- Email: [email protected]
Bacterial adherence on replicas of sodium dodecyl sulfate-polyacrylamide gels
ANALYTICAL
BIOCHEMISTRY
164, 5-11 (1987)
Bacterial Adherence on Replicas of Sodium Dodecyl Sulfate-Polyacrylamide Gels’ ARAPRON Divisions
PRAKOBPHOL,* PATRICIA A. MURRAY,**? AND SUSAN J.FIsHER*$$~
of *Oral Biology and fPeriodontology, School of Dentistry; *Department School of Pharmacy; and 4Department of Anatomy, School of Medicine. San Francisco, California 94143
of Pharmaceutical Chemistry, University of California.
Received August 25, 1986 A method for determining which molecules in a complex mixture of proteins can function as bacterial receptors wasdevised.Salivary proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to nitrocellulose. Bacteria that were metabolically labeled with ‘H or externally labeled with ‘251 were incubated on the nitrocellulose replicas. After 18 h at 4”C, the unbound cells were removed by repeated washing of the replicas, and the bands to which the radiolabeled bacteria bound were visualized by autoradiography. By this technique, Fusobacterium nucleatum, which adheres via carbohydrate residues on receptor molecules, and Staphylococcus aureus, which recognizes the peptide portion of libronectin, were shown to bind specifically to their respective receptors. These results suggest that this method can be useful for profiling bacterial binding to either the carbohydrate or the protein portions of molecules present in complex mixtures, such as those composing biological fluids or tissue substrates. Structural specificities, such as recognition sequences formed by certain oligosaccharides, could be further investigated by adding the appropriate simple sugars, as well as oligosaccharide inhibitors, to the incubation medium. The latter approach is particularly important since most glycoproteins carry multiple N-and O-linked carbohydrate substituents that could serve as bacterial receptors. 0 1987 Academic Press, Inc. KEY WORDS: electrophoresis; bacterial adhesion; glycoproteins: carbohydrates; autoradiography.
The prerequisite first step in the development of a bacterial infection is adherence of the organism to some component of the tissue substrate. Alternatively, greater affinity for molecules present in biological fluids probably facilitates bacterial clearance. These important events are the end result of the interplay of several different factors, which probably operate simultaneously. The mechanisms involved in these processes include mechanical forces as well as more de-
fined interactions involving bacterial recognition of specific receptors on soluble molecules present in biological fluids, on eukaryotic cells, or on extracellular matrix components. With regard to these more defined interactions, elucidation of the structural requirements for the adherence of various bacterial strains to tissue substrates has received considerable attention, since understanding the specific mechanisms involved would offer the possibility of clinical intervention. Although only limited information is available about the structural specificity of bacterial ligands, there is growing evidence that bacteria often bind to substrates via the carbohydrate portions of receptor molecules. For example, Escherichiu coli strains asso-
’ This investigation was supported by USPHS Research Grant DE-07244 from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD. *To whom correspondence should be addressed at HSW 604, University of California, San Francisco, CA 94143-0512. 5
0003-2697/87$3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
6
PRAKOBPHOL,
MURRAY,
ciated with urinary tract infections recognize specific sugars and/or sequences, including D-mannose (l-3), Gal (Yl-4 Gal (4-6), NeuAc3 a2-3 Gal (7), and GlcNAc (8). A lectin on Streptococcus sanguis, an oral pathogen, that specifically recognizes NeuAc a2-3 Gal 81-4 GalNAc sequences has been described (9). Interestingly, there are also exceptions to carbohydrate-mediated bacterial binding. These include the binding of Staphylococcus aureus to fibronectin, which is probably mediated by the peptide portion of the glycoprotein ( lo- 12). A variety of techniques have been used to probe the structural requirements of bacterial ligands. These include inhibition of hemagglutination using a variety of simple saccharides (l-3) and production of monoclonal antibodies against receptors (13). More direct methods of investigation have been hampered by the complexity of the bacterial cell surface. For this reason, more detailed studies have usually assayed the specific inhibition of binding of partially purified receptor molecules to intact bacteria. For example, the cholera toxin glycolipid receptor was identified by assaying the ability of partially purified tissue extracts to inhibit bacterial binding ( 14). These results suggest that successful approaches to studying bacterial adherence involve assays in which the complex bacterial ligands remain intact. In general the receptor molecules, which contain relatively short recognition sequences composed of either carbohydrates or amino acids, appear to survive separation techniques that would alter the conformation of larger molecules. This strategy has led to the interesting observation that fibroblasts could attach to plasma proteins separated on SDS-polyacrylamide gels 3 Abbreviations used: NeuAc, acetylneuraminic acid; GlcNAc, N-acetylglucosamine; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; Bolton-Hunter reagent, N-succinimidyl-hydroxy[‘z51]iodophenyl proprionate; PBS, phosphate-buffered saline; PPO, 2,5-diphenyloxazole; TBS, Tris-buffered saline.
