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Infection and Immunity, April 2005, p. 2400-2410, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2400-2410.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The UspA2 Protein of Moraxella catarrhalis Is Directly Involved in the Expression of Serum Resistance

Ahmed S. Attia,1 Eric R. Lafontaine,1,{dagger} Jo L. Latimer,1 Christoph Aebi,2 George A. Syrogiannopoulos,3 and Eric J. Hansen1*

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas,1 Institute for Infectious Diseases, University of Berne, Berne, Switzerland,2 Department of Paediatrics, University Hospital of Patras, Rion, Patras, Greece3

Received 26 August 2004/ Returned for modification 8 October 2004/ Accepted 29 November 2004


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ABSTRACT
 
Many strains of Moraxella catarrhalis are resistant to the bactericidal activity of normal human serum. Previous studies have shown that mutations involving the insertion of an antibiotic resistance cartridge into the M. catarrhalis uspA2 gene resulted in the conversion of a serum-resistant strain to a serum-sensitive phenotype. In the present study, the deletion of the entire uspA2 gene from the serum-resistant M. catarrhalis strain O35E resulted in a serum-sensitive phenotype and did not affect either the rate of growth or the lipooligosaccharide expression profile of this mutant. Inactivation of the classical complement pathway in normal human serum with Mg2+ and EGTA resulted in the survival of this uspA2 mutant. In contrast, blocking of the alternative complement pathway did not protect this uspA2 mutant from complement-mediated killing. To determine whether the UspA2 protein is directly involved in serum resistance, transformation and allelic exchange were used to replace the uspA2 gene in the serum-resistant strain O35E with the uspA2 gene from the serum-sensitive M. catarrhalis strain MC317. The resultant O35E transformant exhibited a serum-sensitive phenotype. Similarly, when the uspA2 gene from the serum-resistant strain O35E was used to replace the uspA2 gene in the serum-sensitive strain MC317, the MC317 transformant acquired serum resistance. The use of hybrid O35E-MC317 uspA2 genes showed that the N-terminal half of the O35E protein contained a 102-amino-acid region that was involved in the expression of serum resistance. In addition, when the uspA2 genes from strains O35E and MC317 were cloned and expressed in Haemophilus influenzae DB117, only the O35E UspA2 protein caused a significant increase in the serum resistance of the H. influenzae recombinant strain. These results prove that the UspA2 protein is directly involved in the expression of serum resistance by certain M. catarrhalis strains.


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INTRODUCTION
 
Moraxella catarrhalis has gone from being regarded as a relatively harmless commensal organism found in the human nasopharynx to a pathogen which can cause a significant amount of disease (27, 35, 47, 53). In the upper respiratory tract, M. catarrhalis is an important cause of otitis media in infants and young children (27). This unencapsulated, gram-negative bacterium can also cause infectious exacerbations of chronic obstructive pulmonary disease (46, 47) and is an infrequent cause of other diseases, including pneumonia and sinusitis (reviewed in reference 35).

Virtually nothing is known about the virulence mechanisms used by M. catarrhalis to produce disease. The lack of a relevant animal model for otitis media caused by this organism (27) has precluded direct investigation of this process at the experimental level. Numerous M. catarrhalis gene products that could be involved in the colonization of the human nasopharynx by this organism or in its ability to spread into other anatomic niches in the human body have been identified (reviewed in references 27 and 53), and recent studies have highlighted additional gene products (1, 32, 51) that might participate in these processes. However, the functional significance of these gene products in vivo remains to be determined.

One phenotypic trait of M. catarrhalis that has been proposed to correlate with virulence is the ability of some strains of this bacterium to resist complement-mediated killing by normal human serum (NHS) (21). First observed about 20 years ago (reviewed in references 10 and 50), the occurrence of the serum-resistant phenotype has been documented in many subsequent studies of M. catarrhalis isolates (10, 13, 25, 34, 45, 50, 59). The hypothesis that serum resistance might be a virulence factor for M. catarrhalis stemmed from observations that the incidence of complement-resistant M. catarrhalis strains was higher in samples isolated from ill patients (i.e., adults with lower respiratory tract infections) than in samples from healthy adults or children (20, 25). More recent studies indicate, however, that most M. catarrhalis isolates from the nasopharynges of apparently healthy infants and young children are serum resistant (34, 59).

Bacterial resistance to killing by complement can occur by a number of different mechanisms (reviewed in reference 43). In M. catarrhalis, at least five different surface antigens have been shown to date to have some level of involvement, whether direct or indirect, in serum resistance. Mutations affecting the ability of M. catarrhalis to express UspA2 (3), the iron-regulated CopB protein (17), OMP E (36), OMP CD (22), and the Pk lipooligosaccharide epitope (60) have been shown to reduce the serum resistance of the wild-type parent strain to various degrees. In addition, a fur mutant of M. catarrhalis (14) exhibited increased susceptibility to killing by NHS. However, evidence for the direct involvement of any of these gene products in conferring serum resistance on M. catarrhalis has been lacking.

The UspA2 protein is a putative autotransporter macromolecule (18) that forms relatively short, filamentous projections on the surface of M. catarrhalis cells (19, 41) and is a target for biologically active antibodies (11, 33). A small number of M. catarrhalis strains express the closely related UspA2H protein in place of a UspA2 protein (30). Nearly all nasopharyngeal isolates of M. catarrhalis in a recent study (34) possessed a uspA2 or a uspA2H gene. Similarly, in a different study, a uspA2 gene was detected by PCR in a majority of M. catarrhalis isolates (9). Although one study has indicated that the presence of a uspA2 gene did not correlate with complement resistance in M. catarrhalis (9), there are several published studies which indicate that UspA2 (or UspA2H) is involved in the ability of M. catarrhalis to resist serum killing (3, 8, 30). In this report, we demonstrate that the UspA2 protein is directly involved in serum resistance.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and culture conditions. Bacterial strains and plasmids that were 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 in an atmosphere containing 95% air-5% CO2. When necessary, BHI agar was supplemented with kanamycin (15 µg/ml), spectinomycin (15 µg/ml), dihydrostreptomycin sulfate (750 µg/ml), or zeocin (1 µg/ml). Escherichia coli strains were grown on Luria-Bertani agar plates (44) that were supplemented with either kanamycin (50 µg/ml) or spectinomycin (100 µg/ml) when appropriate. Recombinant Haemophilus influenzae strains were cultured on BHI agar plates containing 5% (vol/vol) Levinthal base (5) or in BHI broth containing 10% Levinthal base, both containing ampicillin (10 µg/ml).


