Original Papers

Z. Immun.-Forsch. vol. 154, pp. 197-207 (1978)

Central Institute of Microbiology and Experimental Therapy, Academy of Sciences of GDR, Jena, GDR Institute of Hygiene and Epidemiology, Prague, CSSR

Type-Specific and Non-Type-Specific Reactions of Purified M Protein Preparations

O. KUHNEMUND, J. HAVLICEK, and W. KOHLER

With 7 figures

Received October 6, 1977 . Accepted in Revised Form January 27, 1978

Abstract

M proteins of type 1 and type 12 Streptococcus pyogenes were extracted by means of phage-associated lysin and purified by ion-exchange chromatography on eM and DEAE cellulose. Molecular weight distributions were studied by gel chromatography on Biogel A 0.5 m in a 6 molal' urea solution and by SDS electrophoresis. Serological activities were studied by the complen1.ent-fixation reaction and immunodiffusion and were compared with the estimated molecular weights. Type-specific and non-specific activity was fOlmd to be located on the same polypeptide chain of a size of 2 X 10· dalt,ons (type 1) and 1.5 X 10' daltons (type 12). These serologically active chains are in preparations purified by chromatographic methods accompanied by polypeptides of different sizes which are held together by noncovalent bonds thus forming molecules abov8 4 X 10' daltons.

Introduction

Immunity to infections caused by Streptococcus pyogenes is type specific. The antigen responsible for this type-specific immunity is the so called M protein. Its characteristics have been well described in several reviews (1, 2, 3).

Many authors used different methods of purification of this antigen. The materials which have been obtained by these purification methods show very different properties. Most of them are of a multiple struc­ture and their antigenic properties are not as a rule associated with any clearly defined family of molecules (4). Besides type specificity, sensitive immunological methods allowed detection of non-specific reactions of M proteins (5, 6, 7, 8). These reactions may be caused by one or more components of the «multiple structure» of M proteins.

198 . O. KURNElIIDND, J. HAVLICEK, and VV. KOHLER

Our study aimed at elucidating some of the relations between the antigenic (both type-specific and non-t.ype-specific) properties of purified M protein and its physicochemical behaviour. Phage asso­ciated lysin extracted M protein, purified by ion-exchange chromato­graphy as previously described, was subjected to gel-chromatography and SDS electrophoresis. Preparations of different molecular weights were obtained which reacted both type-specifically and non-type­specifically. It was finally shown that one single polypeptide chain which was most active and abundant in our material was the seat of both the type-specific (i.e. M-protein) antigenic determinant (or deter­minants) and non-type-specific determinant (or determinants) .

Materials and Methods

St?'ains

Stl'eptococC'Us pyogenes (group A), Type 12, No. 22/59; Type I , N o. 40/58 (Czechoslovak National Collection of Type Culture, IHE, Prague, CSSR); Type 19, strain Haskins. The presence of M protein in these strains was checked by their ability to grow in fresh human blood.

Antisera

Antisera were prepared by immunization of rabbits according to the method of LANCEFIELD (9).

Complement-jb:at·ion reaction (CFR)

CFR was perfonned by the micromethod of FULTON and DUlI'lBELL (10) as modified by POKORNY et al. (ll).

Controls for detection of anticomplementary activity were always included. No antigen showed an anticomplementary effect. If a serum was anticomple­mentary, it was mixed with complement 1: 10, incubated at 37 °C for I h and inactivated 1 h at 56 °C. By this procedme all anticomplementary action was abolished.

Cult·ivation

Strains were cultivated in 40 I of semisynthetic medium (12) in a 40 I vessel of the FA 300 fermentation system (LKB Produkter AB Stockholm, Sweden) at 37 °C for 18 h, under regulation of pH (7.2) by the addition of a 20% NaOH solution and the same volume of a 50% dextrose solution.

Cells were harvested by continuous-flow centrifugation (Westfalia Labor­separator type SAOOH).

Yields: 292 g type 1 (wet weight) 375 g type 12 (wet weight)

Ext'l'action

Streptococci of the type 1 and type 12 strains used were extracted by treat­ment with phage-associated lysin as described earlier (13). The extent of lysis was estimated by gram-stained films.

