that the UspA2 protein of FIN2344 somehow interferes, directly or indirectly, with the early stages of complement deposition. This increase in complement deposition on the FIN2344 uspA2 mutant, relative to the wild-type parent strain, was also observed with both C7 and polymerized C9 (Fig. 4). Expression of the FIN2344 UspA2 protein in H. influenzae resulted in serum resistance, but the level of vitronectin binding was much reduced compared to that obtained with the recombinant UspA2 proteins from strains O35E, O12E, and 7169. That this serum resistance phenotype is likely independent of vitronectin binding is reinforced by the observation that vitronectin-depleted NHS was not able to kill the wild-type FIN2344 strain (Fig. 6). Exactly how the FIN2344 UspA2 protein confers serum resistance on this strain is not apparent from the available data. It is possible that the FIN2344 UspA2 protein somehow interferes with complement deposition or that it binds a complement inhibitor or regulator present in NHS. This additional mechanism(s) remains to be identified and reinforces the fact that multiple and sometimes redundant mechanisms may be crucial for a bacterial species to survive in a hostile environment.

    ACKNOWLEDGMENTS

    This study was supported by U.S. Public Health Service grants no. AI36344 to E.J.H., AI054544 to S.R., and AI32725 to P.A.R.

    We thank Robert S. Munson, Jr., for providing the oligonucleotide primers for PCR-based amplification of the ompP2 promoter. We thank John Nelson, Anthony Campagnari, Steven Berk, Frederick Henderson, and Merja Helminen for supplying many of the isolates of M. catarrhalis used in this study.

    REFERENCES

    1. Aebi, C., L. D. Cope, J. L. Latimer, S. E. Thomas, C. A. Slaughter, G. H. McCracken, Jr., and E. J. Hansen. 1998. Mapping of a protective epitope of the CopB outer membrane protein of Moraxella catarrhalis. Infect. Immun. 66:540-548.

    2. Aebi, C., E. R. Lafontaine, L. D. Cope, J. L. Latimer, S. R. Lumbley, G. H. McCracken, Jr., and E. J. Hansen. 1998. Phenotypic effect of isogenic uspA1 and uspA2 mutations on Moraxella catarrhalis O35E. Infect. Immun. 66:3113-3119.

    3. Aebi, C., I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas, G. H. McCracken, Jr., and E. J. Hansen. 1997. A protective epitope of Moraxella catarrhalis is encoded by two different genes. Infect. Immun. 65:4367-4377.

    4. Alexander, H. E. 1965. The Haemophilus group, p. 724-741. In R. J. Dubos and J. G. Hirsch (ed.), Bacterial and mycotic infections of man. J. B. Lippincott Co., Philadelphia, Pa.

    5. Apicella, M. A., R. E. Mandrell, M. Shero, M. E. Wilson, J. M. Griffiss, G. F. Brooks, C. Lammel, J. F. Breen, and P. A. Rice. 1990. Modification by sialic acid of Neisseria gonorrhoeae lipooligosaccharide epitope expression in human urethral exudates: an immunoelectron microscopic analysis. J. Infect. Dis. 162:506-512.

    6. Attia, A. S., E. R. Lafontaine, J. L. Latimer, C. Aebi, G. A. Syrogiannopoulos, and E. J. Hansen. 2005. The UspA2 protein of Moraxella catarrhalis is directly involved in the expression of serum resistance. Infect. Immun. 73:2400-2410.

    7. Berggard, K., E. Johnsson, F. R. Mooi, and G. Lindahl. 1997. Bordetella pertussis binds the human complement regulator C4BP: role of filamentous hemagglutinin. Infect. Immun. 65:3638-3643.

    8. Blom, A. M., K. Berggard, J. H. Webb, G. Lindahl, B. O. Villoutreix, and B. Dahlback. 2000. Human C4b-binding protein has overlapping, but not identical, binding sites for C4b and streptococcal M proteins. J. Immunol. 164:5328-5336.

    9. Blom, A. M., A. Rytkonen, P. Vasquez, G. Lindahl, B. Dahlback, and A. B. Jonsson. 2001. A novel interaction between type IV pili of Neisseria gonorrhoeae and the human complement regulator C4B-binding protein. J. Immunol. 166:6764-6770.

    10. Blom, A. M., B. O. Villoutreix, and B. Dahlback. 2004. Complement inhibitor C4b-binding protein—friend or foe in the innate immune system Mol. Immunol. 40:1333-1346.

    11. Campagnari, A. A., T. F. Ducey, and C. A. Rebmann. 1996. Outer membrane protein B1, an iron-repressible protein conserved in the outer membrane of Moraxella (Branhamella) catarrhalis, binds human transferrin. Infect. Immun. 64:3920-3924.

