10
JOURNAL OF BACTERIOLOGY Vol. 88, No. 4, p. 912-921 October, 1964 Copyright a 1964 American Society for Microbiology Printed in U.S.A. ELECTROPHORETIC SEPARATION OF CONSTITUENTS OF PARTIALLY PURIFIED M PROTEIN OF STREPTOCOCCUS PYOGENES WILLIAM A. PIERCE, JR. Department of Microbiology, Tulane University Medical School, New Orleans, Louisiana Received for publication 6 May 1964 ABSTRACT P1ERCE, WlLLIAM A., JR. (Tulane University, New Orleans, La.). Electrophoretic separation of constituents of partially purified M protein of Streptococcus pyogenes. J. Bacteriol. 88:912-921. 1964.-Partially purified M protein of a group A, type 12 strain of Streptococcus pyogenes was stud- ied by use of chemical, electrophoretic, and im- munological techniques. It was demonstrated in immunodiffusion tests that the antigen contains multiple precipitating components. The type- specific antigen was identified, and evidence was presented that, in some instances at least, the cross-reactions observed between this type 12 M protein and heterologous antisera in immunodiffu- sion tests involve contaminating antigens rather than the component which precipitates with ad- sorbed homologous-typing antiserum. In passive hemagglutination tests where M protein was ad- sorbed to tanned sheep erythrocytes, it was found that antisera suitably adsorbed to show good specificity in capillary precipitin tests neverthe- less still contain cross-reactive antibodies which are detectable by this more sensitive technique. Electrophoresis on starch paste separates some of the components of partially purified M protein, so that a fraction can be obtained which has fewer precipitating antigens, as determined in immuno- diffusion tests, and which is less cross-reactive in passive hemagglutination tests with heterologous unadsorbed antistreptococcal antisera. and reliable test which is reasonably easy to per- form. After the demonstration by Boyden (1951) that streptococcal protein could be adsorbed to tannic acid-treated sheep erythrocytes which were then agglutinable by homologous rabbit antisera, sev- eral investigators attempted to use this method for the measurement of type-specific antistrepto- coccal antibodies (Denny and Thomas, 1953; Holmes, 1958; Quinn and Lowry, 1963). All of the techniques devised thus far suffer from the limitation that numerous cross-reactions occur with human and animal sera. It is necessary, therefore, to adsorb sera prior to testing with heterologous strains of group A streptococci, sometimes with as many as four strains (Quinn and Lowry, 1963). Another solution to the problem of cross-reac- tivity in the hemagglutination test would be the preparation of a noncross-reacting antigen for the sensitization of the tanned erythrocytes. The pres- ent report describes a method for the production of a relatively specific antigen from a type-12 streptococcal strain. This antigen, and the partially purified material from which it was derived, were studied by the techniques of hemag- glutination, immunodiffusion, and zone electro- phoresis. Some of the immunological characteris- tics of these substances are described. Several procedures have been employed for the detection and measurement of type-specific anti- bodies to the group A streptococci, such as passive mouse protection (Lancefield and Todd, 1928), bacteriostasis in whole blood (Rothbard, 1945), long-chain formation (Stollerman and Ekstedt, 1957), complement-fixation (Bone, Braude, and Kleinman, 1957), and bentonite flocculation (Lian and Pierce, 1961). For various reasons, none of these methods has proven to be entirely satisfactory, and there is still need for a sensitive MATERIALS AND METHODS Organisms. Eight strains of Streptococcus pyo- genes (group A) were employed. Type 3 (SS265), type 4 (SS534), and type 6 (SS67) were obtained from the Communicable Disease Center, U.S. Public Health Service, Atlanta, Ga. Type 12 ("Bailey") was kindly supplied by the late L. A. Rantz, formerly at Stanford University School of Medicine. Type 18 (Alll) was obtained through the courtesy of Robert M. Pike, Southwestern Medical School. Type 14(S23) was kindly fur- 912 on October 2, 2017 by guest http://jb.asm.org/ Downloaded from

JOURNAL OF BACTERIOLOGY Vol. No. American Printed U.S.A ... · JOURNAL OF BACTERIOLOGY Vol. 88, No.4, p. 912-921 October, 1964 Copyright a1964 AmericanSocietyfor Microbiology Printedin

  • Upload
    others

  • View
    3

  • Download
    0

Embed Size (px)

Citation preview

Page 1: JOURNAL OF BACTERIOLOGY Vol. No. American Printed U.S.A ... · JOURNAL OF BACTERIOLOGY Vol. 88, No.4, p. 912-921 October, 1964 Copyright a1964 AmericanSocietyfor Microbiology Printedin

JOURNAL OF BACTERIOLOGYVol. 88, No. 4, p. 912-921 October, 1964Copyright a 1964 American Society for Microbiology

Printed in U.S.A.