AND FISHER
and transferred to nitrocellulose filters (15). This process is probably mediated by the tetrapeptide sequence that gives the adhesive properties to fibronectin as well as other plasma and extracellular matrix molecules (16). A similar approach has also been used to investigate the bacterial binding characteristics of glycolipids separated by thin-layer chromatography (6,17). The results of the following study demonstrate that certain elements of the two techniques can be combined to identify molecules that act as bacterial receptors; both metabolically and externally labeled bacteria were shown to bind specifically to replicas of electrophoretically separated salivary glycoproteins. MATERIALS
AND METHODS
Materials. Lactose, galactosamine-HCl, maltose, cellobiose, glucose oxidase, lactoperoxidase, bovine serum albumin Fraction V, PPO, and molecular weight standards for SDS-PAGE were purchased from Sigma Chemical Co. Nitrocellulose membrane (0.45 pm) was obtained from Bio-Rad. Trypticase soy broth was purchased from BBL Microbiology Systems and BectonDickinson Co. and yeast extract from Difco Laboratories. [2,8-3H]Adenine (17.7 Ci/ mmol) was obtained from New England Nuclear. Nalz51 (15.7 Ci/pg iodine) was purchased from Amersham. N-Succinimidyl-hydroxy[ ‘*‘I]iodophenyl proprionate (Bolton-Hunter reagent, 2723 Ci/mmol) was purchased from ICN Biomedical, Inc. X-Omat AR X-ray film was a product of Eastman Kodak Co. SDS-polyacrylamide gel electrophoresis and transfer of proteins. Normal human parotid and submandibular/sublingual salivas were collected separately, as the ductal secretions, into an equal volume of SDS-PAGE loading buffer consisting of 0.1 M phosphate buffer, pH 7.0, containing 6 M urea, 1% SDS, 1% P-mercaptoethanol, and 0.0 15% bromphenol blue. Human plasma fibronectin ( 18) and human cellular (placental) fibronectin (19) were isolated as previously described.
BACTERIAL
ADHERENCE
Proteins were separated on 7.5% slab gels (20). Molecular weight standards were as follows: M, 29,000, carbonic anhydrase; 45,000, ovalbumin; 66,000, bovine plasma albumin; 97,400, phosphorylase b; 116,000, P-galactosidase; 205,000, myosin. In some cases proteins separated on gels were visualized by staining with Coomassie brilliant blue R-250 or silver (21) and glycoproteins by staining with periodic acid-Schiff (22). Proteins were transferred to nitrocellulose membranes according to the procedure of Towbin et al. (23) and were visualized by staining with India ink (24) or amido black (25). After blotting, gels were routinely stained as described above in order to estimate the efficiency of transfer. Preparation of labeled bacteria. E. coli (strain 25922) and S. aurexs (strain 29523) were purchased from the American Type Culture Collection. Fusobacterium nucleaturn (FN2) was the kind gift of Drs. W. Loesche and S. Syed, University of Michigan (Ann Arbor, MI). To prepare metabolically labeled bacteria, the cells were grown to late log phase in trypticase soy broth containing 0.25% yeast extract, 30 pM menadione, 4 pM hemin, 0.65 mM dithiothreitol, and 5 &i/ml [3H]adenine. Radiolabel incorporation was as follows: F. nucleatum, 300 bacteria/cpm; S. aureus, 3800 bacteria/cpm; E. coli, 600 bacteriafcpm. F. nucleatum was also labeled by lactoperoxidase-catalyzed ‘25I iodination (26). Briefly, 2 X 10’ cells were suspended in 1 ml PBS containing 5 pM glucose and 3.6 mU glucose oxidase. Two hundred microcuries of Na’251 and 3.6 mU lactoperoxidase were added to the reaction mixture and the labeling was allowed to proceed for 30 min at room temperature. The cells were washed at least three times in PBS before use in the binding experiments. F. nucleatum incorporated 1 X lo5 cpm/ 10’ cells, approximately 100 bacteria/cpm. S. aureus and E. coli were labeled with 125I by the method of Bolton and Hunter (27). Briefly, 225 PCi of the ‘25I reagent was dried in a conical vial
TO GEL
REPLICAS
7
under a stream of nitrogen. Then, 0.5 ml containing 10” cells suspended in PBS was added to the vial, and the reaction was allowed to proceed for 30 min at 4°C. After the labeling procedure, the cells were washed four times in PBS. Incorporation of radioactivity was as follows: S. aureus, 1000 bacteriafcpm; E. coli, 100 bacteria/cpm.
Attachment of labeled bacteria to nitrocellulose blots. To prevent nonspecific attachment of the cells to the nitrocellulose blots, the replicas were soaked for 1 h at 40°C ( 15) in TBS (50 mM Tris-HCl, 200 mM NaCl, pH 7.4) containing 5% bovine serum albumin, Cells ( 1 X log/ml) were suspended in TBS containing 5% bovine serum albumin and allowed to attach to the replicas for 18 h at 4°C. The blots to which labeled bacteria bound were washed with PBS at least four times, 1 min each, to remove cells that were nonspecifically bound to the membrane. For visualization of bands to which 3H-labeled bacteria were attached, the nitrocellulose membranes were air-dried, dipped in 7% PPO in diethyl ether, removed immediately, and dried again prior to autoradiography. For visualization of bands to which ‘251-labeled bacteria were bound, the blots were air-dried and exposed to X-ray film. RESULTS
Figure 1 illustrates the electrophoretic separation of submandibular/sublingual (lanes 1 and 3) salivas and parotid salivas (lanes 2 and 4), obtained from two subjects. Both types of saliva were collected as the ductal secretions into an equal volume of SDSPAGE loading buffer. When the gel was stained with silver (replica A) many more components were visible than were revealed by staining with Coomassie brilliant blue R-250 (replica B). These included the heavily glycosylated submandibular/sublingual mucins (Mr 150,000 and 500,000) and the proline-rich glycoprotein that is a major component of parotid saliva. Interestingly, glycosylation of this component appears to
PRAKOBPHOL, A 12341234
-
116-.
':
97.4 0
;(
MURRAY,
B
-
66-
2
FIG. 1. SDS-polyacrylamide gel electrophoresis of submandibular/sublingual (lanes I and 3) and parotid salivas (lanes 2 and 4). Silver staining (replica A) revealed many more components than did staining with Coomassie brilliant blue R-250 (replica B). These included the heavily glycosylated submandibular/sublingual mucins (Mr 150,000 and 500,000) and the prolinerich glycoprotein, a major component of parotid saliva. Glycosylation of this component varies significantly among individuals. The maximal differences observed, ranging from M, 60,000-100,000, are shown in lanes 2A and 4A. Arrow indicates origin.