    12. Chapman, A. J., Jr., D. M. Musher, S. Jonsson, J. E. Clarridge, and R. J. Wallace, Jr. 1985. Development of bacterial antibody during Branhamella catarrhalis infection. J. Infect. Dis. 151:878-882.

    13. Chhatwal, G. S., K. T. Preissner, G. Muller-Berghaus, and H. Blobel. 1987. Specific binding of the human S protein (vitronectin) to streptococci, Staphylococcus aureus, and Escherichia coli. Infect. Immun. 55:1878-1883.

    14. Faden, H. 2001. The microbiologic and immunologic basis for recurrent otitis media in children. Eur. J. Pediatr. 160:407-413.

    15. Fernandez, R. C., and A. A. Weiss. 1998. Serum resistance in bvg-regulated mutants of Bordetella pertussis. FEMS Microbiol. Lett. 163:57-63.

    16. Gulig, P. A., G. H. McCracken, Jr., C. F. Frisch, K. H. Johnston, and E. J. Hansen. 1982. Antibody response of infants to cell surface-exposed outer membrane proteins of Haemophilus influenzae type b after systemic Haemophilus disease. Infect. Immun. 37:82-88.

    17. Hays, J. P., A. Ott, C. M. Verduin, A. van Belkum, and S. Kuipers. 2005. Moraxella catarrhalis is only a weak activator of the mannose-binding lectin (MBL) pathway of complement activation. FEMS Microbiol. Lett. 249:207-209.

    18. Heffernan, E. J., S. Reed, J. Hackett, J. Fierer, C. Roudier, and D. Guiney. 1992. Mechanism of resistance to complement-mediated killing of bacteria encoded by the Salmonella typhimurium virulence plasmid gene rck. J. Clin. Investig. 90:953-964.

    19. Helminen, M. E., I. Maciver, J. L. Latimer, L. D. Cope, G. H. McCracken, Jr., and E. J. Hansen. 1993. A major outer membrane protein of Moraxella catarrhalis is a target for antibodies that enhance pulmonary clearance of the pathogen in an animal model. Infect. Immun. 61:2003-2010.

    20. Helminen, M. E., I. Maciver, J. L. Latimer, S. R. Lumbley, L. D. Cope, G. H. McCracken, Jr., and E. J. Hansen. 1993. A mutation affecting expression of a major outer membrane protein of Moraxella catarrhalis alters serum resistance and survival of this organism in vivo. J. Infect. Dis. 168:1194-1201.

    21. Hitchcock, P. J., S. F. Hayes, L. W. Mayer, W. M. Shafer, and S. L. Tessier. 1985. Analyses of gonococcal H8 antigen. Surface location, inter- and intrastrain electrophoretic heterogeneity, and unusual two-dimensional electrophoretic characteristics. J. Exp. Med. 162:2017-2034.

    22. Hoiczyk, E., A. Roggenkamp, M. Reichenbecher, A. Lupas, and J. Heesemann. 2000. Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins. EMBO J. 19:5989-5999.

    23. Hol, C., C. M. Verduin, E. van Dijke, J. Verhoef, and H. van Dijk. 1993. Complement resistance in Branhamella (Moraxella) catarrhalis. Lancet 341:1281.

    24. Hol, C., C. M. Verduin, E. E. A. Van Dijke, J. Verhoef, A. Fleer, and H. van Dijk. 1995. Complement resistance is a virulence factor of Branhamella (Moraxella) catarrhalis. FEMS Immunol. Med. Microbiol. 11:207-212.

    25. Holm, M. M., S. L. Vanlerberg, I. M. Foley, D. D. Sledjeski, and E. R. Lafontaine. 2004. The Moraxella catarrhalis porin-like outer membrane protein CD is an adhesin for human lung cells. Infect. Immun. 72:1906-1913.

    26. Jarva, H., S. Ram, U. Vogel, A. M. Blom, and S. Meri. 2005. Binding of the complement inhibitor C4bp to serogroup B Neisseria meningitidis. J. Immunol. 174:6299-6307.

    27. Joiner, K. A., N. Grossman, M. Schmetz, and L. Leive. 1986. C3 binds preferentially to long-chain lipopolysaccharide during alternative pathway activation by Salmonella montevideo. J. Immunol. 136:710-715.

    28. Jordan, K. L., S. H. Berk, and S. L. Berk. 1990. A comparison of serum bactericidal activity and phenotypic characteristics of bacteremic, pneumonia-causing strains, and colonizing strains of Branhamella catarrhalis. Am. J. Med. 88:28S-32S.