After extraction, the suspensions were centrifuged and the proteins preci­pitated from the supernatants by addition of ammonium sulphate at 0.7 5-fold saturation.

Type- and Non-Type-Specific M Protein Reactions' 199

Purijica#on

Separation of ammonium sulphate precipitates of crude extracts was per­formed on CM-cellulose and on DEAE-cellulose cohmms as described previously (13).

Phage-enzyme-released M protein preparations of type 1 and type 12 strepto­cocci purified by CM-cellulose and DEAE-cellulose ion exchange chromato­graphy were separated on Biogel A 0.5 m 1 ) (2.5 X 82 cm) in a 0.05 molar disodium phosphate buffer, pH 7.0, containing 6 molar urea. The urea was added to eliminate hydrogen bridge bonds.

Six hundred milligrams of each M protein were dissolved in 6 ml of the urea buffer and chromatographed. The eluates were collected in fractions of 3.9 m!. The eluat,es of t,ype 1 as well as type 12 M protein were divided int,o three parts and each was concentrated by ultrafiltration and rcchromatographed on the saIne colUlllll.

Double diff1lsion in agar'ose

The method of OUCHTERLONY (14) was used employing 1 % agarose gel m 0.02 M aethyl barbital, pH 8.6, with 0.02% sodium azide as a preservative.

Analytical SDS electrophoresis

Fractions of the gel chromatographic separation of type 1 and type 12 M protein on Biogel A 0.5 m in 6 M urea solution were selected for comparative SDS electrophoretic runs, which were carried out according to 'VEBER and Os­

BORN (15) in glass tubes (precision glass, 6 mm i.d., 10 cm length) filled with 2.3 ml of gel solution. As reference proteins, bovine serum albumin (Sigma), IgG (Kabi), pepsin (Serva) and lysozym (Calbiochem) were L;sed, all reduced and alkylated as described (16). M Protein preparations were not reduced and alkylated, since comparative studies of preparations from different strains had shown that the SDS patterns of both treated Gnd untreated samples were identical (17).

Preparative SDS electrophores'is

The same method was used for preparative SDS olectrophoresis except that glass tubes of 8 mm diameter were used, filled with 4 ml of gel solution resulting in a gel length of 8 cm. Gels were run in parallel, 2 of them for reference pro­teins, 6 for each M protein sample. The amount of protein applied per gel was 0.18 mg. Five gels were divided in blocks of 4 mm, crushed and eluted with PBS2); the reference gels and 1 sample gel were stained with Amido black 10 B. The eluates were dialyzed to remove SDS and concentrated by means of lyo­philization.

Results

Extraction, purification

The fractionation of phage-associated-lysin extracts of type 1 and type 12 streptococci on OM cellulose by use of three different buffers resulted in three peaks (ICM, lIOM, lIIOM). In both cases, peak IlIOM

1) Bio-Rad Laboratories 2) phosphate-buffered saline

200 . O. KUHNEMUND, J. RAVLICEK, and "V. KOHLER

contained the material with the highest serological activity as was shown in double-diffusion and complement-fixation tests. Peak 10M of both extracts did not precipitate in immunodiffusion. Further purification of peak IIIOlVI of both extracts (types 1 and 12) on DEAE cellulose resulted in two fractions. In both cases, the second fraction ( designated as M 1 IIICM lID EAE and M 12 IIICM lID EAE) showed the highest serological activity.

The yields of lyophilized M protein preparations were, in relation to wet weight of extracted streptococci,

M1 -0.27%, and M12-0.60%.

Immunodiffusion resulted in precipitation against homologous anti­sera as well as in weak precipitation against heterologous antisera (Fig. 1).

Titers in the complement-fixation reaction, calculated to a protein concentration of 1 mg/ml, were comparable in their values (Fig. 2). There was a marked difference between the titers of homologous and heterologous sera with M 1 and M 12 protein preparations.

