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Previous Article | Next Article 
Infection and Immunity, August 2002, p. 4523-4533, Vol. 70, No. 8
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.8.4523-4533.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Nikki J. Wagner,1 Joseph W. St. Geme III,2 and Eric J. Hansen1*
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390,1 The Edward Mallinckrodt Department of Pediatrics and Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 631102
Received 20 February 2002/ Returned for modification 5 April 2002/ Accepted 6 May 2002
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ABSTRACT |
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INTRODUCTION |
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In contrast, there is relatively little known about other surface proteins of M. catarrhalis that might be involved in the ability of this organism to colonize and survive in the nasopharynx (35). The CD outer membrane protein (33) has been shown to bind middle ear mucin in vitro (51), a function that could be involved in the colonization process or in the development of otitis media. The UspA1 protein has been shown to be an adhesin, at least in vitro (38), whereas both the UspA2 protein (38) and outer membrane protein E (50) have been implicated in serum resistance. Both UspA1 and UspA2, consistent with their functional activities, have been localized to the surface of M. catarrhalis, where they are accessible to antibodies (2, 45).
Scott and colleagues (16, 17) correlated both hemagglutination activity and the expression of a 200-kDa protein by some M. catarrhalis isolates with the presence of a fibrillar surface array. In addition, Sasaki and colleagues reported that the 200-kDa protein expressed by M. catarrhalis was subject to phase variation in vitro (K. Sasaki, L. Myers, S. M. Loosmore, and M. H. Klein, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. B/D-306, 1999) and determined the nucleotide sequence of the gene encoding this protein (54). In the present study, we used analysis of mutants to show that this protein, designated Hag (hemagglutinin), is involved not only in hemagglutination but also in autoagglutination and the binding of human immunoglobulin D (IgD) by M. catarrhalis strain O35E. In addition, we determined that the Hag protein, together with the UspA1 and UspA2 proteins (3), all form fibrillar projections on the M. catarrhalis cell surface.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MAbs and Western blot analysis.
Monoclonal antibody (MAb) 17C7, reactive with both the UspA1 and UspA2 proteins of M. catarrhalis strain O35E (3), and MAb 10F3, reactive with the CopB outer membrane protein of this strain (26), have been described. To obtain a MAb specific for the Hag protein, synthetic peptide DNADGNQVNIADIKKDPNSGSSSNR (Hag-1) was synthesized by the Biopolymers Facility at the University of Texas Southwestern Medical Center and covalently bound to keyhole limpet hemocyanin (KLH; Sigma, St. Louis, Mo.) with glutaraldehyde. The sequence of Hag-1 corresponds to an amino acid sequence in the C-terminal one-third of the Hag protein that is present in all Hag proteins whose open reading frames (ORFs) have been sequenced to date (data not shown). The Hag-1-KLH conjugate was used to immunize mice for hybridoma production as previously described (2); MAb 5D2 was shown by enzyme-linked immunosorbent assay to bind a Hag-1-ovalbumin conjugate and was shown by Western blot analysis to bind a 200-kDa M. catarrhalis antigen. Human IgD 
chain myeloma protein (The Binding Site, San Diego, Calif.) was used as the source of IgD for the IgD binding assays. Western blot analysis was performed using either affinity-purified and radioiodinated goat anti-mouse Ig (38) or horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, Pa.) as the secondary antibody to detect mouse MAbs. To detect human IgD, horseradish peroxidase-conjugated goat anti-human IgD (Biosource International, Camarillo, Calif.) was used as the secondary antibody. Horseradish peroxidase-antibody conjugates were detected by chemiluminescence with Western Lightning Chemiluminescence Reagent Plus (New England Nuclear, Boston, Mass.).