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  		TABLE 1. Bacterial strains and plasmids used in this study

Recombinant DNA techniques. Standard molecular biology and recombinant DNA techniques were performed as described previously (44). Restriction endonucleases, calf intestinal alkaline phosphatase, and T4 DNA ligase were obtained from New England Biolabs (Beverly, Mass.). PCR-based amplification of DNA fragments for cloning purposes was performed with Pfu DNA polymerase. For the second cycle of the PCR bridging reactions, ExTaq DNA polymerase (PanVera, Madison, Wis.) was used. The oligonucleotide primers used in this study are listed in Table 2. Chromosomal DNA from M. catarrhalis was isolated with an Easy-DNA kit (Invitrogen, Carlsbad, Calif.). Plasmid DNA was purified from E. coli and H. influenzae with the Qiaprep Spin Miniprep kit (QIAGEN, Valencia, Calif.).


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  		TABLE 2. Oligonucleotide primers used in this study

Construction of a uspA2 deletion mutant. The regions immediately flanking the uspA2 open reading frame (ORF) in M. catarrhalis strain O12E were amplified by PCR using chromosomal DNA from strain O12E as the template. The oligonucleotide primers AA-3-Rev and AA-4-Fw were used to amplify a 0.75-kb upstream region, while the 0.95-kb downstream region was amplified by using primers AA-5-Fw and AA-6-Rev. Both amplicons were digested with SalI and then ligated and used as a template in bridging PCR (i.e., PCR sewing) (31, 40) with the primers AA-4-Fw and AA-6-Rev to generate the 1.7-kb USPA2AB fragment. This fragment was ligated into the pCR2.1 vector (Invitrogen) and transformed into the E. coli Inv{alpha}F' strain (Invitrogen), and the desired recombinant strain was selected on Luria-Bertani agar supplemented with kanamycin. The plasmid containing the USPA2AB insert, designated pAA1, was digested with SalI and then blunt-ended by using the DNA polymerase Pfu (Stratagene, La Jolla, Calif.). The spectinomycin resistance cartridge was excised from pSPECR (57) by digestion with EcoRV and ligated to this blunt-ended plasmid, resulting in pAA2. Nucleotide sequence analysis showed that the spectinomycin resistance cartridge was inserted between the two original flanking regions in the same orientation as the uspA2 gene. Plasmid pAA2 was used to transform the wild-type M. catarrhalis strain O35E in the plate transformation procedure described below, and transformants were selected for spectinomycin resistance. One of these spectinomycin-resistant transformants, designated O35E{Delta}2, was shown by nucleotide sequence analysis to have a complete deletion of the uspA2 ORF.

MAbs and Western blot analysis. Monoclonal antibody (MAb) 17C7, reactive with both M. catarrhalis UspA1 and UspA2 proteins, and MAb 24B5, which binds only UspA1, have been described elsewhere (4, 12). MAb 8E7, which reacts with M. catarrhalis serotype A and C lipooligosaccharides (LOS), has been described previously (23). For detection of proteins, M. catarrhalis whole-cell lysates (4) equivalent to 3 x 107 CFU were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 7.5% (wt/vol) polyacrylamide separating gels, transferred to nitrocellulose membranes (Schleicher & Schuell BioScience, Keene, N.H.), and probed with the appropriate primary antibody. In some Western blot experiments, the whole-cell lysates were diluted to be equivalent to 2 x 106 CFU in order to visualize possible changes in the level of expression of UspA2. The secondary antibody used for Western blot analysis was goat anti-mouse immunoglobulin G (IgG) conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, Pa.). Antigen-antibody complexes were visualized by chemiluminescence by using Western Lightning Chemiluminescence Reagent Plus (New England Nuclear, Boston, Mass.). For detection of LOS, whole-cell lysates were digested with proteinase K at 56°C for 1 h and then subjected to SDS-PAGE with 15% (wt/vol) polyacrylamide separating gels. These gels were then either stained with silver by the method of Tsai and Frasch (52) or used for Western blot analysis.

Characterization of outer membrane proteins. Outer membrane vesicles were extracted from whole cells of M. catarrhalis by using the EDTA-based method described by Murphy and Loeb (37). Proteins present in these vesicles were resolved by SDS-PAGE using 7.5% (wt/vol) polyacrylamide separating gels and stained with Coomassie blue.

Human sera. Blood drawn from healthy adult volunteers was allowed to clot at room temperature. The serum fraction was separated by centrifugation, pooled under aseptic conditions, aliquoted in small volumes, frozen first in a dry ice-ethanol solution, and then stored at –70°C. Complement inactivation was achieved by incubating this NHS at 56°C for 30 min. Factor B-depleted human serum was purchased from a commercial source (Quidel, San Diego, Calif.). To deplete NHS of IgG, a 0.5-ml portion of 40% (vol/vol) NHS diluted in Veronal-buffered saline containing 5 mM MgCl2 and 1.5 mM CaCl2 (VBS++) was mixed with 0.5 ml of GammaBind Plus Sepharose suspension (Amersham Biosciences, Piscataway, N.J.) in a plastic column for 1 h at 4°C with gentle agitation. GammaBind Plus Sepharose is described by the manufacturer as not binding other serum proteins, including IgM, IgE, IgA, and transferrin. The flowthrough from the column was ~20% (vol/vol) NHS depleted of IgG. These IgG-depleted preparations were used immediately in bactericidal assays.

