Hospital,4 State University of New York at Buffalo, Buffalo, New York. Received 22 ... This study was designed to understand the mechanism of mucin-bacterial.
INFECTION AND IMMUNITY, Apr. 1996, p. 1477–1479 0019-9567/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 64, No. 4
Binding between Outer Membrane Proteins of Nontypeable Haemophilus influenzae and Human Nasopharyngeal Mucin MOLAKALA S. REDDY,1* JOEL M. BERNSTEIN,2 TIMOTHY F. MURPHY,3 4 AND HOWARD S. FADEN Department of Oral Biology, School of Dental Medicine,1 Department of Otolaryngology and Pediatrics2 and Division of Infectious Diseases, Departments of Medicine and Microbiology,3 School of Medicine and Buffalo Veterans Administration Hospital, and Department of Pediatrics, Children’s Hospital,4 State University of New York at Buffalo, Buffalo, New York Received 22 September 1995/Returned for modification 27 November 1995/Accepted 23 January 1996
Bacterial colonization of the epithelial cells precedes infection. Mucins of the epithelial cell secretions modulate bacterial colonization. This study was designed to understand the mechanism of mucin-bacterial interactions and in particular binding between nontypeable Haemophilus influenzae and nasopharyngeal mucin(s). In an overlay assay, binding appears to be mediated by outer membrane proteins P2 and P5 of bacteria and sialic acid-containing oligosaccharides of mucin. dition, staining the gel with Coomassie blue revealed the presence of several protein components in pools A and B (Fig. 1b). Immunostaining identified them as serum proteins. For further purification of the high-molecular-weight glycoproteins, pools A and B were combined (87 mg), solubilized in Tris-guanidine buffer (pH 8.5), reduced with dithiothreitol (50 mg for 2 h at 378C), alkylated with iodoacetamide (132 mg for 1 h at room temperature), and fractionated in a column of Sepharose CL-2B (Fig. 1c). Pool 1 (8 mg), pool 2 (13 mg), pool 3 (7 mg), pool 4 (27 mg), and pool 5 (24 mg) were obtained. Although no protein contaminants were detectable in pools 1, 2, and 3 when the gel was stained with Coomassie blue (Fig. 1d), SDS-PAGE of iodinated samples revealed the presence of a trace quantity of protein contaminants in pool 1. A higher carbohydrate content and the presence of a large number of O-glycosidically linked oligosaccharides indicated that pools 2 and 3 were mucins (Table 1). Since their chemical compositions were similar, they were combined and designated as HNM. HNM isolated in this study may represent a mixture of disulfide-linked subunits, as the native mucins are thought to be oligomers of smaller identical subunits. OMPs of NTHi and H. influenzae b (Hib) were isolated as described earlier (5, 9, 12, 13). HNM was radiolabeled by iodination (3). Binding of [125I]HNM to OMPs of NTHi and Hib was examined by an overlay assay (18). In all the strains (28 NTHi and 4 Hib strains) examined, [125I]HNM bound to two OMPs migrating between 45 and 31 kDa. The binding pattern of a representative strain is presented in Fig. 2b, lane 2. On the basis of electrophoretic mobility and immunoblotting, these two proteins were identified as OMPs P2 and P5. Unequivocal identification of OMPs P2 and P5 was made by employing OMPs of bacterial strains and their respective isogenic mutants lacking the respective OMP in the binding assays. Thus, OMPs of Hib DL42 and its isogenic mutant lacking OMP P2 and OMPs of NTHi 1128 and its isogenic mutant lacking OMP P5 were employed in the assay to confirm that OMPs P2 and P5 function as adhesins for HNM. However, in the case of two NTHi strains, [125I]HNM also bound to additional lower-molecular-weight OMPs migrating between 31 and 21.5 kDa. A comparison of the relative concentrations of OMPs P2 and P5 and the lower-molecular-weight OMPs mi-
