Distribution of Cross-reactive Antigens A and B in Streptococcus mutans and Other Oral Streptococci

Transcription

1 Journal of General Miroiology (1980), 118, Printed in Great Britain 383 Distriution of Cross-reative Antigens A and B in Streptoous mutans and Other Oral Streptooi By ROY R. B. RUSSELL Dental Researh Unit, Royal College of Surgeons of Englund, Downe, Orpington, Kent BR6 7JJ (Reeived 6 Deemer 1979) A olletion of strains representing the various ommon speies of oral streptooi was examined for the presene of antigens ross-reating with antigens A and B from Streptoous mutans serotype. Antigens ross-reating with A were found only in S. mutans serotype. Antigens ross-reating with B were found in S. mutans serotypes a,, d, e,fand g and also in strains of S. sanguis. All antigens ross-reating with B were preipitated y antiserum to human heart tissue. INTRODUCTION Reent advanes in the taxonomy of the viridans group of streptooi ommonly found in the oral avity have resulted in general agreement on the oundaries etween the speies Streptoous salivarius, S. mutans, S. milleri, S. sdnguis and S. mitior (also alled S. mitis), though some differenes of opinion remain onerning the relationship etween the last two (Colman, 1976; Faklam, 1977; Hardie & Marsh, 1978). The taxonomi divisions are ased primarily on physiologial harateristis, supported in some ases y studies of ell wall omposition or DNA homology (Colman & Williams, 1972; Coykendall & Munzenmaier, 1978). Studies on the serology of the viridans streptooi, however, have demonstrated extensive ross-reations etween speies, though the ross-reating antigens have not generally een identified (Colman, 1976; Colman & Williams, 1972; Faklam, 1977; Hardie & Bowden, 1976; Hardie & Marsh, 1978). The antigeni struture of S. mutam is of partiular interest eause of the role played y this organism in the initiation of dental aries and the fat that vaines ased on S. mutans an protet experimental animals against this disease (Bowen, 1976). The polysaharide antigens of S. mutans, on whih the serotyping system is ased, have een extensively studied (Linzer et ul., 1976), ut little is yet known aout the protein antigens of the speies (Bratthall & Pettersson, 1976). Reent reports desried the purifiation of two wall-assoiated protein antigens of S. mutans serotype : these antigens, designated A and B, had moleular weights of and , respetively (Russell, 1979a, ). Antigen B was of partiular interest eause it showed antigeni ross-reativity with human heart tissue (Hughes, 1979; Hughes et al., 1980; Russell, 1979). Antiody responses to oth antigens A and B were found in monkeys immunized against dental aries (Cohen et al., 1979), though the role they play in protetion has yet to e eluidated. The present ommuniation desries an investigation into the distriution of these antigens in the various serotypes of S. mutans and in representatives of other streptooal speies /80/ SGM 25 MIC 118

2 384 R. R. B. RUSSELL METHODS Organisms. The following strains of Streptoous mutans were used: serotype u - AHT, OMZ61, NIDR 3720; serotype - FAl,OMZ51, OM271 ; serotype - Ingritt, GS5, NCTC 10449; serotype d - OMZ176, OIHI, B13; serotype e - P4, NCTC 10923, AtlO; serotype f - SEll, 151, 4177; serotype g - K1, OMZ65, TH62. The soures of these strains were desried previously (Russell, 1976) with the exeption of strain AHT whih was otained from D. Bratthall and onfirmed as type a (Russell, 1979d). Streptooi other than S. mutans were kindly provided y G. Colman of this laoratory. Streptoous sunguis strains are listed in Tale 2. Other