ELECTROPHORETIC SEPARATION OFCONSTITUENTS OF PARTIALLY PURIFIEDM PROTEIN OF STREPTOCOCCUS PYOGENES

WILLIAM A. PIERCE, JR.Department of Microbiology, Tulane University Medical School, New Orleans, Louisiana

Received for publication 6 May 1964

ABSTRACT

P1ERCE, WlLLIAM A., JR. (Tulane University,New Orleans, La.). Electrophoretic separation ofconstituents of partially purified M protein ofStreptococcus pyogenes. J. Bacteriol. 88:912-921.1964.-Partially purified M protein of a group A,type 12 strain of Streptococcus pyogenes was stud-ied by use of chemical, electrophoretic, and im-munological techniques. It was demonstrated inimmunodiffusion tests that the antigen containsmultiple precipitating components. The type-specific antigen was identified, and evidence waspresented that, in some instances at least, thecross-reactions observed between this type 12 Mprotein and heterologous antisera in immunodiffu-sion tests involve contaminating antigens ratherthan the component which precipitates with ad-sorbed homologous-typing antiserum. In passivehemagglutination tests where M protein was ad-sorbed to tanned sheep erythrocytes, it was foundthat antisera suitably adsorbed to show goodspecificity in capillary precipitin tests neverthe-less still contain cross-reactive antibodies whichare detectable by this more sensitive technique.Electrophoresis on starch paste separates some ofthe components of partially purified M protein, sothat a fraction can be obtained which has fewerprecipitating antigens, as determined in immuno-diffusion tests, and which is less cross-reactive inpassive hemagglutination tests with heterologousunadsorbed antistreptococcal antisera.

and reliable test which is reasonably easy to per-form.

After the demonstration by Boyden (1951) thatstreptococcal protein could be adsorbed to tannicacid-treated sheep erythrocytes which were thenagglutinable by homologous rabbit antisera, sev-eral investigators attempted to use this methodfor the measurement of type-specific antistrepto-coccal antibodies (Denny and Thomas, 1953;Holmes, 1958; Quinn and Lowry, 1963). All of thetechniques devised thus far suffer from thelimitation that numerous cross-reactions occurwith human and animal sera. It is necessary,therefore, to adsorb sera prior to testing withheterologous strains of group A streptococci,sometimes with as many as four strains (Quinnand Lowry, 1963).Another solution to the problem of cross-reac-

tivity in the hemagglutination test would be thepreparation of a noncross-reacting antigen for thesensitization of the tanned erythrocytes. The pres-ent report describes a method for the productionof a relatively specific antigen from a type-12streptococcal strain. This antigen, and thepartially purified material from which it wasderived, were studied by the techniques of hemag-glutination, immunodiffusion, and zone electro-phoresis. Some of the immunological characteris-tics of these substances are described.

Several procedures have been employed for thedetection and measurement of type-specific anti-bodies to the group A streptococci, such as passivemouse protection (Lancefield and Todd, 1928),bacteriostasis in whole blood (Rothbard, 1945),long-chain formation (Stollerman and Ekstedt,1957), complement-fixation (Bone, Braude, andKleinman, 1957), and bentonite flocculation(Lian and Pierce, 1961). For various reasons,none of these methods has proven to be entirelysatisfactory, and there is still need for a sensitive

MATERIALS AND METHODS

Organisms. Eight strains of Streptococcus pyo-genes (group A) were employed. Type 3 (SS265),type 4 (SS534), and type 6 (SS67) were obtainedfrom the Communicable Disease Center, U.S.Public Health Service, Atlanta, Ga. Type 12("Bailey") was kindly supplied by the late L. A.Rantz, formerly at Stanford University School ofMedicine. Type 18 (Alll) was obtained throughthe courtesy of Robert M. Pike, SouthwesternMedical School. Type 14(S23) was kindly fur-