Nontypeable Haemophilus influenzae (NTHi) normally resides in the nasopharynx and most often causes otitis media and sinusitis (10). In order for the pathogen to maintain a habitat on the mucosa, it must first adhere. A number of studies demonstrated the adherence of H. influenzae to erythrocytes (2), to buccal (8), nasopharyngeal (15), oropharyngeal (4), and tissue culture epithelial cells (23), and to the mucus layer present on the epithelial cells (22). Pilus (4, 15), nonpilus (23), and exposed-surface high-molecular-weight proteins (24) have been implicated in the attachment process to the epithelial cells. On the other hand, neither pili nor the capsule appears to function as an adhesin(s) for nasopharyngeal mucus (22). On the basis of the available literature on respiratory mucus-H. influenzae interactions, it can be concluded that mucins of the nasopharyngeal mucus may function as receptor molecules for NTHi and thus play an important role in the colonization of the nasopharynx (1, 6, 7, 20). In fact, Shuter et al. (21) recently demonstrated the binding of human nasal mucin to the outer membrane proteins (OMPs) of Staphylococcus aureus. Binding appears to be mediated by the carbohydrate moiety of the nasal mucin. In the present study, we purified human nasopharyngeal mucin (HNM) and utilized it in an overlay binding assay to identify the NTHi OMPs that function as adhesins. Experiments to understand the nature of HNM receptors have also been conducted. Nasopharyngeal secretions from 2- to 8-year-old children undergoing an adenoidectomy for either otitis media with effusion or for hypertrophic adenoids causing obstruction were lyophilized and fractionated in a column (1.6 by 95 cm) of Sepharose CL-2B with Tris-guanidine buffer (0.1 M Tris-HCl [pH 7.5] and 6 M guanidine). Chromatography of nasopharyngeal secretions (42 mg) yielded pools A (8 mg), B (6 mg), and C (24 mg) (Fig. 1a). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by periodic acid-Schiff reagent staining indicated the presence of high-molecularweight glycoproteins at the interfaces of the stacking and separating gels in pools A and B (results not presented). In ad* Corresponding author. Mailing address: Department of Oral Biology, 109 Foster Hall, School of Dental Medicine, Buffalo, NY 14214. Phone: (716) 829-2114. Fax: (716) 829-3942. 1477
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INFECT. IMMUN.
FIG. 1. (a) Elution profile of nasopharyngeal secretions in Sepharose CL-2B. (b) SDS-PAGE of the column fractions from panel a. Lane 1, molecular weight markers; lanes 2 through 4, pools A through C, respectively. (c) Elution profile of reduced and alkylated pools A and B in Sepharose CL-2B. (d) SDS-PAGE of the column fractions from panel c. Lane 1, molecular weight markers; lanes 2 through 6, pools 1 through 5, respectively.
grating between 31 and 21 kDa (Fig. 2a, lane 3) in the binding of [125I]HNM (note the band intensity in the autoradiograph; Fig. 2b, lane 3) indicated that binding to the lower-molecularweight components was greater than that to OMPs P2 and P5. These lower-molecular-weight OMPs have not yet been identified. Utilization of purified OMP P6 in the binding assay
TABLE 1. Chemical composition of HNM containing fractions obtained by gel filtration of reduced and alkylated nasal secretions No. of residues per 1,000 amino acids Component
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine S-carboxymethylcysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine N-acetylgalactosamine N-acetylglucosamine Fucose Galactose Mannose Sialic acid a
Tr, present in trace amounts.
Pool 2
Pool 3
70 125 84 90 71 90 89 10 18 61 10 29 86 19 36 22 45 45 170 334 127 238 Tra 110
69 138 85 93 75 73 92 8 22 62 8 24 85 18 35 24 48 41 180 338 142 244 Tr 80
FIG. 2. (a) Various bacterial OMPs Western transferred to the polyvinylidene difluoride membrane were stained with Coomassie blue. (b) Autoradiograph showing the proteins that bound [125I]HNM. Proteins involved in binding are marked with arrowheads in both panels. Lane 1, molecular weight markers; lanes 2 through 7, NTHi 47, NTHi 80, M. catarrhalis 2015 in duplicate, the pilus subunit, and an adhesin of P. aeruginosa, respectively.