Serum bactericidal assay. The ability of M. catarrhalis strains to resist complement-mediated killing by NHS was assessed by use of a liquid-phase assay. Briefly, bacterial cultures that were grown to mid-logarithmic phase were diluted in Veronal-buffered saline containing 0.1% (wt/vol) gelatin to a final concentration of 1 x 105 to 2 x 105 CFU/ml. Subsequently, 10-µl (~1 x 103 to 2 x 103 CFU) portions were added to 10 µl of NHS and 80 µl of VBS++ in 1.5-ml microcentrifuge tubes on ice. Duplicate 10-µl samples were removed from this mixture and spread on appropriate agar plates. The tubes were incubated in a 37°C water bath without shaking for 30 min and then placed on ice, and duplicate 10-µl samples were removed and plated. All experiments were carried out with heat-inactivated NHS as a control. To block the alternative complement activation pathway, factor B-depleted serum was used. To block the classical pathway, NHS was equilibrated in Veronal-buffered saline containing 10 mM MgCl2 and 10 mM EGTA for 15 min on ice before adding the bacteria. To examine the effect of IgG antibodies on serum resistance, a 45-µl portion of the IgG-depleted NHS was mixed with 45 µl of VBS++ to give a final concentration of ~10% NHS for use in the bactericidal assay. To supplement this IgG-depleted serum with IgG antibodies, it was mixed with an equal volume of 20% (vol/vol) heat-inactivated NHS, and this reconstituted serum was used in the bactericidal assay. For assessing the serum resistance of recombinant H. influenzae strains, the same method was utilized, but NHS and heat-inactivated NHS were used at a final concentration of 5% (vol/vol).

Indirect antibody accessibility assay. To detect UspA2 proteins on the surface of bacterial cells, MAb 17C7 was used in the indirect antibody accessibility assay (16). Isogenic uspA1 mutants of M. catarrhalis were constructed by using the plasmid pUSPA1KAN as described previously (3) to avoid interference caused by binding of the UspA1- and UspA2-reactive MAb 17C7 by the UspA1 protein. Briefly, this assay involves incubating bacteria with the MAbs at 4°C, washing away unattached MAbs, and then adding radioiodinated goat anti-mouse immunoglobulin to detect the mouse MAbs bound to the bacterial cell surface.

Identification of streptomycin-resistant mutants of M. catarrhalis. Streptomycin-resistant mutants of O35E and MC317 were obtained by spreading approximately 1010 CFU of each strain on BHI agar plates containing streptomycin and incubating them overnight at 37°C. The development of streptomycin resistance is frequently due to a single nucleotide change in the rpsL gene leading to a single amino acid change in ribosomal protein S12 (38). Nucleotide sequence analysis of the rpsL gene of the streptomycin-resistant O35E mutant (O35E-Smr) confirmed that a single nucleotide change at residue 128 (A to C) resulted in a single altered amino acid (i.e., K43T), whereas in the case of the MC317 streptomycin-resistant mutant (MC317-Smr), there was a single nucleotide change in its rpsL gene at residue 263 (A to G) which resulted in a single altered amino acid (i.e., K88R). A 3-kb amplicon containing the mutated rpsL gene plus flanking sequence was generated by PCR using the oligonucleotide primers Rps-5' and Rps-3' and was used for congression experiments (see below).

Construction of hybrid uspA2 genes. PCR sewing was used to construct hybrid uspA2 genes containing segments from both the O35E and MC317 uspA2 genes. Briefly, the oligonucleotide primers AA-47 and AA-54 were designed to bind to the 5' untranslated region and to the region 3' from the translation termination codon, respectively, of both the O35E and MC317 uspA2 genes. Additional primers (Table 2) were designed to allow amplification of fragments of various lengths from the O35E and MC317 uspA2 genes. In the case of hybrids 1, 2, and 3, the primers at the bridging region were designed to be completely complementary to each other, while in the case of hybrid 4, due to the lack of nucleotide sequence identity between O35E and MC317 in the bridging region, only the first 15 nucleotides (nt) of the oligonucleotide primers (AA-60 and AA-61) were complementary to each other and the rest of the sequence matched the respective DNA template. After verifying the nucleotide sequence of the hybrid PCR products, the products were introduced into the uspA2 mutant MC317.2 by congression as described below; streptomycin-resistant transformants were screened for the loss of the spectinomycin resistance cartridge, and the presence of the hybrid uspA2 gene was verified by nucleotide sequence analysis. Hybrid 5 was obtained as a serendipitous event from a transformation experiment in which the wild-type O35E uspA2 gene was used to transform MC317.2. This transformant was shown to contain a hybrid uspA2 gene in which the first 444 nucleotides were derived from the MC317 uspA2 gene. For the construction of hybrid 6, chromosomal DNA from hybrid 5 was used as the template for the PCR with primers AA-47 and AA-55-Rev to amplify the 5' half of the hybrid 6 gene, and chromosomal DNA from MC317 was used as the template to amplify the 3' half of the hybrid gene using primers AA-55-Fw and AA-54. It should be noted that primers AA-55-Fw and AA-55-Rev both bind twice on the O35E uspA2 ORF (at nt 712 to 730 and 799 to 817); the binding of AA-55-Rev to the first site (nt 712 to 730) was used to obtain the hybrid 6 PCR product, and the binding of AA-55-Fw to the second site (nt 799 to 817) was used to obtain the hybrid 3 PCR product.