The fractions from gel chromatography of M 1 IIICM IIDEAE on Biogel A 0.5 m in buffer containing 6 M urea were tested in gel dif­fusion and divided into three parts (Fig. 3). The third part did not precipitate in gel diffusion at all. The first and second parts (designated as M 1 III/II/I and M 1 III/II/II, respectively), positive in immuno­diffusion, were rechromatographed (Fig. 4). SDS electrophoresis showed that by this gel-chromatographic procedure a separation of

Fig. 1. Immunodiffusion ofM 1 (left) and ofM 12 (right) protein (IIICM IIDEAE) against unabsorbed antisera of different types of group A streptococci. Central wells: M proteins. Number in outer wells: M types of antisera (1: anti type 1, 3: anti type 3 antiserum, etc.).

Type. and Non·Type·Specific M Protein Reactions . 201

1. 1024 o Anti AMI J4

~ An , ."' 12 50

I: SI2

1:255

I :S4

Fig . 2. Complement.fixation reaction titers of lVI 1 and M 12 prot~ against M 1 and M 12 unabsorbed antisera .

00

2.0

1.5

1.0

0.5

10 20 JO o 50 60 70 "0 gO 100 F"RACT

Fig. 3. Gel chromatography of type 1 M protein on Biogel A 0.5 m in 0.05 M Na2HPO •. 2H20 pH 7.0 + 6 M urea (Column: 2.5 X 88 cm; 3.9 ml per frac · tion) .

202 . O. KUHNEllfUND, J. HAVLICEK, and W. KOHLER

58 62 66 7() 74 78 92 86

Fig. 3a shows the serological activity of several fractions tested in immuno­diffusion.

the components according to molecular weight occurred (Fig. 5). Double diffusion tests of the separated fractions showed serological activity against homologous antiserum as well as a weak non-type­specific reaction against a heterologous, type 19 antiserum.

In complement-fixation tests the gel-chromatographed preparations reacted type specifically as well as non-type specifically, as shown in Figure 6. The titers of the tested solutions are calculated to a protein concentration of 1 mg/ml, so that the results are comparable. The highest activity was found in preparation III/II/lIb, having an aver­age molecular weight, according to the calibration of the gel column, of about 8 X 104 •

The gel chromatography of type 12 M protein preparation (M 12 III/II) showed the same results. The eluate was also divided into three parts. Part I and II were rechromatographed and divided as in the experiment with type 1 M protein (see Fig. 4). The immunodiffusion

E

2

os

20 30 40 SO 30 o SO 60 70 F~ACr.

Fig. 4. Rechromatography of peak I (left) and peak II (right) from gel filtration of type 1 M protein on Biogel A 0.5 m (see fig. 3).

Type- and Non-Type-Specific lVI Protein Heactions - 203

40

48 so

S2 53 55 57 59 61 63 65

Fig. 4a. Immunodiffusion test after reclu·omatography of peak II of Figure 3_

patterns as well as the CFR titers of these preparations were very similar to the results in type 1 M protein experiments. Similarly to the gel chromatography experiment with type 1M protein, the highest activity was found in substance M 12 III/II/lIb with an aver­age molecular weight (gel chromatography) of about 8 X 104 .

Fig. 5. SDS Electrophoresis patterns for several fractions of type 1 M protein double chromatographed on Biogel A 0.5 m (see Fig. 4). Reference proteins: BSA, IgG heavy chain, pepsin, IgG light chain, lysozyme.

204 . O. KUHNEMUND, J. HAVLICEK, and W. KOHLER

CFR iller

096

20t.e

1:1021.

Fig. 6. CFR titers of gel filtration fractions of type 1 (left) and type 12 (right) M proteins against type 1 and type 12 antisera. Numbers at bottom of diagrams indicate gel filtration fractions. A and B: original nmterials (AM 1 IIICM IIDEAE and AMI 2 IIICM IIDEAE, resp.).