912

on October 2, 2017 by guest

http://jb.asm.org/

Dow

nloaded from

Page 2: JOURNAL OF BACTERIOLOGY Vol. No. American Printed U.S.A ... · JOURNAL OF BACTERIOLOGY Vol. 88, No.4, p. 912-921 October, 1964 Copyright a1964 AmericanSocietyfor Microbiology Printedin

VOL. 88, 1964 SEPARATION OF STREPTOCOCCAL M PROTEIN COMPONENTS

nished by C. V. Seastone, University of WisconsinMedical School. Types 5 (no. 1) and 36 (no. 94)were isolated at the Student Infirmary, Universityof Wisconsin, from patients with acute folliculartonsillitis. All strains were preserved in the lyoph-ilized or frozen state, and were found to pro-duce moderate to large amounts of type-specificantigen as determined by the method of Rothbardand Watson (1948).

Antisera. Rabbits were immunized by the in-travenous injection of heat-killed vaccines ac-cording to a standard procedure (Wadsworth,1947). Sera were preserved with 0.2% phenol andstored at 5 C. The adsorbed homologous typingsera were obtained through the courtesy of JohnF. Winn, Diagnostic Reagents Section, Communi-cable Disease Center, Atlanta, Ga. These serawere freed from cross-reacting antibodies by ad-sorption of 4 parts of serum with 1 part of packed,heat-killed streptococci of heterologous type. Atleast three adsorptions were required for each lotof antiserum.

Antigens. Cultures for the preparation of anti-gens were grown in 30- to 60-liter lots in Todd-Hewitt Broth (Difco) at 37 C for 24 hr.

Fractions were prepared by the method ofLancefield and Perlmann (1952), modified inthat most batches of cells were extracted onlytwice, and a final ribonuclease concentrationof 0.0025 mg/ml was used to depolymerizenucleic acids. Three fractions were prepared fromthe extracts by ammonium sulfate precipitation.One contained protein precipitating at 0.3 satura-tion (S-1), and a second contained proteinprecipitating between 0.3 and 0.6 saturation (S-2).In a few instances, all protein precipitating at 0.6saturation was used and, since this precipitatepresumably included all of the protein found inthe other two fractions, it was designated(S-1:S-2).The fractions were dialyzed against distilled

water, lyophilized, and stored at 5 C in vacuumdesiccators over Drierite.

Protein determination. Protein was estimated byultraviolet light absorption with the method ofLayne (1957).

Precipitin test. The capillary tube precipitinmethod of Swift, Wilson, and Lancefield (1943)was used.

Hemagglutination. The technique of Stavitsky(1954a) was used with the following modifications.

Sheep erythrocytes were treated with 1:40,000tannic acid for 10 min at 25 C rather than at37 C. Sensitization of tanned cells was done atpH 7.2 at a temperature of 25 C for 20 min, 1volume of 2.5% erythrocytes (v/v) being mixedwith 1 volume of antigen. After sensitization andwashing, the cells were resuspended to a final0.5% concentration. For the final test, 0.3 ml of0.5% sensitized cells was added to 0.3 ml of eachantiserum dilution.The activity of each antigen was determined by

sensitizing erythrocytes with decreasing amountsof protein and testing them against a standardhomologous, unadsorbed antiserum diluted1:2,560. The least amount of antigen giving 2+hemagglutination was considered to be one unit.Two units were employed to sensitize cells to beused for the titration of antisera. Antisera werediluted in fourfold steps in 1:100 normal rabbitserum.

Immunodiffusion. Petri dishes (inner diameter,50 mm) were leveled and filled with 7.0 ml of 0.8%Ionagar No. 2 (Oxoid) containing 1.0% sodiumazide, the latter functioning both as preservativeand buffer (Feinberg, 1957). Wells were punchedon 12-mm centers with a no. 3 cork borer andlined with agar. Antigens were employed at aconcentration of 1.0 mg of protein per ml inbuffered saline. Antiserum was undiluted. Ob-servations were made after 12 days of incubationat 37 C in closed moist chambers. Bands weredesignated numerically, beginning with the bandfarthest from the antigen reservoir. After reading,each plate was filled with buffered saline andallowed to stand at room temperature throughfour daily changes of buffered saline. The agar wasthen partially dried at 37 C for 4.5 hr, transferredto a glass slide, and thoroughly dried at 37 C. Theprecipitin bands were stained for 15 min withamidoschwarz (Crowle, 1961), the backgroundthen being cleared with 2.0% acetic acid.