AND FISHER
nents could be detected using amido black (data not shown). The only detectable binding of metabolically 3H-labeled F. nucleatum was to the proline-rich glycoprotein (replica B, lanes 2 and 4) M, 60,000 or 100,000, depending on the individual from whom the saliva was collected. No binding of F. nucleatum to other parotid components or to any submandibular/sublingual glycoproteins was observed. Binding of this bacterium, which recognizes terminal galactose residues (P. Murray et al., manuscript in preparation), could be totally inhibited by the addition of 5 nM lactose to the incubation buffer (replica C). Addition of 5 or 100 nrvt glucose, maltose, cellobiose, or galactosamine did not inhibit bacterial binding, indicating the specificity of the adherence. Binding of lactoperoxidase-catalyzed lz51labeled F. nucleatum to SDS gel replicas (Fig.
A 12
change during prolonged periods of parotid secretion (28). We also observed substantial differences, independent of the time of secretion, in the estimated molecular weight of this component isolated from six different individuals whose saliva was used for this study. The maximal differences, ranging from M, 60,000- 100,000, are shown in lanes 2A and 4A. Binding of metabolically ‘H-labeled F. nucleatum to salivary glycoproteins transferred to nitrocellulose is illustrated in Fig. 2. In all cases (replicas A-C) lanes 1 and 3 contained submandibular/sublingual saliva and lanes 2 and 4 contained parotid saliva, collected as previously described. Staining of replica A with India ink showed a pattern similar to that obtained when SDS gels of submandibular/sublingual and parotid salivas were stained with silver (Fig. lA), except that highly glycosylated components appeared as negatively staining bands. These compo-
B 3
4
1234
c
12
3
4
205-
: 0 ; 5
11697.466-
4529-
FIG. 2. Binding of metabolically labeled F. nucleatum to nitrocellulose replicas of electrophoretically separated salivary glycoproteins. In all cases (replicas A-C) lanes 1 and 3 contained submandibular/sublingual saliva and lanes 2 and 4 contained parotid saliva. Replica A was stained with India ink and showed a pattern similar to that obtained when SDS gels of submandibular/sublingual and parotid salivas were stained with silver (Fig. 1A). ‘H-labeled F. nucleatum bound to the proline-rich glycoprotein (replica B, lanes 2 and 4) IV, 60,000 or 100,000. Binding of this bacterium, which recognizes terminal galactose residues, could be totally inhibited by the addition of 5 nM lactose to the incubation buffer (replica C). Arrow indicates origin.
BACTERIAL
ADHERENCE
3) revealed a similar pattern of attachment; the most heavily labeled band was the proline-rich glycoprotein (Mr 100,000; replica B, lane 2). In addition, specific attachment to two lower molecular weight parotid glycoproteins (Mr 38,000 and 45,000) was also consistently observed. No additional submandibular/sublingual components (replica B, lane 1) capable of binding the bacterium were revealed. These results suggest that the sensitivity of the technique can be improved if the bacteria used in the binding assay are labeled with 12?, since this method results in a substantial increase in the specific activity of radiolabel incorporation. However, the level of nonspecific binding may vary greatly depending on the method of ‘25I iodination. For example, “‘I-labeling of F. nucleatum using either chloramine T or the BoltonHunter reagent abolished the ability of the bacterium to bind specifically to galactose residues (data not shown), suggesting substantial structural alteration of the bacterial A
B
TO GEL REPLICAS
205': 0
7
116-
-2
97.4-
5
66-
FIG. 4. Binding of “51-labeled S. aureus and 1251-labeled E. coli to human tissue and plasma fibronectin. In all cases lane I contained tissue (placental) fibronectin and lane 2 contained plasma fibronectin. Substantial binding of S. aureus (replica A) to tissue fibronectin (lane 1) was observed, whereas no binding to plasma fibronectin (lane 2) could be detected. In addition, no binding of ‘251-labeled E. coli (replica B) to either tissue or plasma fibronectin was observed. Arrow indicates origin.
ligand that recognizes galactose-containing receptor molecules. In contrast, S. aureus labeled with ‘25I by 205the method of Bolton and Hunter (27) retained the ability to bind specifically to fibro116': 97.4nectin (Fig. 4). For this experiment binding 0 66to both human tissue and plasma fibro25 nectin was assessed. As we previously obF served ( 19) the two fibronectins had differ45ent electrophoretic characteristics. On 7.5% 29 SDS-polyacrylamide gels, human placental fibronectin migrated as a somewhat diffuse band of subunit M, 240,000 and human plasma fibronectin as a distinct doublet of M, FIG. 3. Binding of lactoperoxidase-catalyzed ‘251-la220,000. Following electrophoresis, both beled F. nucleutum to replicas of electrophoretically sepglycoproteins were transferred to nitrocelluarated salivary glycoproteins. In both cases (replicas A and B) lane 1 contained submandibular/sublingual sa- lose. The efficiency of transfer was estimated liva and lane 2 contained parotid saliva. Replica A was to be the same, since 10 pg of each was elecstained using 0.1% India ink in PBS containing 5% trophoretically separated and silver staining Tween. The most heavily labeled band was the prolineof the gel after transfer revealed no remainrich glycoprotein (M, 100,000; replica B, lane 2). In ading fibronectin. dition, specific attachment to two lower molecular Using a soluble assay, we have shown that weight parotid glycoproteins (Mr 38,000 and 45,000) was also consistently observed. Arrow indicates origin. the binding of tissue fibronectin to S. aureus 12
12
10
PRAKOBPHOL,
MURRAY,
AND FISHER