Preparative SDS electrophoresis

In view of their high serological activity preparations lVI 1 III/II/lIb and lVI12 III/II/lIb were selected for preparative SDS electrophoresis. The patterns of bands and the corresponding complement-fixation reaction titers for both electrophoresis separation experiments are shown in Figure 7. In spite of several bands having been obtained, as may be seen on the photographs of stained gels, type 1 as well as type 12 experiments displayed only one sharp peak in the complement­fixation reaction, corresponding to one band of the electrophoretic pattern. In titration experiments with homologous as well as hetero­logous antisera, the maxima of serological activity appeared exactly in the same fraction. The maximum in serological activity corresponded to a molecular weight of 2 X 104 in type 1 experiment and of 1.5 X 104

in type 12 experiment.

Discussion

Serological type specificity is an intrinsic property of lVI protein. This property has been used in most isolation and purification ex­periments as a control. In addition to this type specificity, more or

CF .. 16l.

132

Type- and Non-Type-Specific M Protein Reactions . 205

J:I m nCE lib 'f' . 8

[) ~ An A~2~ ·8 III

2mt liCE Db

32

2 , 6 8 16 18 20 20

Fig. 7. CFR titers of samples isolated by preparative SDS electrophoresis. Abscissa: gel fraction number. Reference protein: BSA, IgG heavy chain, pepsin, IgG light chain, lysozyme.

less non-type-specific reactivity has been found in M proteins. "\iVID­DaWSON and MAXTED (5, 18) suggested that the non-type-specific serological reactivity of their M protein preparation, also found in the complement-fixation reaction, is caused by an «M-associated protein». Because of the complex SDS-electrophoresis pattern of M protein preparations, it is impossible to say whether their type-specific and non-type-specific moieties are located on the same polypeptide chain or not. Therefore we decided to use preparative SDS electrophoresis to have a possibility of testing the serological properties of separated polypeptide chains. In both experiments (type 1 as well as type 12) we found serological reactivity (type-specific as well as non-type­specific) situated at the same spot on the gels, corresponding to one single band. This suggests that these two moieties are connected by covalent bonds, i.e. are located on the same polypeptide chain. The ability of the different fractions to absorb opsonic antibodies has not been tested. The maximum of this activity and the maximum of the CFR titer need not necessarily coincide.

The molecular weights of M proteins reported in the literature differ in a broad range, depending on the method of preparation (2, 19, 20, 21, 22). In gel chromatography experiments, for preparations to be used in preparative SDS electrophoresis molecular weights of about 8 X 104 in the presence of 6M urea have been found. This is a con­siderable difference from the molecular weights of the serologically active polypeptides found in preparative SDS electrophoresis experi­ments (2 X 104 type 1, 1.5 X 104 type 12). No serological activity was found in the gel chromatography experiments below the 4 X 104

206 . O. KUHNEMUND, J. HAVLICEK, and W. KOHLER

molecular weight boundary. This suggests that the preparations isolated by gel chromatography consisted of several polypeptide chains, held together by non-covalent bonds. These complexes do not seem to be oligomers of the same poly-peptide chain but rather agglomerates of polypeptides of different sizes, as shown by SDS electrophoresis.

There is a difference between the molecular weights of the sero­logically active polypeptides of type 1 and 12 strains used, as was shown in SDS electrophoresis experiments. More experiments will have to be performed in order to decide whether this difference is due to type specificity of M proteins or is caused by variation between individual strain.

Acknowledgement

We wish to thank Prof. Dr. J. SJOQUIST as well as Dr. J. SJODAHL, Uppsala, for their stimulating discussions and for amino acid analysis.

References

1. LANCE FIELD , R. C. 1962. Current knowlcdge of type-specific M antigens of group A streptococci. J. Immllloi. 89: 307.

2. Fox, E. N. 1974. M proteins of group A streptococci. Bact. Rev. 38: 57. 3. HAVLICEK, J. 1974. Streptococcus pyogenes: M protein and type-specific

vaccine. Cs. Epidemiol. Mikrobiol. Imnmnol. 23: 114-127. 4. Fox, E. N. and M. K. WITTNER. 1965. The multiple molecular structure

of the M proteins of group A streptococci. Proc. Nat,. Acad. Sci. (Wash.) 54: 1118.

5. WIDDOWSON, J. P., "V. R. MAXTED, :cmd A. M. PINNEY. 1971. An Masso· ciated protein antigen (MAP) of group A streptococci. J. Hyg. 69: 553.