Starch-block electrophoresis. The method em-ployed for zone electrophoresis in starch pastewas based on that described by Kunkel andSlater (1952). Potato starch (E. H. Sargent andCo., Chicago, Ill.) was prepared for use by treat-ing 1,700 g successively with 3,800 ml of 20%,2,200 ml of 10%, and 2,100 ml of 5% ethyl al-cohol, allowing the bulk of the starch to settleduring each washing. After four washes with2,000-ml portions of distilled water, a final thick

913

on October 2, 2017 by guest

http://jb.asm.org/

Dow

nloaded from

Page 3: JOURNAL OF BACTERIOLOGY Vol. No. American Printed U.S.A ... · JOURNAL OF BACTERIOLOGY Vol. 88, No.4, p. 912-921 October, 1964 Copyright a1964 AmericanSocietyfor Microbiology Printedin

J. BACTERIOL.

slurry was made in borate-sulfate buffer. Theslurries were usable for 10 days if kept in the cold.

Borate-sulfate buffer (pH 8.6; 0.1 M) wasprepared by mixing 1,200 ml of 0.5 M boric acid,160 ml of 1 N NaOH, 150 ml of 1 M Na2SO4, and4,400 ml of distilled water, adjusting the pH to8.6 if necessary with additional 1 N NaOH andadding distilled water to a final volume of 6 liters.The apparatus for electrophoresis was con-

structed of Lucite according to a design by GeraldS. Berenson of the Department of Medicine,Louisiana State University School of Medicine.The starch bed was 1.2 cm deep, 10 cm wide, and25 cm long. Each sample was taken up in an ap-propriate weight of clean, dry starch and insertedinto a trough (1 by 7 cm) cut normal to the longaxis of the bed and midway between the two endsand the sides. The buffer reservoirs were eachfilled with 1,500 ml of borate-sulfate buffer.Chambers for circulation of coolant were pro-vided above and below the bed, through whichrefrigerated water was circulated during each runso that the starch temperature was maintainedat 13 C or lower. Voltage gradients varied be-tween 3.4 to 3.7 v/cm, and were maintained for17 hr with an input of 150 v from a constantvoltage power source. Current ranged from 22 to32 ma in various runs. After electrophoresis, frac-tions were displaced from segments (1 by 10 cm)

TABLE 1. Serological activity of typefraction S-1:S-2 in hemagglutii

and precipitin tests*

Antiserum

Serologicaltype of

Rabbit no. iununnz-ing

strepto-coccus

436 12447 3766 4771 5764 616 14

775 1873 36

Precipitin test

++

+

* Antigen concentration in precipil70 ,ug of protein per ml. Antigen conce

sensitization of erythrocytes was 2 ugper ml.

t Highest serum dilution tested.

of starch through sintered-glass funnels with two5.0-ml volumes of saline buffered at pH 7.2 with0.025 M phosphate. The average protein recoverywas 63 %.

Starch-gel electrophores. Starch-gel electro-phoresis was conducted in a vertical apparatussimilar to that described by Smithies (1959), withthe use of the discontinuous system of buffers ofPoulik (1957). The gels were prepared fromhydrolyzed starch (Connaught Medical ResearchLaboratories, Toronto, Ontario, Canada). Elec-trophoresis was performed at room temperaturefor 7 hr in a voltage gradient of 3.7 v/cm and acurrent density at 30 ma.

After each run, the gel (18 by 12.8 by 0.6 cm)was cut into two sections (18 by 12.8 by 0.3 cm)which were stained for 15 min with saturatedamidoschwarz (Smithies, 1955). The gels werewashed twice with methyl alcohol-glacial aceticacid (9:1), allowed to stand overnight in a thirdapplication of this wash, and then washed oncewith distilled water-glacial acetic acid (9.5:1).After maximal pattern development, the gelswere cleared by heating in glycerol at 70 to 80 Cfor 15 min. Such gels, when transferred to freshglycerol, could be stored for months without ap-preciable change in patterns.