is several times greater than the binding of binding characteristics, previously studied plasma fibronectin (S. Fisher et al., manuusing soluble assays, were retained when the script submitted for publication). Interestattachment of bacteria to molecules that had ingly, we observed a similar difference in this been electrophoretically separated and transstudy when we investigated the ability of S. ferred to nitrocellulose was assayed. In addiaweus to bind to tissue and plasma fibro- tion to duplicating the results of soluble nectins transferred to nitrocellulose (Fig. 4, assays, this technique circumvents technical replica A); substantial binding to tissue fi- difficulties inherent in many such assays. For bronectin (lane 1) was observed, whereas no example, we found that the proline-rich glybinding to plasma fibronectin (lane 2) could coprotein, a major component of parotid sabe detected. Since binding of ‘251-labeled liva, is difficult to label without modifying plasma fibronectin to S. aureus was pre- the carbohydrate portion of the molecule viously detected using a soluble assay, these using methods such as treatment with galacresults suggest that in this particular case the tose oxidase followed by reduction with sensitivity of this assay is less than can be NaB3H4. Accordingly, the problems assoachieved by other techniques. Consistent ciated with using soluble assays to evaluate with the results of other laboratories (29), as the bacterial binding characteristics of this well as with those observed in our soluble glycoprotein were compounded. In contrast, assay, no binding of ‘251-labeled E. coli (rep- bacterial attachment mediated by the carbolica B) was observed to either tissue (lane 1) hydrate portion of the proline-rich glycoproor plasma (lane 2) fibronectin. Identical retein was readily assayed using replicas of SDS sults were obtained when metabolically lagels. In addition, this approach also allowed beled cells were used for the binding experithe rapid comparison of the relative roles of ments (data not shown). various highly glycosylated salivary components, such as the submandibular/sublingual DISCUSSION mucins and the proline-rich glycoprotein, in the binding of F. nucleatum. The results of this study suggest a method Technical difficulties most often encounby which the complex interactions occurring between bacteria and their substrate attach- tered arose from the nonspecific adherence ment sites can be studied. Adherence of bac- of bacteria to nitrocellulose blots after exterteria to replicas of electrophoretically sepa- nal solid-phase labeling with ‘251. Presumof bacterial ligands rerated molecules allows a rapid assessment of ably, modification sulted in altered substrate specificity. As a the relative contribution of candidate attachresult, metabolic labeling produced the most ment molecules to the adherence or clearconsistent results. However, we were able to ance processes. This capability is particularly label all the bacteria used in this study by one important since most sites of bacterial atof several 12SI-Iabeling techniques, although tachment in vivo are composed of complex mixtures of such molecules; detailed analyses different techniques had to be used for different bacteria. Variations in the uptake of that utilize either purified bacterial ligands label by various bacteria in vitro, combined or substrate components are not usually with the inability to introduce radioactive feasible. The utility of this method was shown for label using external ‘251-labeling techniques, could present significant problems. In such two different bacteria: F. nucleatum, which recognizes the carbohydrate portion of sub- cases detection of bound bacteria using antisera specific for bacterial surface compostrate molecules such as salivary glycoproteins, and S. aureus, which recognizes the nents, followed by addition of a labeled secpeptide portion of fibronectin. In both cases ond antibody, could be employed.
BACTERIAL
ADHERENCE
The method described in this paper could form an important portion of a rational experimental design for the rapid characterization of bacterial binding to complex biological substrates. For example, saliva could be delipidated and the bacterial binding characteristics of the individual glycolipid components, separated by thin-layer chromatography, determined as previously described by Hansson et al. (17). Likewise, bacterial attachment to the remaining salivary proteins and glycoproteins, separated by SDS-PAGE and transferred to nitrocellulose, could also be determined. In this manner structural specificities, such as recognition sequences formed by certain oligosaccharide sequences, could be further investigated by adding the appropriate simple sugars, as well as oligosaccharide inhibitors, to the incubation medium. The latter approach is particularly important since most glycoproteins carry multiple N- and O-linked carbohydrate substituents that could serve as bacterial receptors. ACKNOWLEDGMENT We thank Mr. Charles Hoover for assistance with the bacterial cultures.
REFERENCES 1. Beachey. E. H. (1980) Bacterial Adherence. Receptors and Recognition, Series B, Vol. 8, Chapman & Hall, New York. 2. Jones, G. W., and Isaacson, E. (1983) CRC Crit. Rev. Microbial. 34, 229-260. 3. Firon, N., Ofek, I., and Sharon, N. (1984) Infect, Zmmun. 43, 1088-1090. 4. Kallenius, G., Molby, R., Svensson, S. B., Winberg, J., Lunblad, A., Svensson, S., and Cedergren, B. (1980) FEMS Microbial. Left. 7, 297-302. 5. O’Hanley, P., Low, D., Romero, I., Lark, D., Vosti, K., Falkow, S., and Schoolnik, G. (1985) N. Engl. J. Med. 313,414-420. 6. Bock, K., Breimer, M. E., Brignole, A., Hanson, G. C., Karlsson, K.-A., Larson, G., Leffler, H., Samuelsson, B., Stromberg, N., Svanborg Eden, C., and Thurin, J. (1985) J. Biol. Chem. 260, 8545-8551.