TEM. After M. catarrhalis cells were grown for 3 days on the Sorbarod filter, the BHI growth medium was replaced by transmission electron microscopy (TEM) prefixative consisting of 75 mM lysine monohydrochloride (Sigma), 2% (vol/vol) paraformaldehyde (Electron Microscopy Sciences, Fort Washington, Pa.), and 2.5% (vol/vol) glutaraldehyde (Sigma) in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences). Prefixative was pumped onto the filter at 0.1 ml/min for 1 h at 37°C. Then, TEM fixative consisting of 2% paraformaldehyde and 2.5% (vol/vol) glutaraldehyde in 0.1 M sodium cacodylate was pumped onto the filters for 2 h at a rate of 0.1 ml/min at 37°C. The filter was then rinsed three times for 10 min in 0.1 M sodium cacodylate buffer at room temperature after which 1% osmium tetroxide in this buffer (Electron Microscopy Sciences) was added and the filter was rocked gently for 90 min. The filter was then washed with distilled water, dehydrated with ethanol, and embedded in Spurr resin (Polysciences, Warrington, Pa.), which was polymerized at 60°C overnight. Sections for TEM were cut at 80 nm with a diamond knife (Micro Star, Huntsville, Tex.) and picked up on copper 200-mesh thin-bar grids (Electron Microscopy Sciences). The grids were stained with uranyl acetate and lead citrate and observed with a JEOL 1200EX II transmission electron microscope.
Cryoimmunoelectron microscopy. Biofilm-grown M. catarrhalis cells in the Sorbarod filter were pre-fixed as described above except that the prefixative was composed of 2% paraformaldehyde, 0.2% glutaraldehyde, and 75 mM lysine monohydrochloride in phosphate-buffered saline (PBS), pH 7.3. The cells on the filter were then fixed with 2% paraformaldehyde and 0.2% glutaraldehyde in PBS, pH 7.3, at 37°C for 2 h as described above. Subsequently, they were embedded in 10% gelatin as described previously (57) except that the gelatin was not fixed. After centrifugation, the gelatin was solidified on ice and the blocks were prepared for ultramicrotomy and infused with 2.3 M sucrose. Ultrathin sections were obtained and immunolabeled as described previously (58) with minor modifications. In particular, 10% (vol/vol) goat serum was used in the blocking buffer in place of 1% bovine serum albumin and immunolabeling was carried out with a 1:1 dilution of MAb 5D2 or a 1:10 dilution of MAb 17C7 for 2 h and a 1:15 dilution of goat anti-mouse IgG-18-nm colloidal gold (Jackson ImmunoResearch Laboratories) for 1 h. Sections were stained with uranyl acetate and embedded in methyl cellulose according to a modification of the method of Tokuyasu (62) introduced by Griffiths et al. (21). Samples were viewed and photographed with a Zeiss 902 electron microscope.
PCR. PCR was performed with either XL (Perkin-Elmer Biosystems, Foster City, Calif.) or ExTaq (PanVera, Madison, Wis.) DNA polymerase according to the manufacturers' instructions. Purified chromosomal DNA (Easy-DNA kit; Invitrogen) was used as the template for PCR. Oligonucleotide primers P1 (5'-TTGCCCCATATCTGTACG-3') and P2 (5'-GGTCATGGTGAAAGAGAATC-3') were used to amplify a 7-kb product containing the hag gene from strain O35E. Oligonucleotide primers P3 (5'-AGAATGATGATGCCTACGAG-3') and P2 were used to amplify the hag gene from strain O12E.
Nucleotide sequence analysis. PCR products were sequenced with a model 373A or model 377 automated DNA sequencer (Perkin-Elmer Biosystems). DNA sequence information was analyzed by using the MacVector analysis package (version 6.5; Oxford Molecular Group, Campbell, Calif.).