Plate transformation and allelic exchange. Replacement of a mutated uspA2 gene in M. catarrhalis with a different wild-type or hybrid uspA2 gene was accomplished by using congression (39) in a modification of a plate transformation method previously described (26). Briefly, the wild-type or hybrid uspA2 gene (~0.5 µg of DNA) and the 3-kb PCR amplicon containing the appropriate (i.e., O35E or MC317) mutated rpsL gene (~25 ng of DNA) were mixed with two to three colonies of the recipient strain on a BHI plate in an area approximately 1 cm in diameter. The plate was incubated at 37°C for 6 h, after which the bacterial growth was suspended in BHI broth, serially diluted, and plated on BHI plates containing streptomycin. Streptomycin-resistant transformants were then screened for loss of the antibiotic resistance cartridge (i.e., a spectinomycin resistance cartridge in MC317.2 and a zeocin resistance cartridge in O35E.2ZEO) that had been present in the recipient strain's uspA2 gene. Nucleotide sequence analysis was used to identify those transformants in which the entire mutated uspA2 gene had been replaced by the uspA2 gene used for transformation.

Cloning and expression of uspA2 genes in H. influenzae. Plasmid pGJB103M was used as the vector for cloning M. catarrhalis uspA2 genes in H. influenzae. This plasmid is a modified version of pGJB103 (6) and has an additional 1.3-kb insertion containing an SphI site and an AvrII site into which the M. catarrhalis uspA2 genes were cloned. Both the O35E and MC317 uspA2 genes were PCR amplified with the primers AA-47 and AA-54 together with M. catarrhalis chromosomal DNA as the template. Each PCR product and pGJB103M were digested with both SphI and AvrII; these digested PCR products were individually ligated into pGJB103M and used to electroporate H. influenzae Rd strain DB117 (48). Ampicillin-resistant colonies were screened for the expression of UspA2 by using MAb 17C7 in a colony blot-radioimmunoassay (15). Several MAb-reactive clones were obtained but were found to have very low expression of the UspA2 proteins. To increase the expression of the uspA2 genes in H. influenzae, bridging PCR was used to insert a kanamycin (kan) gene promoter in front of the O35E and MC317 uspA2 genes in pGJB103M. The kan promoter region was PCR amplified with the primers kan-pro-5'-SphI and kan-pro-3', with the plasmid pLS88 (58) as the template. The uspA2 genes were PCR amplified with primers AA-62 and AA-54. Primer AA-62 binds to the same region in the 5' untranslated region of the uspA2 genes as AA-47, but it lacks an SphI site and contains instead 14 nt that are complementary to the last 14 nt of the kan promoter. After PCR, ligation, and restriction enzyme digestion, the uspA2 genes fused to the kan promoter were cloned as described above. The resultant recombinant plasmids expressing relatively high levels of the UspA2 proteins were designated pAA-35U2-kp and pAA-317U2-kp. As a negative control, the kan promoter region was PCR amplified with the primers kan-pro-5'-SphI and kan-pro-3'-AvrII and cloned into pGJB103M to yield pAA-kp.

Nucleotide sequence analysis. The nucleotide sequences of DNA inserts in plasmids and PCR fragments were determined by use of an ABI PRISM 377 sequencer (Applied Biosystems, Foster City, Calif.). The sequences were analyzed by using SeqEd version 1.03 (Applied Biosystems) and MacVector (version 6.5; Oxford Molecular Group, Campbell, Calif.).

Nucleotide sequence accession number. The nucleotide sequence of the MC317 uspA2 gene was deposited at GenBank and assigned accession no. AY730666.


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RESULTS
 
Construction of a uspA2 deletion mutant. To construct a uspA2 deletion mutant, the uspA2 ORF was deleted from strain O35E as described in Materials and Methods. Nucleotide sequence analysis of the resultant uspA2 deletion mutant O35E{Delta}2 confirmed the replacement of the uspA2 ORF with the spectinomycin resistance cartridge. This analysis also showed that there was a putative ORF for a predicted metR homolog located a short distance downstream from uspA2; this ORF was oriented in the opposite direction from uspA2. The nucleotide sequence of this putative ORF was found to have been altered by recombination in this uspA2 deletion mutant. Therefore, to confirm that this downstream ORF was not involved in conferring serum resistance on M. catarrhalis, we tested a mutant of O35E that had a transposon inserted in this ORF and showed that it was as serum-resistant as the wild-type parent strain (data not shown).

Phenotypic characterization of the uspA2 deletion mutant O35E{Delta}2. There was no apparent difference in the colony morphologies of the wild-type parent strain O35E and the O35E{Delta}2 mutant (data not shown). In addition, the extents and rates of growth of these two strains were very similar if not identical (data not shown). Western blot analysis of whole-cell lysates of the wild-type strain (Fig. 1A, lane 1) and the O35E{Delta}2 mutant (Fig. 1A, lane 2) with MAb 17C7, which binds both UspA2 and UspA1 (4), showed that the mutant lacked the high-molecular-weight aggregate that represents the UspA2 protein. At the same time, there was no obvious change in the expression of the UspA1 protein by this mutant (Fig. 1B, lane 2), as revealed by Western blot analysis using the UspA1-specific MAb 24B5 (12). Examination of the proteins present in EDTA-extracted outer membrane vesicles of both the wild type (Fig. 1C, lane 1) and the mutant (Fig. 1C, lane 2) revealed that the mutant lacked the high-molecular-weight UspA2 band that was present in the wild-type parent strain. However, no other differences were visible in these outer membrane protein profiles. It has been shown previously that changes in the LOS of M. catarrhalis can affect its susceptibility to killing by NHS (60). However, there was no apparent change in the SDS-PAGE migration patterns of the LOS from O35E and O35E{Delta}2, as detected either by silver staining (Fig. 1D) or by Western blot analysis using the LOS-reactive MAb 8E7 (Fig. 1E).