TO GEL REPLICAS
11
7. Harkkinen, J., Finne, J., Achtman, M., Vaisanen, V., and Korhonen, T. K. (1983) Biochem. Biophys. Res. Commun. 111,456-461. 8. Vaisanen-Rhen, V., Korhonen, T. K., and Finne, J. (1983) FEES Left. 159,233-236. 9. Murray, P. A., Levine, M. J., Tabak, L. A., and Reddy, M. S. (1982) Biochem. Biophys. Rex Commun. 106,390-396. 10. Kuusela, P.. Vartio, T., Vuento, M., and Myhre, E. B. (1984) Infect. Zmmun. 45,433-436. 11. Kuusela, P., Vartio, T., Vuento, M., and Myhre, E. B. (1985) Znfect. Zmmun. 50, 77-8 1. 12. Mosher, D. F., and Proctor, R. A. (1981) Science 209,927-929. 13. Greaves, M. (1984) Monoclonal Antibodies to Receptors. Receptors and Recognition, Series B, Vol. 17, Chapman & Hall, London. 14. Eidels, L., Proia, R. L., and Hart, D. A. (1983) Microbiol. Rev. 41, 596-620. 15. Hayman, E. G., Engvall, E., A’Heam, E., Barnes, D., Pierschbacher, M., and Ruoslahti, E. (1982) J. Cell Biol. 95, 20-23. 16. Pierschbacher, M., and Ruoslahti, E. (1984) Nature (London) 309,30-33. 17. Hansson, G., Karlsson, K.-A., Larson, G., Stromberg, N., and Thurin, J. (1985) Anal. Biochem. 146, 158-163.
18. Engvall, E., and Ruoslahti, E. (1977) Znt. J. Cancer 20, 1-5. 19. Zhu, B. C.-R., Fisher, S. J., Pande, H., Calaycay, J., Shively, J. E., and Laine. R. A. (1984) J. Biol. Chem. 259,3962-3970. 20. Laemmli, U. K. (1970) Nature (London) 277, 680-685. 21. Morrissey, J. H. (1981) Anal. Biochem. 117, 307-3 10. 22. Wada, H. G., Haas, P., and Sussman, H. (1977) J. Biol. Chem. 254, 12629-12635. 23. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA X,4350-4354. 24. Hancock, K., and Tsang, V. (1983) Anal. Biochem. 133, 157-162.
25. Schaffer, W., and Weissmann, G. (1973) Anal. Biothem. 56,502-5 14. 26. Hubbard, A. L., and Cohn, Z. A. (1972) J. Cell Biol. 55,390-406. 27. Bolton, A. E., and Hunter, W. M. (1973) Biochem. J. 133,529-539. 28. Levine, M. J.. Ellison, S. A., and Bahl, 0. P. (1973) Arch. Oral Biol. 18, 827-837. 29. Proctor, R. A., Prendergast, E.. and Mosher, D. F. (1982) Blood 59,68 l-687.
BIOCHEMISTRY
164, 5-11 (1987)
Bacterial Adherence on Replicas of Sodium Dodecyl Sulfate-Polyacrylamide Gels’ ARAPRON Divisions
PRAKOBPHOL,* PATRICIA A. MURRAY,**? AND SUSAN J.FIsHER*$$~
of *Oral Biology and fPeriodontology, School of Dentistry; *Department School of Pharmacy; and 4Department of Anatomy, School of Medicine. San Francisco, California 94143
of Pharmaceutical Chemistry, University of California.
Received August 25, 1986 A method for determining which molecules in a complex mixture of proteins can function as bacterial receptors wasdevised.Salivary proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to nitrocellulose. Bacteria that were metabolically labeled with ‘H or externally labeled with ‘251 were incubated on the nitrocellulose replicas. After 18 h at 4”C, the unbound cells were removed by repeated washing of the replicas, and the bands to which the radiolabeled bacteria bound were visualized by autoradiography. By this technique, Fusobacterium nucleatum, which adheres via carbohydrate residues on receptor molecules, and Staphylococcus aureus, which recognizes the peptide portion of libronectin, were shown to bind specifically to their respective receptors. These results suggest that this method can be useful for profiling bacterial binding to either the carbohydrate or the protein portions of molecules present in complex mixtures, such as those composing biological fluids or tissue substrates. Structural specificities, such as recognition sequences formed by certain oligosaccharides, could be further investigated by adding the appropriate simple sugars, as well as oligosaccharide inhibitors, to the incubation medium. The latter approach is particularly important since most glycoproteins carry multiple N-and O-linked carbohydrate substituents that could serve as bacterial receptors. 0 1987 Academic Press, Inc. KEY WORDS: electrophoresis; bacterial adhesion; glycoproteins: carbohydrates; autoradiography.
The prerequisite first step in the development of a bacterial infection is adherence of the organism to some component of the tissue substrate. Alternatively, greater affinity for molecules present in biological fluids probably facilitates bacterial clearance. These important events are the end result of the interplay of several different factors, which probably operate simultaneously. The mechanisms involved in these processes include mechanical forces as well as more de-
fined interactions involving bacterial recognition of specific receptors on soluble molecules present in biological fluids, on eukaryotic cells, or on extracellular matrix components. With regard to these more defined interactions, elucidation of the structural requirements for the adherence of various bacterial strains to tissue substrates has received considerable attention, since understanding the specific mechanisms involved would offer the possibility of clinical intervention. Although only limited information is available about the structural specificity of bacterial ligands, there is growing evidence that bacteria often bind to substrates via the carbohydrate portions of receptor molecules. For example, Escherichiu coli strains asso-
’ This investigation was supported by USPHS Research Grant DE-07244 from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD. *To whom correspondence should be addressed at HSW 604, University of California, San Francisco, CA 94143-0512. 5
0003-2697/87$3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
6
PRAKOBPHOL,
MURRAY,
ciated with urinary tract infections recognize specific sugars and/or sequences, including D-mannose (l-3), Gal (Yl-4 Gal (4-6), NeuAc3 a2-3 Gal (7), and GlcNAc (8). A lectin on Streptococcus sanguis, an oral pathogen, that specifically recognizes NeuAc a2-3 Gal 81-4 GalNAc sequences has been described (9). Interestingly, there are also exceptions to carbohydrate-mediated bacterial binding. These include the binding of Staphylococcus aureus to fibronectin, which is probably mediated by the peptide portion of the glycoprotein ( lo- 12). A variety of techniques have been used to probe the structural requirements of bacterial ligands. These include inhibition of hemagglutination using a variety of simple saccharides (l-3) and production of monoclonal antibodies against receptors (13). More direct methods of investigation have been hampered by the complexity of the bacterial cell surface. For this reason, more detailed studies have usually assayed the specific inhibition of binding of partially purified receptor molecules to intact bacteria. For example, the cholera toxin glycolipid receptor was identified by assaying the ability of partially purified tissue extracts to inhibit bacterial binding ( 14). These results suggest that successful approaches to studying bacterial adherence involve assays in which the complex bacterial ligands remain intact. In general the receptor molecules, which contain relatively short recognition sequences composed of either carbohydrates or amino acids, appear to survive separation techniques that would alter the conformation of larger molecules. This strategy has led to the interesting observation that fibroblasts could attach to plasma proteins separated on SDS-polyacrylamide gels 3 Abbreviations used: NeuAc, acetylneuraminic acid; GlcNAc, N-acetylglucosamine; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; Bolton-Hunter reagent, N-succinimidyl-hydroxy[‘z51]iodophenyl proprionate; PBS, phosphate-buffered saline; PPO, 2,5-diphenyloxazole; TBS, Tris-buffered saline.