Construction of isogenic mutants. Strain O35E.118CAT, an isogenic uspA1 mutant version of M. catarrhalis strain O35E, has been described (39). Isogenic uspA2 mutants were produced by using oligonucleotide primers 5'-CGGGATCCTTCTCCCCCTAAAAATCGCTGT-3' and 5'-AGGGATCCCGCTGTATGCCGCTACTCGCAGCT-3' (BamHI sites are underlined) for the PCR-based amplification of a 2.6-kb fragment containing an incomplete uspA2 ORF from wild-type M. catarrhalis strain P44; this fragment was cloned into pCR2.1 (Invitrogen). This uspA2 sequence was then subcloned as an EcoRI fragment into pBluescript KS(+) (Stratagene, La Jolla, Calif.). A 0.4-kb BglII fragment was deleted from the middle of the uspA2 sequence, and a 0.5-kb Zeocin resistance cassette was ligated into this site to create plasmid pELU244ZEO. This plasmid was electroporated into M. catarrhalis strain O35E to produce uspA2 mutant O35E.2ZEO by allelic exchange. uspA1 uspA2 double mutant O35E.ZC was constructed by using plasmid pELU1CAT (39) to electroporate uspA2 mutant O35E.2ZEO and by identifying a transformant resistant to both chloramphenicol and Zeocin.
With the working assumption that the 200-kDa protein of M. catarrhalis strain 4223 (Sasaki et al., Abstr. 99th Gen. Meet. Am. Soc. Microbiol., 1999) was likely the same protein described phenotypically by Fitzgerald and coworkers (16), the nucleotide sequence of the gene from M. catarrhalis strain 4223 encoding the 200-kDa protein (54) was used to design oligonucleotide primers for the PCR-based amplification of the hag gene. Isogenic hag mutants were constructed by using oligonucleotide primers 5'-ATTCTAGAGCTCAGGGTGATGCCTCGATTGCC-3' and 5'-ATTCTAGATGGAAGAAGCGGATACCTTGTTC-3' (XbaI sites are underlined) together with M. catarrhalis strain P44 chromosomal DNA to amplify a 5.5-kb fragment from within the hag ORF, which was subsequently cloned into the XbaI site in pUC19 (New England Biolabs, Beverly, Mass.). A spectinomycin resistance cartridge (63) was then ligated into the EcoRV site in the hag fragment to make plasmid pELHGSPEC. uspA1 uspA2 hag triple mutant O35E.ZCS was constructed by electroporating uspA1 uspA2 mutant O35E.ZC with pELHGSPEC and identifying a transformant resistant to chloramphenicol, Zeocin, and spectinomycin. The other hag mutants used in this study (Table 1) were constructed by electroporating wild-type and mutant strains of M. catarrhalis with pELHGSPEC.
Hemagglutination assays. Overnight 4-ml cultures of M. catarrhalis were centrifuged at 7,500 x g for 8 min and resuspended in PBS to a density of 300 Klett units with a Klett-Summerson colorimeter (Klett Mfg. Co., New York, N.Y.). A 50-µl portion of this suspension and serial twofold dilutions of this suspension were added in triplicate to a 96-well U-bottom Costar polypropylene plate (Fisher Scientific Co., Pittsburgh, Pa.). Citrated human blood (Rockland, Gilbertsville, Pa.) was centrifuged at 1,000 x g, and a 2% (vol/vol) suspension of erythrocytes in PBS was prepared. A 50-µl portion of the erythrocyte suspension was then added to each well, and the microtiter plate was gently agitated on a Vortex mixer for 30 s. Hemagglutination was recorded photographically after 15 min.
Autoagglutination assays. M. catarrhalis cells scraped from the surface of BHI agar plates were suspended in 1 ml of PBS (pH 7.3). Portions of this suspension were added to 4 ml of PBS to attain a density of 400 Klett units in a glass tube. Autoagglutination was measured as the decrease in Klett units over time.
Adherence assays. The ability of M. catarrhalis to attach to Chang human conjunctival epithelial cells in vitro was measured as described previously (38).