indicated that it was not a member of the lower-molecularweight adhesins. From the observations discussed above, it can be concluded that only selected OMPs of NTHi bind HNM and that the binding is not concentration dependent. To examine the selectivity and specificity of HNM-bacterial interactions, binding studies employing OMPs of two other pathogens, Moraxella catarrhalis (11) and Pseudomonas aeruginosa (18), were performed. Of the several OMPs of M. catarrhalis, [125I]HNM bound to a single OMP with a size of ;57 kDa (Fig. 2a and b, lanes 4 and 5). The 57-kDa component was identified as the CD protein of M. catarrhalis (19). Similarly, binding of [125I]HNM to two proteins of P. aeruginosa, an adhesin to tracheobronchial mucin (18), and a subunit of P. aeruginosa pili was examined. [125I]HNM bound to the adhesin to tracheobronchial mucin and not to the pilus subunit (Fig. 2a and b, lanes 6 and 7). We also observed that of the various proteins (phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and lysozyme) employed as protein markers, [125I]HNM bound only to lysozyme (results not presented). From the observations discussed above, it can be concluded that only a few OMPs participate in bacterial binding to mucins, and therefore binding may involve specific interactions. Preliminary experiments to identify the HNM receptor(s) for OMPs P2 and P5 of NTHi were conducted. Asialo-[125I] HNM was prepared by mild acid hydrolysis of [125I]HNM and utilized in the binding studies. Removal of sialic acid resulted in a loss of binding of [125I]HNM to OMPs P2 and P5, indicating a role for sialic acid-containing oligosaccharides in bacterial binding. This role was confirmed by inhibition studies. Following Western transfer, the polyvinylidene difluoride membrane was incubated with O-glycosidically linked oligosaccharides isolated from fetuin (3.2 mmol) prior to the addition of [125I]HNM. Fetuin O-glycosidically linked oligosaccharides inhibited the binding of [125I]HNM to OMPs P2 and P5. These results are in agreement with those of several other studies that found that mucin oligosaccharides function as receptors for bacteria (6, 14, 16, 17, 25). In conclusion, a nasopharyngeal mucin(s) subunit(s) isolated from nasopharyngeal secretions was utilized in an overlay binding assay to determine the mechanism of binding between
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mucin and OMPs of NTHi. OMPs P2 and P5 and unidentified lower-molecular-weight OMPs of NTHi appear to function as adhesins for HNM. Sialic acid-containing oligosaccharides of HNM appear to be the receptors for NTHi. Further characterization of the mucin receptors and bacterial adhesins is in progress. The results may lead to a better understanding of the mechanism of NTHi colonization of the nasopharynx and in turn to the development of potential vaccines to prevent infection.
9.
10.
11.
12.
This work was supported by Public Health Service grants DE11064 from the National Institute of Dental Research to M.S.R., AI19641 from the National Institute of Allergy and Infectious Diseases to T.F.M., and 19679 from the National Institute of Child Health and Human Development to H.S.F. and a grant from the Cystic Fibrosis Foundation to M.S.R. We thank M. J. Levine for his valuable suggestions during the course of this study. We also thank Eric J. Hansen for providing Hib strains DL42 and DL42/2F42, L. O. Bakaletz for providing NTHi strain 1128 and its isogenic mutant lacking OMP P5, Kyungchoel Yi and Alan Lesse for providing monoclonal antibody 2C7, and Jeffrey Surface and Charmaine Kirkham for technical assistance. REFERENCES 1. Davies, J., I. Carlstedt, A.-K. Nilsson, A. Hakansson, H. Sabharwal, L. V. Alphen, M. A. Ham, and C. Svanborg. 1995. Binding of Haemophilus influenzae to purified mucins from the human respiratory tract. Infect. Immun. 63:2485–2492. 2. Gilsdorf, J. R., H. Y. Chang, K. W. McCrea, and L. O. Bakaletz. 1992. Comparison of hemagglutinating pili of Haemophilus influenzae type b with similar structures of nontypeable H. influenzae. Infect. Immun. 60:374–379. 3. Greenwood, F. C., W. M. Hunter, and J. S. Glover. 1963. The preparation of 131 I-labeled human growth hormone of high specific radioactivity. Biochem. J. 89:114–123. 4. Guerina, N. G., S. Langermann, H. W. Clegg, T. W. Kessler, D. A. Goldmann, and J. R. Gilsdorf. 1982. Adherence of piliated Haemophilus influenzae type b to human oropharyngeal cells. J. Infect. Dis. 146:564. 5. Haase, E. M., A. A. Campagnari, J. Sarwar, M. Shero, M. Wirth, C. U. Cumming, and T. F. Murphy. 1991. Strain-specific and immunodominant surface epitopes of the P2 porin protein of nontypeable Haemophilus influenzae. Infect. Immun. 59:1278–1284. 6. Krivan, H. C., D. D. Roberts, and V. Ginsburg. 1988. Many pulmonary pathogenic bacteria bind specifically to the carbohydrate sequence GalNAcb1, 4Gal found in some glycolipids. Proc. Natl. Acad. Sci. USA 85:6157–6161. 7. Kubiet, M., and R. Ramphal. 1995. Adhesion of nontypeable Haemophilus influenzae from blood and sputum to human tracheobronchial mucins and lactoferrin. Infect. Immun. 63:899–902. 8. Lampe, R. M., E. O. Mason, Jr., S. L. Kaplan, C. L. Umstead, M. D. Yow,
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