AND FISHER
and transferred to nitrocellulose filters (15). This process is probably mediated by the tetrapeptide sequence that gives the adhesive properties to fibronectin as well as other plasma and extracellular matrix molecules (16). A similar approach has also been used to investigate the bacterial binding characteristics of glycolipids separated by thin-layer chromatography (6,17). The results of the following study demonstrate that certain elements of the two techniques can be combined to identify molecules that act as bacterial receptors; both metabolically and externally labeled bacteria were shown to bind specifically to replicas of electrophoretically separated salivary glycoproteins. MATERIALS
AND METHODS
Materials. Lactose, galactosamine-HCl, maltose, cellobiose, glucose oxidase, lactoperoxidase, bovine serum albumin Fraction V, PPO, and molecular weight standards for SDS-PAGE were purchased from Sigma Chemical Co. Nitrocellulose membrane (0.45 pm) was obtained from Bio-Rad. Trypticase soy broth was purchased from BBL Microbiology Systems and BectonDickinson Co. and yeast extract from Difco Laboratories. [2,8-3H]Adenine (17.7 Ci/ mmol) was obtained from New England Nuclear. Nalz51 (15.7 Ci/pg iodine) was purchased from Amersham. N-Succinimidyl-hydroxy[ ‘*‘I]iodophenyl proprionate (Bolton-Hunter reagent, 2723 Ci/mmol) was purchased from ICN Biomedical, Inc. X-Omat AR X-ray film was a product of Eastman Kodak Co. SDS-polyacrylamide gel electrophoresis and transfer of proteins. Normal human parotid and submandibular/sublingual salivas were collected separately, as the ductal secretions, into an equal volume of SDS-PAGE loading buffer consisting of 0.1 M phosphate buffer, pH 7.0, containing 6 M urea, 1% SDS, 1% P-mercaptoethanol, and 0.0 15% bromphenol blue. Human plasma fibronectin ( 18) and human cellular (placental) fibronectin (19) were isolated as previously described.
BACTERIAL
ADHERENCE
Proteins were separated on 7.5% slab gels (20). Molecular weight standards were as follows: M, 29,000, carbonic anhydrase; 45,000, ovalbumin; 66,000, bovine plasma albumin; 97,400, phosphorylase b; 116,000, P-galactosidase; 205,000, myosin. In some cases proteins separated on gels were visualized by staining with Coomassie brilliant blue R-250 or silver (21) and glycoproteins by staining with periodic acid-Schiff (22). Proteins were transferred to nitrocellulose membranes according to the procedure of Towbin et al. (23) and were visualized by staining with India ink (24) or amido black (25). After blotting, gels were routinely stained as described above in order to estimate the efficiency of transfer. Preparation of labeled bacteria. E. coli (strain 25922) and S. aurexs (strain 29523) were purchased from the American Type Culture Collection. Fusobacterium nucleaturn (FN2) was the kind gift of Drs. W. Loesche and S. Syed, University of Michigan (Ann Arbor, MI). To prepare metabolically labeled bacteria, the cells were grown to late log phase in trypticase soy broth containing 0.25% yeast extract, 30 pM menadione, 4 pM hemin, 0.65 mM dithiothreitol, and 5 &i/ml [3H]adenine. Radiolabel incorporation was as follows: F. nucleatum, 300 bacteria/cpm; S. aureus, 3800 bacteria/cpm; E. coli, 600 bacteriafcpm. F. nucleatum was also labeled by lactoperoxidase-catalyzed ‘25I iodination (26). Briefly, 2 X 10’ cells were suspended in 1 ml PBS containing 5 pM glucose and 3.6 mU glucose oxidase. Two hundred microcuries of Na’251 and 3.6 mU lactoperoxidase were added to the reaction mixture and the labeling was allowed to proceed for 30 min at room temperature. The cells were washed at least three times in PBS before use in the binding experiments. F. nucleatum incorporated 1 X lo5 cpm/ 10’ cells, approximately 100 bacteria/cpm. S. aureus and E. coli were labeled with 125I by the method of Bolton and Hunter (27). Briefly, 225 PCi of the ‘25I reagent was dried in a conical vial
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under a stream of nitrogen. Then, 0.5 ml containing 10” cells suspended in PBS was added to the vial, and the reaction was allowed to proceed for 30 min at 4°C. After the labeling procedure, the cells were washed four times in PBS. Incorporation of radioactivity was as follows: S. aureus, 1000 bacteriafcpm; E. coli, 100 bacteria/cpm.