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RESULTS |
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Effect of the hag mutation on hemagglutination ability. To determine whether the 200-kDa protein described by Scott and coworkers (46) as being associated with the hemagglutination ability of some M. catarrhalis strains was encoded by the hag gene, we constructed an isogenic hag mutant version of M. catarrhalis strain O35E. The hag mutant, O35E.HG (Fig. 1B, lane 4), did not express any detectable Hag protein, whereas the wild-type parent strain, O35E (Fig. 1B, lane 1), expressed a readily detectable 200-kDa antigen that bound Hag-specific MAb 5D2. This hag mutant (Fig. 1A, lane 4) also expressed wild-type levels of both UspA1 and UspA2. The wild-type parent strain (Fig. 2A) caused hemagglutination of human erythrocytes. In contrast, hag mutant O35E.HG did not cause hemagglutination (Fig. 2B).
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Construction of mutants deficient in expression of surface-exposed proteins. Previous studies from this laboratory indicated that both UspA1 and UspA2 are present on the bacterial cell surface (2), and it was apparent from the functional activity of the Hag protein (e.g., hemagglutination) that it was also surface exposed. In preliminary experiments examining the growth of M. catarrhalis in biofilms, we were able to use TEM of biofilm-derived cells to detect the presence of a dense layer of projections extending from the cell surface of wild-type strain O35E (Fig. 7A). To determine whether the Hag, UspA1, and UspA2 proteins were present in this surface array on the wild-type parent strain, we first constructed an M. catarrhalis strain O35E mutant that was unable to express these three proteins. This uspA1 uspA2 hag triple mutant (Fig. 1, lane 8) expressed none of these proteins at a detectable level, whereas the wild-type parent strain expressed all three macromolecules, as determined by Western blot analysis of whole-cell lysates (Fig. 1, lane 1). TEM of biofilm-derived cells of this uspA1 uspA2 hag mutant (Fig. 7B) revealed that it appeared to possess no surface projections except for some bleb-like structures.
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Cryoimmunoelectron microscopy was used to confirm that all three of these proteins were expressed on the M. catarrhalis cell surface. The uspA2 hag double mutant, which expressed only the UspA1 protein, bound the UspA1- and UspA2-reactive MAb 17C7 (Fig. 8A) and did not bind the Hag-specific MAb 5D2 (Fig. 8B). The uspA1 hag double mutant, which expressed only UspA2, also bound MAb 17C7 (Fig. 8C) and did not bind MAb 5D2 (Fig. 8D). Finally, the uspA1 uspA2 double mutant, which expressed only the Hag protein, bound the Hag-specific MAb 5D2 (Fig. 8F) but did not react with MAb 17C7 (Fig. 8E). In all cases, MAb-mediated binding of the antibody-conjugated gold particles was localized almost exclusively to the surface of the bacterial cell or to the area immediately exterior to the cell surface.
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DISCUSSION |
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Our previous studies had indicated that both UspA1 and UspA2 were exposed on the surface of M. catarrhalis (2, 14) and that both of these proteins had regions that were likely to form coiled coils (14), which could project from the cell surface. The reported association of the 200-kDa protein with a fibrillar layer on some strains of M. catarrhalis (17) prompted us to investigate whether the UspA1 and UspA2 proteins could also be involved in the formation of this fibrillar layer. Examination by TEM of a triple mutant lacking the ability to express the Hag, UspA1, and UspA2 proteins (Fig. 7B) revealed that there were no detectable fibrillar projections on the surface of this strain. We then examined the surface phenotype of double mutants which could express only one of each of these three proteins. These studies revealed that a uspA2 hag mutant with a functional uspA1 gene (Fig. 7C) and a uspA1 uspA2 mutant with a functional hag gene (Fig. 7E) both expressed fibrillar structures that were sparsely distributed on the bacterial cell surface. In contrast, the uspA1 hag mutant, which could express UspA2 (Fig. 7D), had a much denser layer of shorter projections on its cell surface. Close inspection of the wild-type parent strain (Fig. 7A) reveals the presence of two layers of projections. It is of interest to note that Wistreich and Baker (64) reported that the single fimbriate strain of M. catarrhalis used in their study expressed two fimbrial types, with one being much longer than the other.