6. MAXTED, W. R., and J. P. WIDDOWSON. 1972. The protein antigens of group A streptococci, p. 251 ff. in: L. W. WANNAMAKER and J. N. MATSEN (ed.), Streptococci and streptococcal diseases, Academic Press Inc., New York.

7. BEACHEY, E. H., 1. OFEK, U. CUNNINGHAM, and A. BISNO. 1974. Evaluation of micro complement fixation tests for antibodies against group A strepto­coccal M and M associated antigens in rabbit and human sera. Appl. Micro­bioI. 27: l.

8. BEACHEY, E. H., 1. OFEK, and A. BISNO. 1973. Studies of non type specific antigens associated with streptococcal M proteins in sera of patients with rheumatic fever. J. 1mmunol. Ill: 1361.

9. LANCE FEILD , R. C. 1938. A microprecipitin technique for classifying hemo­lytic streptococci and improved methods for producing antisera. Proc. Soc. Exp. BioI. (N. Y.) 38: 473.

10. FULTON, F., and K. R. DUMBELL. 1949. The serological comparison of strains of influenza viruses. J. gen. Microbiol. 3: 97.

11. POKORNY, J., M. ZASTERA, and B. CUBIK. 1972. A proposal for standard­ization of the complement fixation microtechnique for toxoplasmosis sero­diagnostics. Cs. Epidemiol. Mikrobiol. 1m.munol. 21: 225.

12. GERLACH, D., und W. KOHLER. Verfahren zur Gewinnung von Strepto­kokkenstoffwechselprodukten. WP 007 gj174180.

Type- and Non-Type-Specific M Protein Reactions . 207

13. KUHNEMUND, 0., und VV. KOHLER. 1976. Untersuchungen zur Immun­biologie von M-Proteinen des Streptococcus pyogenes. 1. Mitteilung: Iso­lierung und Reinigung des M Proteins aus Streptokokken del' Gruppe A (Strept,ococcus pyogenes) Typ 1. Allergie u. Immunol. 22: 17l.

14. OUCHTERLONY, O. 1949. Antigen-antibody refwtions in gels. Acta path. Micro bioI. Scand. 26: 507.

15. VVEBER, K., and M. OSBORN. 1969. The reliability of molecular weight determination by dodecyl sulfate polyacrylamide gel electrophoresis .. J. BioI. Chern. 244: 4406.

16. GOLLWITZER, R., R. TIMPEL, U. BECKER, and H. FURTHMAYR. Chelnical and immunological properties of reduced and alkylated polypeptide chains of bovine fibrinogen. Europ. J. Biochem. 28: 497.

17. KUHNEMUND, 0., >md J. HAVLICEK. Studies of specific reactions of M pro­tein preparations. Abstr. VI Internat. Symposium on Strept. pyogenes, Prague 1975.

18. WIDDOWSON, JEAN P., W. R. MAXTED, and A. M. PINNEY. 1976. Immuno­logical heterogeneity among the M associated protein antigens of group A streptococci. J. Med. Microbiology 9: 73.

19. RUSSELL, H., and RICHARD B. FACKLAM. 1975. Guanidine extraction of streptococcal M protein. Infect. and Immunity 12: 679.

20. CUNNINGHAM, M., and E. H. BEACHEY. 1975. Immunochemical properties of streptococcal M protein purified by isoelectric focusing. J. Immunol. ll5: 1002.

21. FISCHETTI, V. A., E. C. GOTTSCHLICH, G. SIVIGLIA, and J. B. ZABRISKIE. 1976. Streptococcal M protein extracted by non-ionic detergent. J. expo Med. 144: 32.

22. HAVLICEK, J. 1975. M protein of type 12 Streptococcus pyogenes isolation by electrofocusing and some molec,llar weight dependent properties. Path. Microbiol. 42: 147.

Dr. O. KUHNEMUND, ZentraJinstitut fUr Mikrobiologie und Experimentelle Therapie, Akademie del' Wissenschaften del' DDR, Beuthenbergstra13e 11, DDR-69 Jena