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  		FIG. 1. Comparison of the phenotypes of the wild-type M. catarrhalis strain O35E and the uspA2 mutant O35E{Delta}2. (A) Western blot analysis of whole-cell lysates of O35E (lane 1) and O35E{Delta}2 (lane 2) using MAb 17C7 as the primary antibody. This MAb binds both UspA2 (indicated by bracket) and UspA1 (indicated by arrow). (B) Western blot analysis of these whole-cell lysates using the UspA1-specific MAb 24B5 as the primary antibody. (C) Proteins present in outer membrane vesicles from these two strains were resolved by SDS-PAGE and stained with Coomassie blue. The absence of UspA2 in the O35E{Delta}2 mutant is indicated by the arrow. Whole-cell lysates digested with proteinase K were resolved by SDS-PAGE, and LOS was visualized either by staining with silver (D) or by Western blot analysis (E) using the LOS-reactive MAb 8E7 as the primary antibody. Protein molecular mass markers (in kilodaltons) are present on the left sides of panels A, B, and C.

Killing of the serum-sensitive uspA2 deletion mutant O35E{Delta}2 involves the classical complement pathway. As expected from the results obtained with a different uspA2 mutant (3), the use of 10% NHS in a serum bactericidal assay showed that the uspA2 deletion mutant O35E{Delta}2 (Fig. 2A, column 2b) was exquisitely serum sensitive, whereas the wild-type strain O35E (Fig. 2A, column 1b) was completely resistant. These two strains survived equally well in heat-inactivated NHS (Fig. 2A, columns 1a and 2a). To selectively block the alternative complement pathway, NHS depleted of factor B (49) was used in the serum killing assay. The wild-type parent strain O35E (Fig. 2A, column 1c) was able to survive in the factor B-depleted serum, whereas the O35E{Delta}2 mutant (Fig. 2A, column 2c) was readily killed by the same serum. This result suggested that the alternative complement pathway was probably not involved in the killing of this serum-sensitive mutant. To selectively block the classical pathway, the bactericidal assay was carried out in the presence of Mg2+ and in the absence of Ca2+ (24); NHS was selectively depleted of Ca2+ by using the chelating agent EGTA. Strains O35E (Fig. 2A, column 1d) and O35E{Delta}2 (Fig. 2A, column 2d) survived equally well under these assay conditions, a result which indicated that killing of this uspA2 mutant by NHS is accomplished via the classical pathway of complement activation.



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  		FIG. 2. Killing of wild-type and mutant strains of M. catarrhalis by human sera. (A) Effect of various sera on selected strains. The wild-type strain O35E (1), the uspA2 mutant O35E{Delta}2 (2), and the wild-type strain MC317 (3) were incubated for 30 min at 37°C in VBS++ containing 10% (vol/vol) heat-inactivated NHS (a), 10% (vol/vol) NHS (b), or 10% (vol/vol) factor B-depleted human serum (c) or in VBS containing 10 mM MgCl2, 10 mM EGTA, and 10% (vol/vol) NHS (d). Portions of these reaction mixtures were plated at time zero and after 30 min, and the percentage of survival is calculated relative to the original inoculum. The data presented here are the means of results from three independent experiments plus the standard errors. (B) Involvement of IgG in killing of the uspA2 mutant O35E{Delta}2. Wild-type strain O35E (1) and the uspA2 mutant O35E{Delta}2 (2) were incubated with the following sera: 10% (vol/vol) heat-inactivated NHS (a), 10% (vol/vol) NHS (b), 10% (vol/vol) IgG-depleted NHS (c), and 10% (vol/vol) IgG-depleted NHS mixed with heat-inactivated NHS as a source of IgG (d). The data presented here are the means of results from three independent experiments plus the standard errors.

Human IgG is essential for the killing of the O35E{Delta}2 uspA2 mutant by NHS. To investigate the role of IgG antibody in the killing of the uspA2 deletion mutant, NHS depleted of IgG was used in the serum bactericidal assay. Western blot analysis of this NHS after adsorption with GammaBind Plus Sepharose showed that this treatment removed almost all of the IgG from this serum (data not shown). There was no obvious difference in the survival rates of the wild-type O35E strain in heat-inactivated NHS (Fig. 2B, column 1a), NHS (Fig. 2B, column 1b), IgG-depleted serum (Fig. 2B, column 1c), and IgG-depleted serum supplemented with heat-inactivated NHS (as a source of IgG) (Fig. 2B, column 1d). In contrast, the uspA2 mutant O35E{Delta}2 survived in both the heat-inactivated NHS (Fig. 2B, column 2a) and the IgG-depleted serum (Fig. 2B, column 2c) but was readily killed by both NHS (Fig. 2B, column 2b) and the IgG-depleted serum supplemented with heat-inactivated NHS (Fig. 2B, column 2d). The latter serum mixture was used to confirm that the IgG depletion step had not inactivated the complement components required for serum killing. These results indicated that the killing of the serum-sensitive O35E{Delta}2 mutant via the classical pathway is IgG antibody dependent.

Identification of a serum-sensitive M. catarrhalis strain that expresses UspA2. M. catarrhalis strain MC317 was one of the relatively few isolates obtained from the nasopharynges of infants and children that proved to be serum sensitive (34). In our hands, strain MC317 was readily killed by NHS (Fig. 2A, column 3b). In addition, this strain was also sensitive to killing by factor B-depleted serum, with 82% of the initial inoculum being killed (Fig. 2A, column 3c). Nucleotide sequence analysis of the uspA2 gene from MC317 revealed an ORF with 1,953 nucleotides that encoded a predicted protein containing 650 amino acids. Alignment of the deduced amino acid sequences of the UspA2 proteins from O35E and MC317 (Fig. 3A) revealed significant regions of sequence identity between the two proteins, especially in the putative signal peptide and the C-terminal portion of these proteins. Overall, these two proteins have 68% identity. However, the MC317 UspA2 protein contains 74 more amino acids than that of strain O35E.