RESULTS

Properties of partially purified type 12 M pro-12 (Baitey) tein. Several lots of the various fractions weretation made from the type 12 strain. In a typical prep-

aration, 127 g (wet weight) of cells yielded 38 mgof S-1 and 155 mg of S-2. The 280:260 m,u ultra-violet absorption ratios of all of the preparations

HA titer ranged from 0.69 to 1.07, lower than the ratios re-(reciprocal) ported by Lancefield and Perlmann (1952) and by

Barkulis and Jones (1957) for type 1 and type 14proteins, suggesting incomplete removal of

2,560t nucleic acids during purification of the type-1240 antigen. The electrophoretic data also indicated40 this.160 The fractions exhibited good serological ac-160 tivity, as few as 5 ,ug of protein per ml giving

2,560 visible capillary tube precipitation with ho-160 mologous antisera. They precipitated with homol-160 ogous and heterologous unadsorbed antisera

tin test was in Ouchterlony plates with the formation of mul-bntration for tiple bands, as many as four being seen occasion-g of protein ally with the S-1:S-2 preparations, even in the

heterologous reactions. Strong cross-reactivitywas observed in hemagglutination tests. Table 1

914 PIERCE

on October 2, 2017 by guest

http://jb.asm.org/

Dow

nloaded from

Page 4: JOURNAL OF BACTERIOLOGY Vol. No. American Printed U.S.A ... · JOURNAL OF BACTERIOLOGY Vol. 88, No.4, p. 912-921 October, 1964 Copyright a1964 AmericanSocietyfor Microbiology Printedin

\-OL. 88, 1964 SEPARATION OF STlREP'TOCOCCAL M PItOTEIN COMPONENTS

shows the results obtained when fraction S-1 :S-2was tested by these two techniques with unad-sorbed antisera. A solution containing 70 ,ug ofprotein per ml was only slightly cross-reactive inthe precipitin test, whereas erythrocytes sensi-tized with a solution containing 2 ,ug of plroteinper ml exhibited marked cross-reactivity. Ad-sorbed typing sera which are type-specific inprecipitin tests exhibited cross-reactivity in thehemagglutination test (Table 2). The lattertechnique, therefore, provides a more rigoroustest of the type-specificity of antistreptococcalantisera, a conclusion implicit in the previous ob-servations of Denny and Thomas (1953) andHolmes (1958).The S-1 and S-2 fractions were examined by

starch-gel electrophoresis. Quantities of 350 to400 ,ug were placed in appropriate wells in the gel,and electrophoresis was carried out as described.Five components were found in S-1, whereas S-2formed two broad zones (Fig. 1). Component 1 ofS-2 was not always seen, and was usually indis-tinct, sometimes being obscured by the ratherdense track of stainable material that followedthe buffer front that moves anodically in thisbuffer system (Poulik, 1960). Occasionally, it ap-peared that component 2 of S-2 was composed ofthree segments, but in most runs it moved as anapparently single entity. S-2 did not contain sig-nificant amounts of protein having the samemobility as components 2 of S-1.

In immunodiffusion tests, all the antigens wereobserved to cross-react extensively with unad-sorbed sera. Cross-reactions were not seen withadsorbed typing sera. Figure 2 illustrates the re-actions of an S-1:S-2 preparation with homol-ogous and heterologous antisera. It can be seenthat each of the three heterologous unadsorbed

TABLE 2. Cross-reactivity of adsorbed typing serain hemtagglutination tests with type 12 (Bailey)

fraction S-1:S-2*

Antiserum type HA titer (reciprocal)

12 163,8403 1606 64014 16018 16036 160

* Antigen concentration for sensitization oferythrocytes was 8,ug of protein per ml.

s-ij III I11 54 3 2

0

S-2 U2

FIG. 1. Starch-gel elect?ophoi-esis patternstype 12 (Bailey) fractions S-1 and S-2.

+

of

#4312.