A recent report from Hoiczyk et al. (32), which appeared while our study was in progress, indicated that a uspA1 uspA2 mutant version of strain O35E that had been constructed previously by our laboratory (2) lacked filamentous projections. There are two possible explanations for the reported absence of filamentous projections on this particular uspA1 uspA2 mutant. The first is that the sparsely distributed surface projections associated with expression of the hag gene may simply have been destroyed in the preparation of their samples for TEM. The second possibility would involve a mutation in the hag gene. Sasaki and coworkers (Abstr. 99th Gen. Meet. Am. Soc. Microbiol., 1999) reported the presence of nine consecutive G residues near the 5' end of the ORF encoding the 200-kDa protein of M. catarrhalis strain 4223. A spontaneous change in the number of G residues from nine to eight, likely as the result of slipped-strand mispairing (30), caused lack of expression of the 200-kDa protein. The latter possibility seems less likely to have occurred in uspA1 uspA2 mutant O35E.12 (2), however, in view of the fact that the corresponding region of the hag gene of strain O35E has only six G residues and this shorter poly(G) tract would be less likely to undergo slipped-strand mispairing.
Inactivation of the hag gene in M. catarrhalis strain O35E resulted in the loss of both hemagglutination (Fig. 2) and autoagglutination (Fig. 3) ability. This result confirmed that the Hag protein is responsible for hemagglutination by strain O35E. In addition, the propensity of this strain to form large aggregates (i.e., autoagglutination) was eliminated by the hag mutation. Interestingly, loss of the ability to express Hag was not associated with a decreased ability to attach to several human epithelial cell lines, including Chang conjunctival epithelial cells (Fig. 4) and HEp-2 cells. This finding confirms an earlier report by Scott and colleagues (18) which indicated that the hemagglutination activity of M. catarrhalis isolates appeared to be independent of their ability to attach to HEp-2 cells in vitro. Whether the Hag protein could be involved in the attachment of M. catarrhalis to other cell types or to respiratory tract mucin (51) remains to be determined.
The lack of autoagglutination by the hag mutant (Fig. 3) mimics that described for a spontaneous mutant or variant version of M. catarrhalis strain 4223 that was isolated by Murphy and colleagues (37). These workers selected a nonclumping variant of strain 4223 by sequential passage in broth. This variant lacked detectable expression of a 200-kDa protein and also had reduced expression of the high-molecular-weight outer membrane protein (i.e., UspA2) as well as altered surface accessibility of some but not all of the surface epitopes of its outer membrane protein CD and its lipooligosaccharide molecule (37). It is likely that the 200-kDa protein missing from this variant is Hag, although this needs to be confirmed by Western blot analysis. The existence of a spontaneous frameshift mutant of strain 4223 that lacked the ability to express the 200-kDa protein (Sasaki et al., Abstr. 99th Gen. Meet. Am. Soc. Microbiol., 1999) raises the possibility that the nonclumping variant isolated by broth passage (37) may have had the same type of mutation in the hag ORF. The other alterations in the phenotype of this nonclumping variant could have been the effect of the lack of expression of the 200-kDa protein on the surface architecture of the outer membrane. Alternatively, this variant of strain 4223 may have possessed additional genetic changes.