    29. Karalus, R., and A. Campagnari. 2000. Moraxella catarrhalis: a review of an important human mucosal pathogen. Microbes Infect. 2:547-559.

    30. Lafontaine, E. R., L. D. Cope, C. Aebi, J. L. Latimer, G. H. McCracken, Jr., and E. J. Hansen. 2000. The UspA1 protein and a second type of UspA2 protein mediate adherence of Moraxella catarrhalis to human epithelial cells in vitro. J. Bacteriol. 182:1364-1373.

    31. Li, D. Q., F. Lundberg, and A. Ljungh. 2001. Binding of vitronectin and clusterin by coagulase-negative staphylococci interfering with complement function. J. Mater. Sci. Mater. Med. 12:979-982.

    32. Liu, S., D. S. Thaler, and A. Libchaber. 2002. Signal and noise in bridging PCR. BMC Biotechnol. 2:13.

    33. Luke, N. R., S. Allen, B. W. Gibson, and A. A. Campagnari. 2003. Identification of a 3-deoxy-D-manno-octulosonic acid biosynthetic operon in Moraxella catarrhalis and analysis of a KdsA-deficient isogenic mutant. Infect. Immun. 71:6426-6434.

    34. McMichael, J. C., M. J. Fiske, R. A. Fredenburg, D. N. Chakravarti, K. R. VanDerMeid, V. Barniak, J. Caplan, E. Bortell, S. Baker, R. Arumugham, and D. Chen. 1998. Isolation and characterization of two proteins from Moraxella catarrhalis that bear a common epitope. Infect. Immun. 66:4374-4381.

    35. McQuillen, D. P., D. P. Gulati, and P. A. Rice. 1994. Complement mediated bacterial killing assays. Methods Enzymol. 236:137-147.

    36. Meier, P. S., R. Troller, I. N. Grivea, G. A. Syrogiannopoulos, and C. Aebi. 2002. The outer membrane proteins UspA1 and UspA2 of Moraxella catarrhalis are highly conserved in nasopharyngeal isolates from young children. Vaccine 20:1754-1760.

    37. Meyer, T. F., N. Mlawer, and M. So. 1982. Pilus expression in Neisseria gonorrhoeae involves chromosomal rearrangement. Cell 30:45-52.

    38. Morgan, B. P. 2000. Complement methods and protocols. Humana Press, Totowa, N.J.

    39. Munson, R. S., Jr., and R. W. Tolan. 1989. Molecular cloning, expression, and primary sequence of outer membrane protein P2 of Haemophilus influenzae type b. Infect. Immun. 57:88-94.

    40. Murphy, T. F., A. L. Brauer, B. J. Grant, and S. Sethi. 2005. Moraxella catarrhalis in chronic obstructive pulmonary disease: burden of disease and immune response. Am. J. Respir. Crit. Care Med. 172:195-199.

    41. Murphy, T. F., A. L. Brauer, N. Yuskiw, and T. J. Hiltke. 2000. Antigenic structure of outer membrane protein E of Moraxella catarrhalis and construction and characterization of mutants. Infect. Immun. 68:6250-6256.

    42. Nordstrm, T., A. M. Blom, A. Forsgren, and K. Riesbeck. 2004. The emerging pathogen Moraxella catarrhalis interacts with complement inhibitor C4b binding protein through ubiquitous surface proteins A1 and A2. J. Immunol. 173:4598-4606.

    43. Pausa, M., V. Pellis, M. Cinco, P. G. Giulianini, G. Presani, S. Perticarari, R. Murgia, and F. Tedesco. 2003. Serum-resistant strains of Borrelia burgdorferi evade complement-mediated killing by expressing a CD59-like complement inhibitory molecule. J. Immunol. 170:3214-3222.

    44. Pearson, M. M., E. R. Lafontaine, N. J. Wagner, J. W. St. Geme III, and E. J. Hansen. 2002. A hag mutant of Moraxella catarrhalis strain O35E is deficient in hemagglutination, autoagglutination, and immunoglobulin D-binding activities. Infect. Immun. 70:4523-4533.

    45. Peng, D., B. P. Choudhury, R. S. Petralia, R. W. Carlson, and X. X. Gu. 2005. Roles of 3-deoxy-D-manno-2-octulosonic acid transferase from Moraxella catarrhalis in lipooligosaccharide biosynthesis and virulence. Infect. Immun. 73:4222-4230.

    46. Podack, E. R., W. P. Kolb, and H. J. Muller-Eberhard. 1977. The SC5b-7 complex: formation, isolation, properties and subunit composition. J. Immunol. 119:2024-2029.