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  		FIG. 3. Comparison of UspA2 proteins from M. catarrhalis strains O35E and MC317. (A) Alignment of the deduced amino acid sequences. Identical amino acids are shaded in dark gray, while conserved amino acids are shaded with light gray. This figure was generated by using the ClustalW Alignment program in MacVector (version 6.5). (B) Relative amounts of UspA2 exposed on the surface of the following strains as measured by the indirect antibody accessibility assay: the M. catarrhalis uspA1 uspA2 mutant O35E.12 (1), the uspA1 mutant O35E.1 (2), and the uspA1 mutant MC317.1 (3). The counts per minute of radioiodinated goat anti-mouse IgG bound to MAb 17C7 on the bacterial cell surface (a) are plotted on the y axis. MAb 3F12, a murine IgG MAb specific for the major outer membrane protein of H. ducreyi (28), was used as the negative control (b). These data represent the means of results from two independent experiments plus standard deviations.

Western blot analysis showed that MC317 expressed a UspA2 protein that binds MAb 17C7 (data not shown). To determine whether UspA2 is exposed on the surface of MC317, we used the indirect antibody accessibility assay with MAb 17C7 as the primary antibody. A uspA1 mutant of MC317 (MC317.1) was constructed for use in this assay to eliminate expression of the UspA1 protein which also binds MAb 17C7. The results of the indirect antibody accessibility assay showed that the UspA2 protein of MC317.1 (Fig. 3B, column 3a) is readily accessible to MAb 17C7, albeit at a slightly lower level than the UspA2 protein of the O35E.1 strain (Fig. 3B, column 2a). Both strains bound MAb 17C7 at much higher levels than did the negative control strain, the uspA1 uspA2 double mutant O35E.12 (Fig. 3B, column 1a). MAb 3F12, a murine IgG MAb specific for the major outer membrane protein of Haemophilus ducreyi (28), was used as a control for nonspecific antibody binding (Fig. 3B, columns 1b to 3b). These results indicate that the serum-sensitive phenotype of the MC317 strain was not caused by either a lack of expression or a lack of surface exposure of the UspA2 protein. The uspA1 mutant of MC317 also had a much reduced ability to attach to Chang conjunctival epithelial cells in vitro (data not shown), comparable to that seen with uspA1 mutants of other M. catarrhalis strains.

Exchange of uspA2 genes between O35E and MC317. To determine whether differences in the primary amino acid sequences of the UspA2 proteins from O35E and MC317 were responsible for their different phenotypes with respect to serum killing, we exchanged uspA2 genes between these two strains by using transformation and congression as described in Materials and Methods. Western blot analysis using MAb 17C7 did not reveal apparent differences in the levels of expression of UspA2 by the streptomycin-resistant O35E-Smr strain (Fig. 4A, lane 1) and the MC317-Smr strain (Fig. 4A, lane 4); these streptomycin-resistant mutants served as control strains for this set of experiments. In addition, the transformants O35E/317U2 (i.e., O35E expressing the MC317 UspA2 protein) (Fig. 4A, lane 3) and MC317/35U2 (i.e., MC317 expressing the O35E UspA2 protein) (Fig. 4A, lane 6) expressed levels of UspA2 protein similar to those of the O35E-Smr and MC317-Smr strains.



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  		FIG. 4. Effect of uspA2 gene exchange on killing of M. catarrhalis by NHS. (A) Western blot analysis using MAb 17C7 to probe whole-cell lysates of the following strains: O35E-Smr, lane 1; the uspA2 mutant O35E.2ZEO, lane 2; transformant strain O35E/317U2 (i.e., O35E expressing the MC317 UspA2 protein), lane 3; MC317-Smr, lane 4; the uspA2 mutant MC317.2, lane 5; and transformant strain MC317/35U2 (i.e., MC317 expressing the O35E UspA2 protein), lane 6. The whole-cell lysates used in this Western blot experiment were diluted to be equivalent to 2 x 106 CFU in order to visualize possible changes in the level of expression of UspA2. The bracket indicates the region containing UspA2 and its degradation products. Molecular mass markers are shown to the left in kilodaltons. The MAb 17C7-reactive antigen that migrated near the 105-kDa marker is UspA1. (B) Resistance of these six strains (in the same order as in panel A) to killing by either 10% (vol/vol) heat-inactivated NHS (a) or 10% NHS (b). The data presented here are the means of results from three independent experiments plus the standard errors.

These two transformants, together with their respective control strains and the corresponding uspA2 insertion mutants (which were used to construct the transformant strains), were tested in bactericidal assays. All six of these strains survived equally well in the heat-inactivated NHS (Fig. 4B, columns 1a to 6a). The O35E transformant expressing the MC317 UspA2 protein (O35E/317U2) (Fig. 4B, column 3b) was readily killed by NHS, as was MC317-Smr (Fig. 4B, column 4b). On the other hand, the MC317 transformant expressing the O35E UspA2 protein (MC317/35U2) (Fig. 4B, column 6b) had a level of serum resistance that was only slightly less than that of the O35E-Smr strain (Fig. 4B, column 1b). Both uspA2 mutants were, as expected, sensitive to killing by NHS (Fig. 4B, columns 2b and 5b). These results show that differences in the amino acid sequences of these two UspA2 proteins are likely responsible for the observed difference in serum resistance of O35E and MC317.

Cloning and expression of M. catarrhalis uspA2 genes in H. influenzae. To establish that UspA2 was directly involved in the expression of serum resistance by strain O35E, the uspA2 genes from O35E and MC317 were cloned and expressed in H. influenzae DB117. A constitutive kan promoter was inserted in front of these cloned M. catarrhalis genes to increase expression of UspA2 (Fig. 5A, lanes 2 and 3). When tested for the ability to resist killing by 5% (vol/vol) NHS, the recombinant H. influenzae strain expressing the O35E UspA2 protein (Fig. 5B, column 2b) was more serum resistant than the recombinant expressing the MC317 UspA2 protein (Fig. 5B, column 3b). There was no difference between the level of serum resistance of the H. influenzae recombinant strain expressing the MC317 UspA2 protein (Fig. 5B, column 3b) and that of the strain which carried only the kan promoter within the plasmid (Fig. 5B, column 1b).