It is of interest that the majority of the IgD-binding activity associated with the Hag protein (Fig. 5A) did not migrate to the same position in the separating gel as did the Hag antigen which bound the Hag-specific MAb 5D2 (Fig. 5B). Most of the MAb-reactive Hag protein migrated to a position just beneath the 216-kDa marker, whereas the IgD-binding activity migrated much more slowly, with an apparent molecular mass well in excess of 216 kDa. This behavior of the Hag protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is similar to that described for UspA1 and UspA2, both of which form very large aggregates or complexes when analyzed by SDS-PAGE (14). It should also be noted that the IgD-binding activity of the Mid protein, described by Forsgren et al. (19), had two different forms in SDS-PAGE, with one form migrating near the 200-kDa marker and the other form barely entering the separating gel. It appears from our data that IgD preferentially binds to the multimeric form of the Hag protein from strain O35E; the structural basis for this recognition pattern remains to be determined.
The biologic significance of two (i.e., hemagglutination and autoagglutination) of the three different phenotypes associated with expression of the Hag protein by M. catarrhalis strain O35E has been determined for a number of different organisms. Hemagglutination in gram-negative bacteria is often associated with expression of pili or nonpilus adhesive proteins that promote attachment to and colonization of host mucosal surfaces (40, 41, 44, 55). The M. catarrhalis hag mutant did not display any decrease in its ability to attach to a number of human cell lines in vitro, although it is possible that lack of Hag expression could affect attachment to cell types not included in the present study. Autoagglutination, often caused either by certain types of pili or by outer membrane proteins, has been used as a marker of virulence or attachment ability for several gram-negative pathogens including such diverse organisms as Vibrio cholerae (13), Yersinia enterocolitica (56), and Fusobacterium nucleatum (24). The lack of a relevant animal model for testing the virulence of M. catarrhalis precludes determination of whether the hag mutant is truly less virulent than its wild-type parent strain.
The biologic relevance of IgD-binding activity is more difficult to ascertain. It has been known for many years that M. catarrhalis readily binds soluble human IgD (20), and Forsgren and colleagues have reported recently that the Mid protein binds IgD-expressing B cells (19). Soluble IgD is present in the nasopharynges of healthy children (59), thus providing the opportunity for this Ig to be bound by M. catarrhalis. Whether the interaction between IgD and the Hag protein might somehow augment the ability of M. catarrhalis to colonize the nasopharynx or cause otitis media remains to be determined.
The N-terminal and C-terminal regions of the 200-kDa protein from strain 4223 (54), the Hag proteins of strains O35E and O12E, and the Mid protein of strain Bc5 (19) have nearly perfect identity, with significant amino acid sequence differences being found in the more-central regions of these proteins. This situation is similar to that described individually for the UspA1 and UspA2 proteins of M. catarrhalis, where the N and C termini of each type of macromolecule are virtually identical among different strains (14). If the four 200-kDa proteins described immediately above are truly all Hag proteins, it appears that there is a clustering of amino acid sequence polymorphisms between the highly conserved N and C termini. This is similar to the mosaic structures of other surface-exposed proteins from different pathogens including Neisseria gonorrhoeae (23) and Neisseria meningitidis (31), where mosaic genes have been proposed to result from horizontal genetic exchange. The fact that M. catarrhalis can be transformed in vitro (11) makes this possibility feasible.
The IgD-binding activity of the Hag protein described in this study makes it likely that Hag and the Mid protein described by Forsgren et al. (19) are the same macromolecule with strain-specific amino acid sequence differences. Definitive proof that Hag and Mid are the same protein is dependent on the demonstration that an isogenic mid mutant version of M. catarrhalis strain Bc5 has lost its abilities to bind human IgD and hemagglutinate human erythrocytes.
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ACKNOWLEDGMENTS |
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We thank John D. Nelson, Timothy F. Murphy, Steven L. Berk, Frederick W. Henderson, and Paul Roy for providing the M. catarrhalis strains used in this study. We also thank both Dennis Belotto and Marilyn Levy for expert technical assistance in performing TEM and cryoimmunoelectron microscopy, respectively.
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FOOTNOTES |
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Present address: Department of Microbiology and Immunology, Medical College of Ohio, Toledo, OH 43614-5806.
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REFERENCES |
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