Attachment of labeled bacteria to nitrocellulose blots. To prevent nonspecific attachment of the cells to the nitrocellulose blots, the replicas were soaked for 1 h at 40°C ( 15) in TBS (50 mM Tris-HCl, 200 mM NaCl, pH 7.4) containing 5% bovine serum albumin, Cells ( 1 X log/ml) were suspended in TBS containing 5% bovine serum albumin and allowed to attach to the replicas for 18 h at 4°C. The blots to which labeled bacteria bound were washed with PBS at least four times, 1 min each, to remove cells that were nonspecifically bound to the membrane. For visualization of bands to which 3H-labeled bacteria were attached, the nitrocellulose membranes were air-dried, dipped in 7% PPO in diethyl ether, removed immediately, and dried again prior to autoradiography. For visualization of bands to which ‘251-labeled bacteria were bound, the blots were air-dried and exposed to X-ray film. RESULTS
Figure 1 illustrates the electrophoretic separation of submandibular/sublingual (lanes 1 and 3) salivas and parotid salivas (lanes 2 and 4), obtained from two subjects. Both types of saliva were collected as the ductal secretions into an equal volume of SDSPAGE loading buffer. When the gel was stained with silver (replica A) many more components were visible than were revealed by staining with Coomassie brilliant blue R-250 (replica B). These included the heavily glycosylated submandibular/sublingual mucins (Mr 150,000 and 500,000) and the proline-rich glycoprotein that is a major component of parotid saliva. Interestingly, glycosylation of this component appears to
PRAKOBPHOL, A 12341234
-
116-.
':
97.4 0
;(
MURRAY,
B
-
66-
2
FIG. 1. SDS-polyacrylamide gel electrophoresis of submandibular/sublingual (lanes I and 3) and parotid salivas (lanes 2 and 4). Silver staining (replica A) revealed many more components than did staining with Coomassie brilliant blue R-250 (replica B). These included the heavily glycosylated submandibular/sublingual mucins (Mr 150,000 and 500,000) and the prolinerich glycoprotein, a major component of parotid saliva. Glycosylation of this component varies significantly among individuals. The maximal differences observed, ranging from M, 60,000-100,000, are shown in lanes 2A and 4A. Arrow indicates origin.
AND FISHER
nents could be detected using amido black (data not shown). The only detectable binding of metabolically 3H-labeled F. nucleatum was to the proline-rich glycoprotein (replica B, lanes 2 and 4) M, 60,000 or 100,000, depending on the individual from whom the saliva was collected. No binding of F. nucleatum to other parotid components or to any submandibular/sublingual glycoproteins was observed. Binding of this bacterium, which recognizes terminal galactose residues (P. Murray et al., manuscript in preparation), could be totally inhibited by the addition of 5 nM lactose to the incubation buffer (replica C). Addition of 5 or 100 nrvt glucose, maltose, cellobiose, or galactosamine did not inhibit bacterial binding, indicating the specificity of the adherence. Binding of lactoperoxidase-catalyzed lz51labeled F. nucleatum to SDS gel replicas (Fig.
A 12
change during prolonged periods of parotid secretion (28). We also observed substantial differences, independent of the time of secretion, in the estimated molecular weight of this component isolated from six different individuals whose saliva was used for this study. The maximal differences, ranging from M, 60,000- 100,000, are shown in lanes 2A and 4A. Binding of metabolically ‘H-labeled F. nucleatum to salivary glycoproteins transferred to nitrocellulose is illustrated in Fig. 2. In all cases (replicas A-C) lanes 1 and 3 contained submandibular/sublingual saliva and lanes 2 and 4 contained parotid saliva, collected as previously described. Staining of replica A with India ink showed a pattern similar to that obtained when SDS gels of submandibular/sublingual and parotid salivas were stained with silver (Fig. lA), except that highly glycosylated components appeared as negatively staining bands. These compo-
B 3
4
1234
c
12
3
4
205-
: 0 ; 5
11697.466-
4529-
FIG. 2. Binding of metabolically labeled F. nucleatum to nitrocellulose replicas of electrophoretically separated salivary glycoproteins. In all cases (replicas A-C) lanes 1 and 3 contained submandibular/sublingual saliva and lanes 2 and 4 contained parotid saliva. Replica A was stained with India ink and showed a pattern similar to that obtained when SDS gels of submandibular/sublingual and parotid salivas were stained with silver (Fig. 1A). ‘H-labeled F. nucleatum bound to the proline-rich glycoprotein (replica B, lanes 2 and 4) IV, 60,000 or 100,000. Binding of this bacterium, which recognizes terminal galactose residues, could be totally inhibited by the addition of 5 nM lactose to the incubation buffer (replica C). Arrow indicates origin.
BACTERIAL
ADHERENCE
3) revealed a similar pattern of attachment; the most heavily labeled band was the proline-rich glycoprotein (Mr 100,000; replica B, lane 2). In addition, specific attachment to two lower molecular weight parotid glycoproteins (Mr 38,000 and 45,000) was also consistently observed. No additional submandibular/sublingual components (replica B, lane 1) capable of binding the bacterium were revealed. These results suggest that the sensitivity of the technique can be improved if the bacteria used in the binding assay are labeled with 12?, since this method results in a substantial increase in the specific activity of radiolabel incorporation. However, the level of nonspecific binding may vary greatly depending on the method of ‘25I iodination. For example, “‘I-labeling of F. nucleatum using either chloramine T or the BoltonHunter reagent abolished the ability of the bacterium to bind specifically to galactose residues (data not shown), suggesting substantial structural alteration of the bacterial A
B
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7
116-
-2
97.4-
5
66-
FIG. 4. Binding of “51-labeled S. aureus and 1251-labeled E. coli to human tissue and plasma fibronectin. In all cases lane I contained tissue (placental) fibronectin and lane 2 contained plasma fibronectin. Substantial binding of S. aureus (replica A) to tissue fibronectin (lane 1) was observed, whereas no binding to plasma fibronectin (lane 2) could be detected. In addition, no binding of ‘251-labeled E. coli (replica B) to either tissue or plasma fibronectin was observed. Arrow indicates origin.