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  		FIG. 5. Effect of expression of the M. catarrhalis UspA2 protein on serum resistance of H. influenzae DB117. (A) Western blot analysis using MAb 17C7 to probe whole-cell lysates of H. influenzae DB117 carrying the following plasmids: pAA-kp (negative control), lane 1; pAA-35U2-kp, lane 2; and pAA-317U2-kp, lane 3. Molecular mass markers (in kilodaltons) are shown to the left. (B) Resistance of these three strains (in the same order as above) to killing by either 5% (vol/vol) heat-inactivated NHS (a) or 5% NHS (b). The data presented here are the means of results from three independent experiments plus the standard errors. The asterisk indicates that the difference between column 2b and column 3b is statistically significant (P value, 0.002) as determined by the use of a paired t test.

Construction and expression of hybrid uspA2 genes in M. catarrhalis. In order to determine whether there is a certain region or domain within the O35E UspA2 protein that is essential for the serum resistance phenotype, PCR sewing was used to construct hybrid uspA2 genes containing different fragments of the O35E and MC317 uspA2 ORFs. These hybrid uspA2 genes were introduced into the MC317.2 uspA2 mutant by transformation and congression. The basic approach involved introducing increasingly greater proportions of the O35E UspA2 protein (starting at the C-terminal end) and then assaying for gain of function (i.e., serum resistance) when the hybrid genes were expressed in a serum-sensitive background (i.e., MC317.2).

A schematic representation of the six hybrids used in this study is presented in Fig. 6A. All six hybrid UspA2 proteins were expressed at similar levels (Fig. 6B, lanes 2 to 7), as revealed by Western blot analysis. All six hybrid strains survived equally well in heat-inactivated NHS (Fig. 6C, columns 2a to 7a). As shown before, the MC317-Smr strain (Fig. 6C, column 1b) was serum sensitive, and the O35E-Smr strain was serum resistant (Fig. 6C, column 9b). When tested in the serum bactericidal assay, the first hybrid UspA2 protein that was found to increase the serum resistance of MC317.2 was hybrid 4 (Fig. 6C, column 5b). The addition of more O35E UspA2 sequence in hybrid 5 (Fig. 6C, column 6b) increased the serum resistance of this strain to a level approaching that obtained with the entire O35E UspA2 protein in MC317.2 (Fig. 6C, column 8b). To eliminate the possibility that the serum-sensitive hybrid strains did not have surface-exposed UspA2 proteins, we constructed uspA1 mutants of the serum-sensitive hybrid strains and probed these with the UspA2-reactive MAb 17C7 in the indirect antibody accessibility assay. The results of these experiments showed that these uspA1 mutants bound amounts of the radioiodinated goat anti-mouse IgG probe that ranged from 80 to 134% of that bound by the uspA1 mutant of the fully serum-resistant hybrid 6 (data not shown). Therefore, all of these hybrid strains, regardless of their serum resistance phenotype, expressed similar amounts of surface-exposed UspA2 protein.



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  		FIG. 6. Comparison and analysis of MC317-O35E hybrid UspA2 proteins. (A) Schematic representation of the six hybrids. Open segments and upper numbers represent the amino acid sequence from MC317; filled segments and lower numbers represent the sequence from O35E. Numbering begins with the first residue of the complete protein (containing the signal peptide). All constructs are drawn to scale. (B) Western blot analysis using MAb 17C7 to probe whole-cell lysates of the following strains: MC317-Smr, lane 1; hybrid 1, lane 2; hybrid 2, lane 3; hybrid 3, lane 4; hybrid 4, lane 5; hybrid 5, lane 6; hybrid 6, lane 7; MC317/35U2, lane 8; and O35E-Smr, lane 9. The whole-cell lysates used in this Western blot experiment were diluted to be equivalent to 2 x 106 CFU in order to visualize any change in the level of expression of UspA2. The bracket indicates the region containing UspA2 and its degradation products. The MAb 17C7-reactive antigen that migrated near the 105-kDa marker is UspA1. Molecular mass markers are shown to the left (in kilodaltons). (C) Resistance of these nine strains (in the same order as in panel B) to killing by either 10% (vol/vol) heat-inactivated NHS (a) or 10% NHS (b). The data presented here are the means of results from three independent experiments plus the standard errors.

These results suggested that the region of the O35E UspA2 protein between amino acids (aa) 143 and 273 was essential for converting MC317.2 to a serum-resistant phenotype. To confirm this, hybrid 6 was constructed in which the O35E region extending from aa 143 to 244 was used to replace the equivalent region in the MC317 UspA2 protein. When tested in the serum bactericidal assay, MC317.2 expressing the hybrid 6 UspA2 protein (Fig. 6C, column 7b) expressed a level of serum resistance equivalent to that obtained with both MC317.2 expressing the O35E UspA2 protein (Fig. 6C, column 8b) and O35E-Smr (Fig. 6C, column 9b).


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DISCUSSION
 
Previous studies from this laboratory showed that inactivation of the uspA2 gene or the very similar uspA2H gene of serum-resistant M. catarrhalis strains (3, 30) resulted in sensitivity to killing by complement-sufficient NHS. Whether the loss of expression of UspA2 (or UspA2H) itself was directly responsible for the altered phenotype or whether the absence of UspA2 affected or altered the expression of some other surface antigen that is directly responsible for serum resistance could not be determined in these earlier studies. To address this issue, we cloned and expressed the uspA2 genes from both a serum-resistant M. catarrhalis strain and a serum-sensitive M. catarrhalis strain in a heterologous background (i.e., H. influenzae). When tested in a serum bactericidal assay, the UspA2 protein from the serum-resistant strain, but not the UspA2 protein from the serum-sensitive strain, conferred increased serum resistance on the H. influenzae recombinant strain.