strains used were S. mitior FW9, FW63 and FW75, S. milleri IS8, IS57, 7180, S198, FW702 and , and S. salivarius FW43 and BU104. Media and ulture onditions. Bateria were grown in Todd-Hewitt roth (Oxoid) for 20 h at 37 "C. Extration methods. Bateria from a ulture grown for 20 h in 20 ml Todd-Hewitt roth were olleted y entrifugation. For aid extration, the pellet was heated with 50 p1 0.2 M-HC~ at 100 "C for 10 min. For autolave extration, the pellet was heated with 50 pl0.9 % (w/v) NaCl at 121 "C for 15 min. For extration y mutanolysin, the pellet was suspended in 0.01 M-Tris/maleate/NaOH uffer (ph 7-0) ontaining mutanolysin M-1 enzyme (Yokogawa et al., 1975; 200 pg m1-l) and inuated for 3 h at 37 "C. The extrated slurries were used for immunodiffusion without further entrifugation. Mutanolysin was generously provided y M. Yokogawa, Dainippon Pharmaeutial Company, Japan. Preparation of antiseru. Typing sera were prepared in raits y a ourse of intravenous injetions of heat-killed ells of S. mutans as desried y Bratthall (1970). The prodution of antiserum to pure antigens A and B has een desried (Russell, 1979a, 6) and rait antiserum to human heart tissue was kindly provided y Dr M. Hughes, Wellome Researh Laoratories (Hughes, 1979; Hughes et al., 1980). Serologial methods. Agglutination of roth ultures grown for 16 h was tested y mixing a drop of ulture with 5pI antiserum on a mirosope slide and roking it gently. Agglutination, when it ourred, was apparent within 5 min. Immunodiffusion was performed in 1 % (w/v) agarose in 0-05 M-Tris/HCl uffer (ph 7-5) on glass slides. Samples of 20 pl antigen preparation or antiserum were plaed in 4 mm diameter wells 4 mm apart. Slides were dried, washed in saline and the immunopreipitates were stained with Coomassie lue as desried y Axelsen et al. (1973). Preparations of antigens. Lipoteihoi aid and serotype polysaharides were extrated from ateria with hot aqueous phenol (Westphal & Jann, 1965). The antigens were then purified y gel filtration on a olumn of Sepharose 6B followed y ion-exhange hromatography on DEAE-Sephadex, essentially as desried y Linzer et al. (1976). Lipoteihoi aid was prepared from strain Ingritt, and serotype polysaharides from AHT, FAl, Ingritt and K1. RESULTS Antiodies in typing sera Antigens A and B were purified from S. mutans strain Ingritt (serotype ) and antigenially idential proteins were deteted in strains of serotypes e andf ; serotype strains ontained an antigen ross-reating with A while strains of serotypes d and g ontained antigens ross-reating with B (Russell, 1979a, ). The ourrene of these antigens in more than one serotype suggested that antiodies to antigens A or B might e a ause of lak of speifiity of typing sera. A olletion of sera prepared in this laoratory for serotyping purposes was therefore examined y immunodiffusion for the presene of antiodies to a numer of defined antigens. All the typing sera were prepared y intravenous injetion of raits with heat-killed whole ells and 10 out of 12 sera had the desired anti-polysaharide antiodies, though four were non-speifi due to the presene of antiodies to lipoteihoi aid (Tale 1). Preipitin lines of polysaharide or lipoteihoi aid appeared sooner and were stronger than those ontaining protein antigens when slides were viewed on a darkground lightox, ut slides stained with Coomassie lue learly revealed the presene of preipitates to antigen B in six of the sera and to antigen A in three sera (Tale 1). Figure 1 shows the reations of antiserum against strain GS5 with extrats from representatives from all seven serotypes.