ligand that recognizes galactose-containing receptor molecules. In contrast, S. aureus labeled with ‘25I by 205the method of Bolton and Hunter (27) retained the ability to bind specifically to fibro116': 97.4nectin (Fig. 4). For this experiment binding 0 66to both human tissue and plasma fibro25 nectin was assessed. As we previously obF served ( 19) the two fibronectins had differ45ent electrophoretic characteristics. On 7.5% 29 SDS-polyacrylamide gels, human placental fibronectin migrated as a somewhat diffuse band of subunit M, 240,000 and human plasma fibronectin as a distinct doublet of M, FIG. 3. Binding of lactoperoxidase-catalyzed ‘251-la220,000. Following electrophoresis, both beled F. nucleutum to replicas of electrophoretically sepglycoproteins were transferred to nitrocelluarated salivary glycoproteins. In both cases (replicas A and B) lane 1 contained submandibular/sublingual sa- lose. The efficiency of transfer was estimated liva and lane 2 contained parotid saliva. Replica A was to be the same, since 10 pg of each was elecstained using 0.1% India ink in PBS containing 5% trophoretically separated and silver staining Tween. The most heavily labeled band was the prolineof the gel after transfer revealed no remainrich glycoprotein (M, 100,000; replica B, lane 2). In ading fibronectin. dition, specific attachment to two lower molecular Using a soluble assay, we have shown that weight parotid glycoproteins (Mr 38,000 and 45,000) was also consistently observed. Arrow indicates origin. the binding of tissue fibronectin to S. aureus 12
12
10
PRAKOBPHOL,
MURRAY,
AND FISHER
is several times greater than the binding of binding characteristics, previously studied plasma fibronectin (S. Fisher et al., manuusing soluble assays, were retained when the script submitted for publication). Interestattachment of bacteria to molecules that had ingly, we observed a similar difference in this been electrophoretically separated and transstudy when we investigated the ability of S. ferred to nitrocellulose was assayed. In addiaweus to bind to tissue and plasma fibro- tion to duplicating the results of soluble nectins transferred to nitrocellulose (Fig. 4, assays, this technique circumvents technical replica A); substantial binding to tissue fi- difficulties inherent in many such assays. For bronectin (lane 1) was observed, whereas no example, we found that the proline-rich glybinding to plasma fibronectin (lane 2) could coprotein, a major component of parotid sabe detected. Since binding of ‘251-labeled liva, is difficult to label without modifying plasma fibronectin to S. aureus was pre- the carbohydrate portion of the molecule viously detected using a soluble assay, these using methods such as treatment with galacresults suggest that in this particular case the tose oxidase followed by reduction with sensitivity of this assay is less than can be NaB3H4. Accordingly, the problems assoachieved by other techniques. Consistent ciated with using soluble assays to evaluate with the results of other laboratories (29), as the bacterial binding characteristics of this well as with those observed in our soluble glycoprotein were compounded. In contrast, assay, no binding of ‘251-labeled E. coli (rep- bacterial attachment mediated by the carbolica B) was observed to either tissue (lane 1) hydrate portion of the proline-rich glycoproor plasma (lane 2) fibronectin. Identical retein was readily assayed using replicas of SDS sults were obtained when metabolically lagels. In addition, this approach also allowed beled cells were used for the binding experithe rapid comparison of the relative roles of ments (data not shown). various highly glycosylated salivary components, such as the submandibular/sublingual DISCUSSION mucins and the proline-rich glycoprotein, in the binding of F. nucleatum. The results of this study suggest a method Technical difficulties most often encounby which the complex interactions occurring between bacteria and their substrate attach- tered arose from the nonspecific adherence ment sites can be studied. Adherence of bac- of bacteria to nitrocellulose blots after exterteria to replicas of electrophoretically sepa- nal solid-phase labeling with ‘251. Presumof bacterial ligands rerated molecules allows a rapid assessment of ably, modification sulted in altered substrate specificity. As a the relative contribution of candidate attachresult, metabolic labeling produced the most ment molecules to the adherence or clearconsistent results. However, we were able to ance processes. This capability is particularly label all the bacteria used in this study by one important since most sites of bacterial atof several 12SI-Iabeling techniques, although tachment in vivo are composed of complex mixtures of such molecules; detailed analyses different techniques had to be used for different bacteria. Variations in the uptake of that utilize either purified bacterial ligands label by various bacteria in vitro, combined or substrate components are not usually with the inability to introduce radioactive feasible. The utility of this method was shown for label using external ‘251-labeling techniques, could present significant problems. In such two different bacteria: F. nucleatum, which recognizes the carbohydrate portion of sub- cases detection of bound bacteria using antisera specific for bacterial surface compostrate molecules such as salivary glycoproteins, and S. aureus, which recognizes the nents, followed by addition of a labeled secpeptide portion of fibronectin. In both cases ond antibody, could be employed.
BACTERIAL
ADHERENCE
The method described in this paper could form an important portion of a rational experimental design for the rapid characterization of bacterial binding to complex biological substrates. For example, saliva could be delipidated and the bacterial binding characteristics of the individual glycolipid components, separated by thin-layer chromatography, determined as previously described by Hansson et al. (17). Likewise, bacterial attachment to the remaining salivary proteins and glycoproteins, separated by SDS-PAGE and transferred to nitrocellulose, could also be determined. In this manner structural specificities, such as recognition sequences formed by certain oligosaccharide sequences, could be further investigated by adding the appropriate simple sugars, as well as oligosaccharide inhibitors, to the incubation medium. The latter approach is particularly important since most glycoproteins carry multiple N- and O-linked carbohydrate substituents that could serve as bacterial receptors. ACKNOWLEDGMENT We thank Mr. Charles Hoover for assistance with the bacterial cultures.
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