The fact that some strains of M. catarrhalis can resist serum killing was first described some 20 years ago by Winn and Morse (reviewed in references 10 and 50), and several recent studies indicate that the vast majority of M. catarrhalis isolates are resistant to serum killing (13, 34, 45). The identity of the gene product(s) responsible for this phenotype has been the subject of much research interest over the past decade, and it was first suggested by Verduin et al. (54) that a proteinaceous substance on the surface of M. catarrhalis was responsible for complement resistance. Initial studies attempting to identify either the proposed resistance factor or the mechanism of resistance utilized serum-resistant and serum-sensitive clinical isolates (8, 54, 55), and only in the past few years have isogenic parent-mutant pairs been tested (3, 14, 22, 36, 60). These studies indicated that there were several mutations affecting the expression of outer membrane proteins (14, 17, 22, 36) or LOS (60) that could alter serum resistance of M. catarrhalis to various degrees.

The results of the present study showed that UspA2 is directly involved in the expression of serum resistance by some strains of M. catarrhalis. That UspA2 might be involved in serum resistance was also predicted by another independent study using phage antibodies to characterize serum-resistant and serum-sensitive isolates of M. catarrhalis. Phage antibodies that selectively bound serum-resistant strains were shown to bind high-molecular-weight outer membrane protein (29), which is identical to UspA2, in Western blot analysis (8). Whether the serum-sensitive M. catarrhalis strains that failed to bind these phage antibodies also did not express a UspA2 protein was not reported (8).

Two recent studies have suggested that complement-resistant M. catarrhalis strains comprise a distinct subpopulation or lineage within this species (9, 56). One group of workers used pulsed-field gel electrophoresis, a nonribosomal PCR restriction fragment length polymorphism (RFLP) procedure, and random amplification of polymorphic DNA analysis to divide 47 serum-resistant and 28 serum-sensitive strains into two groups, with the serum-resistant strains falling into a clonal group (56). The other laboratory applied probe-generated RFLP and single-adapter amplified fragment length polymorphism analyses to characterize 90 M. catarrhalis strains, resulting in a dendrogram that had two main branches (9). The vast majority of complement-resistant strains examined in the latter study clustered into one of the two main branches. Interestingly, PCR-based analysis indicated that equal percentages of complement-sensitive and complement-resistant strains had a uspA2 gene. This finding raises the possibility that these complement-sensitive stains expressed a UspA2 protein similar to that of MC317, which was unable to confer serum resistance on M. catarrhalis.

That some M. catarrhalis strains express a UspA2 protein that cannot confer serum resistance is reminiscent of the situation with some strains of serum-resistant and serum-sensitive Neisseria gonorrhoeae, which differ in their expression of a particular type of porin protein (42). Gene exchange experiments involving the uspA2 genes of strains O35E and MC317 and the consequent change in the serum sensitivity of the transformant strains (Fig. 4) suggested that differences within the primary amino acid sequences of these two UspA2 proteins were responsible for these different susceptibilities to killing by NHS. This hypothesis was confirmed by the finding that the region of the O35E UspA2 protein between aa 143 and 244 was sufficient to convert strain MC317 to serum resistance when expressed in the equivalent position within the MC317 UspA2 protein.

Exactly how the UspA2 protein confers serum resistance on some M. catarrhalis strains remains to be determined. A previous study from another laboratory comparing serum-sensitive and serum-resistant M. catarrhalis isolates used an indirect method (i.e., a bystander hemolytic assay) to show that M. catarrhalis strains activated the classical complement pathway in an IgG-dependent manner (54). Use of the uspA2 deletion mutant in the present study in a direct bactericidal assay confirmed both that the classical complement pathway was essential for killing of this mutant (Fig. 2A) and that serum IgG antibody was also necessary (Fig. 2B). It is possible that UspA2 could bind an inhibitor or regulator of the classical complement cascade. One group has suggested that the binding of the serum protein vitronectin by UspA2 (i.e., high-molecular-weight outer membrane protein) (33) interferes with the complement cascade (reviewed in reference 8). More recently, another laboratory has proposed that both UspA2 and the UspA1 protein bind C4bp, a soluble inhibitor of the classical pathway (reviewed in reference 7). Alternatively, UspA2 might form a shield or protective layer on the surface of M. catarrhalis, preventing serum bactericidal antibodies from binding to certain antigens on the surface of the outer membrane. The fact that UspA2 forms what appears to be a very dense layer of projections that extend from the bacterial cell surface (19, 41) gives some credence to the latter possibility. In this situation, the UspA2 protein might also be able to physically hinder insertion of the membrane attack complex. However, the fact that some UspA2-directed antibodies are bactericidal for M. catarrhalis (11, 33) is not consistent with the latter hypothesis. It is most likely that some combination of the possibilities cited above is responsible for the ability of UspA2 to confer serum resistance on some strains of M. catarrhalis. Ongoing efforts in this laboratory are focused on elucidating the relevant mechanism(s).


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ACKNOWLEDGMENTS
 
This study was supported by U.S. Public Health Service grant no. AI36344 to E.J.H.

We thank Anthony Campagnari for expert advice and assistance in LOS analysis. We appreciate the helpful comments of the members of the Hansen laboratory in the development of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9048. Phone: (214) 648-5974. Fax: (214) 648-5905. E-mail: eric.hansen{at}utsouthwestern.edu. Back

Editor: D. L. Burns

{dagger} Present address: Department of Microbiology, Medical College of Ohio, Toledo, OH 43614-5806. Back


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Infection and Immunity, April 2005, p. 2400-2410, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2400-2410.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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