3 Cross-reative antigens in oral streptooi 385 Fig. 1 Fig. 2 Fig. 1. lmmunodiffusion of antiserum raised against whole ells of strain GS5 (entre well) and autolave extrats of representatives of the different S. mutans serotypes. The line lose to the antiserum well is due to antiody to serotype polysaharide, while the other lines are due to antiody to antigen B. Fig. 2. Immunodiffusion of antisera raised against SDS-extrated ells of different S. mutans serotypes and pure antigen B from strain Ingritt. 1, anti-fa1 (6); 2, anti-om261 (a); 3, antilngritt (); 4, anti-kl (g). Tale 1. Antiodies present in sera prepared for serotyping S. mutans Eah serum ame from a different rait immunized with heat-killed S. mutans. Sera were tested y two-dimensional immunodiffusion against four purified antigens. The polysaharide antigen used was that prepared from a strain of the same serotype as the immunizing strain. Immunizing strain AHT OMZ61 FA1 FA1 FA 1 GS5 Ingritt Ingritt Tngri t t K1 K1 K1 Serotype a n K K s I - Antigen" h PS LTA A B * PS, Serotype-speifi polysaharide; LTA, lipoteihoi aid. Presene of antigen B in serotype a Evidene from the typing sera indiated that antiserum raised against the serotype u strain OMZ61 reated with antigen B, although no ross-reating antigen had previously een deteted in ulture filtrates, autolave or aid extrats of strains of this serotype (Russell, 1979 ). Antigen B from strain Ingritt was originally haraterized as an antigen whih remained on the ateria even after oiling in sodium dodeyl sulphate (SDS) (Russell, 1979). That an analogous antigen existed in serotype u was demonstrated y using SDS-extrated ells of OMZ61 to immunize a rait. The resultant antiserum preipitated antigen B (Fig. 2). The same approah failed to detet any suh antigen in strain FA1 (serotype ). Further evidene for the presene of antigen B in serotype a strains was provided y the oservation of the slide agglutination of serotype a strains y antiserum raised against pure antigen B (Tale 2)

4 386 R. R. B. RUSSELL S. mutans" Serotype n d e f g S. sanguis Type1 Tale 2. Detetion of antigens reating with antiserum to antigen B Detetion method r--- h \ Autolave Mutanolysin Strain Agglutination extrat extrat Type I1 MWK NCTC 7865 NCTC 7869 NCTC S159 + Bu174 NCTC * Three representatives of eah serotype were examined; no variations were found within a serotype. i- Autoagglutination in the asene of serum. Extration of antigen B with mutanolysin The aove results indiated that strains of serotype a possessed an antigen resemling antigen B, ut that it was not as readily released as the orresponding antigen from other serotypes. An alternative method for release of antigen was therefore sought. Mutanolysin is an enzyme from Streptornyes gloisporus whih has een reported to lyse all serotypes of S. mutuns and other streptooi (Hamada et al., 1978). We onfirmed these oservations and found that digestion with mutanolysin effiiently released antigen B from all strains known to ontain it, inluding those of serotype a (Tale 2). No ross-reating antigen was released from any of the three serotype strains. Antigens in other streptooi The methods found suitale for deteting antigen in the S. mutans strains were applied to a olletion of representative strains of the other streptooi ommonly found in the mouth. An antigen ross-reating with B was deteted in six out of seven strains of S. sanguis (Tale 2). No suh antigen was found in the strains of S. mitior, S. milleri or S. salivarius. The presene of an antigen ross-reating with antigen A from strain Ingritt was previously reported in serotype strains (Russell, 1979 a). The present survey onfirmed this finding, ut failed to detet a ross-reating antigen in any of the other strains examined. Heart ross-reativity Antigen B from S. mutans strain Ingritt has een shown to e preipitated y rait antiserum raised against human heart tissue (Hughes, 1979; Hughes et ul., 1980; Russell, 1979). In immunodiffusion the same anti-heart serum reated with all those extrats of S. mutdns and S. sanguis whih ontained antigen ross-reating with antigen B. + DISCUSSION The antigens involved in ross-reativity etween oral streptooi have not een identified. Lipoteihoi aid and dextran are elieved to e responsile for some ross-