    47. Podack, E. R., K. T. Preissner, and H. J. Muller-Eberhard. 1984. Inhibition of C9 polymerization within the SC5b-9 complex of complement by S-protein. Acta Pathol. Microbiol. Immunol. Scand. Suppl. 284:89-96.

    48. Prasadarao, N. V., A. M. Blom, B. O. Villoutreix, and L. C. Linsangan. 2002. A novel interaction of outer membrane protein A with C4b binding protein mediates serum resistance of Escherichia coli K1. J. Immunol. 169:6352-6360.

    49. Preissner, K. T., E. R. Podack, and H. J. Muller-Eberhard. 1985. The membrane attack complex of complement: relation of C7 to the metastable membrane binding site of the intermediate complex C5b-7. J. Immunol. 135:445-451.

    50. Preissner, K. T., and D. Seiffert. 1998. Role of vitronectin and its receptors in haemostasis and vascular remodeling. Thromb. Res. 89:1-21.

    51. Ram, S., M. Cullinane, A. M. Blom, S. Gulati, D. P. McQuillen, B. G. Monks, C. O'Connell, R. Boden, C. Elkins, M. K. Pangburn, B. Dahlback, and P. A. Rice. 2001. Binding of C4b-binding protein to porin: a molecular mechanism of serum resistance of Neisseria gonorrhoeae. J. Exp. Med. 193:281-295.

    52. Rautemaa, R., G. A. Jarvis, P. Marnila, and S. Meri. 1998. Acquired resistance of Escherichia coli to complement lysis by binding of glycophosphoinositol-anchored protectin (CD59). Infect. Immun. 66:1928-1933.

    53. Rivas, G., K. C. Ingham, and A. P. Minton. 1994. Ca(2+)-linked association of human complement C1s and C1r. Biochemistry 33:2341-2348.

    54. Setlow, J. K., D. C. Brown, M. E. Boling, A. Mattingly, and M. P. Gordon. 1968. Repair of deoxyribonucleic acid in Haemophilus influenzae. J. Bacteriol. 95:546-558.

    55. Soto-Hernandez, J. L., S. Holtsclaw-Berk, L. M. Harvill, and S. L. Berk. 1989. Phenotypic characteristics of Branhamella catarrhalis strains. J. Clin. Microbiol. 27:903-908.

    56. Tan, T. T., T. Nordstrm, A. Forsgren, and K. Riesbeck. 2005. The respiratory pathogen Moraxella catarrhalis adheres to epithelial cells by interacting with fibronectin through ubiquitous surface proteins A1 and A2. J. Infect. Dis. 192:1029-1038.

    57. Thern, A., L. Stenberg, B. Dahlback, and G. Lindahl. 1995. Ig-binding surface proteins of Streptococcus pyogenes also bind human C4b-binding protein (C4BP), a regulatory component of the complement system. J. Immunol. 154:375-386.

    58. Tschopp, J., A. Chonn, S. Hertig, and L. E. French. 1993. Clusterin, the human apolipoprotein and complement inhibitor, binds to complement C7, C8, and the b domain of C9. J. Immunol. 151:2159-2165.

    59. Verduin, C. M., C. Hol, A. Fleer, H. van Dijk, and A. van Belkum. 2002. Moraxella catarrhalis: from emerging to established pathogen. Clin. Microbiol. Rev. 15:125-144.

    60. Verduin, C. M., M. Jansze, C. Hol, T. E. Mollnes, J. Verhoef, and H. van Dijk. 1994. Differences in complement activation between complement-resistant and complement-sensitive Moraxella (Branhamella) catarrhalis strains occur at the level of membrane attack complex formation. Infect. Immun. 62:589-595.

    61. Wetzler, L. M., E. C. Gotschlich, M. S. Blake, and J. M. Koomey. 1989. The construction and characterization of Neisseria gonorrhoeae lacking protein III in its outer membrane. J. Exp. Med. 169:2199-2209.

    62. Wolf, B., M. Kools-Sijmons, C. Verduin, L. C. Rey, A. Gama, J. Roord, J. Verhoef, and A. van Belkum. 2000. Genetic diversity among strains of Moraxella catarrhalis cultured from the nasopharynx of young and healthy Brazilian, Angolan and Dutch children. Eur. J. Clin. Microbiol. Infect. Dis. 19:759-764.

    63. Zaleski, A., N. K. Scheffler, P. Densen, F. K. N. Lee, A. A. Campagnari, B. W. Gibson, and M. A. Apicella. 2000. Lipooligosaccharide Pk (Gal1-4Gal1-4Glc) epitope of Moraxella catarrhalis is a factor in resistance to bactericidal activity mediated by normal human serum. Infect. Immun. 68:5261-5268.(Ahmed S. Attia, Sanjay Ra)

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