5 Cross-reative antigens in oral streptooi 387 reations (Bratthall, 1969; Chorpenning et dl., 1975), and Hardie & Bowden (1976) presented preliminary evidene that a protein antigen was ommon to S. mutuns and S. sanguis. I reently haraterized antigen B of S. mutans strain Ingritt as a wall-assoiated protein with an apparent moleular weight of and the present report shows that six of the seven serotypes of S. mutans ontain this or a losely similar antigen, as do strains of S. sangtris. It is perhaps surprising that S. mutans serotype strains laked the antigen as serotypes and are losely related genetially (Coykendall, 1974) and have already een shown to have antigenially ross-reating gluosyltransferase (Russell, 1979 ) and antigen A (Russell, 1979~). The possiility remains that a ross-reating antigen might e disovered if a wider range of growth onditions and extration methods were introdued. Cross-reating antigen B was deteted in strains of oth S. sanguis type I and S. SdF?gUiS type 11 ut not in strains of S. mitior. Some authors have suggested S. sanguis type I1 strains should e lassified as S. mitior from whih they differ only in the aility to synthesize a water-solule gluan (Coykendall & Munzenmaier, 1978), and while this might suggest that the antigen ommon to all the S. sanguis strains is gluosyltransferase, lear evidene has een presented that gluosyltransferase and antigen B from S. mutuns strain Ingritt are distint entities (Russell, 1979). The most widely employed tehnique for produing sera for typing S. mutans involves intravenous injetion of whole ateria into raits and it is generally reognized that one or more asorption steps are needed to produe serotype-speifi sera. The results presented in Tale 1 indiate that antigens A and B may frequently e the auses of the non-speifiity of sera. A knowledge of antigeni determinants whih may e shared etween speies is essential in any studies in whih attempts are made to assess levels of antiody to a partiular organism. For instane, numerous attempts have een made to orrelate levels of antiody to S. inutans with aries experiene, ut it is now lear that if whole ateria are used in immunoassay proedures not only speifi antiody will e deteted, ut also antiody to suh non-speifi antigens as lipoteihoi aid (Deker et at., 1972; Markham et al., 1973) and antigen B. Charaterization of the antigens involved in heart ross-reativity and their distriution is also needed for appreiation of the hazards involved in the development of streptooal vaines. Furthermore, a knowledge of the antigeni relationships of different oral streptooi may help provide information on the mehanism y whih immunity to dental aries is otained, as it is lear that immunization with S. mutnns may indue antiodies apale of reating with other speies. 1 am grateful to D. Bratthall for supplying S. mutuns AHT, to G. Colman for providing many aterial strains and some of the typing sera, to M. Hughes for the anti-heart serum and to M. Yokogawa for his generous gift of mutanolysin. AXELSEN, N. H., KROLL, J. & WEEKE, B. (1973). A Manual of Quantitative Immunoeletrophoresis. Oslo : Universitatetsforlaget. BOWEN, W. H. (1976). Relevane of aries vaine investigations in rodents, primates and humans : ritial assessment. In Immunologial Aspets of Dental Caries, pp Edited y W. H. Bowen, R. J. Geno & T. C. O'Brien. London: Information Retrieval Limited. BRATTHALL, D. (1969). Immunodiffusion studies on the serologial speifiity of streptooi resemling Streptoous mutans. Odontologisk rev.y 20, R E F E R E N C, E S BRATTHALL, D. (1970). Demonstration of five serologial groups of streptooal strains resemling Streptoous mutans. Odontologisk revy 21, BRATTHALL, D. & PETTERSSON, B.-M. (1976). Common and unique antigens of Streptoous mutans. Journal of Dental Researh 55 (Speial Issue A), A60-A64. CHORPENNING, F. W., COOPER, H. R. & ROSEN, S. (1975). Cross-reations of Streptoous mutans due to ell wall teihoi aid. Infetion and Immunity 12, COHEN, B., COLMAN, G. & RUSSELL, R. R. B. (1979).

6 388 R. R. B. RUSSELL Immunization against dental aries : further mutans with heart-tissue antigens. In Pathogeni studies. British Dental Journal 147, Streptooi, pp Edited y M. T. Parker. COLMAN, G. (1976). The viridans streptooi. In Chertsey : Reedooks. Seleted Topis in Clinial Bateriology, pp HUGHES, M., MACHARDY, S. M., SHEPPARD, A. J Edited y J. de Louvois. London: Bailliere & WOODS, N. C. (1980). Evidene for an immuno- Tindall. logial relationship etween Streptoous mutans COLMAN, G. & WILLIAMS, R. E. 0. (1972). and human ardia tissue. Infetion and Immunity Taxonomy of some human viridans streptooi. 27, In Streptooi and Streptooal Diseases, pp. LINZER, R., GILL, K. & SLADE, H. D. (1976) Edited y L. W. Wannamaker & J. M. Chemial omposition of Streptoous mutans Matsen. New York & London: Aademi Press. type antigen: omparison to type a, and d COYKENDALL, A. L. (1974). Four types of Strepto- antigens. Journal of Dental Reserh 55 (Speial ous mutans ased on their geneti, antigeni Issue A), A109-Al15. and iohemial harateristis. Journal of General MARKHAM, J. L., KNOX, K. W. & SCHAMSCHULA, Miroiology 83, R. G. (1973). Antiodies to teihoi aids in man. COYKENDALL, A. L. & MUNZENMAIER, A. J. (1978). Arhives of Oral Biology 18, Deoxyri onulei aid ase sequene studies on RUSSELL, R. R. B. (1976). Classifiation of Streptogluan-produing and gluan-negative strains of ous muttlts strains y SDS gel eletrophoresis. Streptoous mitior. International Journal of Miroios Letters 2, Systemati Bateriology 28, RUSSELL, R. R. B. (1979a). Cell-wall protein antigens DECKER, G. P., CHORPENNING, F. W. & FREDERICK, of Streptoous mutans. In Pathogeni Strepto- G. T. (1972). Naturally ourring antiodies to oi, pp Edited y M. T. Parker. aillary teihoi aids. Journal of Immunology Chertsey : Reedooks. 108, RUSSELL, R. R. B. (1979 ). Wall-assoiated protein FACKLAM, R. R. (1977). Physiologial differentiation antigens of Streptoous mutans. Journal of of viridans streptooi. Journal of Clinial General Miroiologj) 114, Miroiology 5, RUSSELL, R. R. B. (1979~). Glyosyltransferases of HAMADA, S. TORII, M., KOTANI, S., MASUDA, N., Streptoous mutuns strain Ingritt. Miroios 00SHIM4, T., YOKOGAWA, K. & KAWATA, s. 23, (1978). Lysis of Streptoous mutans ells with RUSSELL, R. R. B. (1979d). Comparison of Streptomutanolysin, a lyti enzyme prepared from a ous mutans AHT with strains of serotypes a and ulture liquor of Streptomyes gloisporus g y iohemial and eletrophoreti methods. Arhives of Oral Biology 23, Arhives of Oral Biology 24, HARDIE, J. M. & BOWDEN, G. H. (1976). Some WESTPHAL, 0. & JANN, K. (1965). Baterial liposerologial ross-reations etween Streptoous polysaharide extration with phenol-water and mutans, S. sanguis and other dental plaque further appliations of the proedure. Methods in streptooi. Journal of Dental Researh 55 Carohydrate Chemistry 5, (Speial Issue C), C50-C58. YOKOGAWA, K., KAWATA, S., TAKEMURA, T. Rr HARDIE, J. M. & MARSH, P. D. (1978). Streptooi YOSHIMURA, Y. (1 975). Purifiation and properties and the human oral flora. In Streptooi, pp. of lyti enzymes from Streptomyes gloisporus Edited y F. A. Skinner & L. B. I 829. Agriultural and Biologial Chemistry 39, Quesnel. London : Aademi Press HUGHES, M. (I 979). Cross-reations of Streptoous