-43-

F1G. 2. Ouchterlony plate. Center well, type 12(Bailey) fraction S-1:S-2; wells no. 1 and no. 4,homologouis unadsorbed antiserum (rabbit no. 438);well no. 2, type 36 unadsorbed antiserumn (rabbitno. 444); well no. 3, type 18 unadsorbed antiserum(rabbit no. 774); well no. 5, type 3 unadsorbedZ anti-serum (rabbit no. 447); and well no. 6, homologoustyping antisertumtt.

antisera precipitated with the antigen and, in thecase of the type-36 antiserum, four zones of pre-cipitation developed. Zone 2 (with reference tothe homologous unadsorbed antiserum) repre-sents a component of the Bailey fraction whichshares reactive groupings with componentspresent in group A streptococci of types 3, 18, and36, since it precipitated with antisera preparedagainst these types. Only the adsorbed ho-mologous typing antiserum did not react withthis component. The precipitate formed by thetyping antiserum presumably represents the reac-tion between the type-specific component ofS-1:S-2 and its antibody. An antibody with thisspecificity was also present in the homologous

911'a

on October 2, 2017 by guest

http://jb.asm.org/

Dow

nloaded from

Page 5: JOURNAL OF BACTERIOLOGY Vol. No. American Printed U.S.A ... · JOURNAL OF BACTERIOLOGY Vol. 88, No.4, p. 912-921 October, 1964 Copyright a1964 AmericanSocietyfor Microbiology Printedin

J. BACTERIOL.

unadsorbed antiserum. The antisera to strepto-coccal types 3, 18, and 36 did not precipitate withthe type-specific component of the antigen.

Figure 3 shows that antisera to streptococcaltypes 3, 14, and 36 failed to precipitate with thespecific component of S-1, although two of thesesera precipitated with other components of thefraction. Similar results were obtained with frac-tion S-2.No cross-reactions were seen between the type-

specific component of theM protein preparationsand any of the heterologous unadsorbed antiseratested. It can be concluded that cross-reactionsoften involve the nontype-specific components ofM protein preparations and that these compo-nents are distinct from the specific component. Itis possible that in certain instances the specificantigens of two group A strains of differing typemight contain one or more identical determinantgroups, and therefore would be expected to ex-

hibit cross-reactivity in immunodiffusion tests.In Fig. 4, S-1 and S-2 fractions are compared

in terms of their reactivity with homologous un-adsorbed and adsorbed antisera. At least threeantigenic components were found in S-1 whenunadsorbed antiserum was used. Component 2appeared to be type-specific, although it should

FIG. 3. Ouchterlony plate. Center well, type 12(Bailey) fraction S-1; wells no. 1, 8, and 6, homolo-gous typing antiserum; well no. 2, type 86 unad-sorbed antiserum (rabbit no. 44); well no. 4, type 14unadsorbed antiserum (rabbit no. 482); well no. 6,type unadsorbed antiserum (rabbit no. 447).

S1S2

I2.~~~~~~~~~~~FIG. 4. Ouchterlony plate. Center well, homolo-

gous unadsorbed antiserum (rabbit no. 8); peripheralwells, numbered clockwise: no. 1 and 4, homologoustyping serum; no. 2, type 12 (Bailey) fraction S-1;no. 8, type 12 (Bailey) fraction S-2.

300-

E 200

00 I

+-* a + + +- - - *PRECIPTATION WIH H0M0LOO UADSORUEO AITSERUM

1.3 120? 0.7 0 OS oJ 0.7 065Os6s0.m0.280/260 ULTRAVIOLET ASOAPTION 6a0

-1i -s -6 -4 -2 0 42 44 +6 +6 +10 +2

FIG. 5. Starch-paste electrophoresis of type 12(Bailey) fraction S-2. See text for details.

be noted that one lot of homologous adsorbedantiserum precipitated with component 3 as wellas with component 2. The specific antigen of S-2was shown to be component 1, which gave reac-tions of identity with antigens 1 and 2 of S-1.Component 1 of S-1 was probably present also inS-2, although its precipitate was obscured by thezone of precipitation formed by the specific com-ponent. Components 3 of S-1 and 2 of S-2 showeda reaction of identity. This antigen did not pre-cipitate with the typing antiserum, and thereforewas not the specific antigen. It was observed inother tests, not shown, to cross-react with het-erologous u.nadsorbed antisera.

Starch-block electrophoresis. Data from a repre-sentative starch-block electrophoresis experi-ment are shown in Fig. 5. The S-1 and S-2fractions gave similar patterns. The S-1:S-2

IE.mEm --=

916 PIERCE

500

400

on October 2, 2017 by guest

http://jb.asm.org/

Dow

nloaded from

Page 6: JOURNAL OF BACTERIOLOGY Vol. No. American Printed U.S.A ... · JOURNAL OF BACTERIOLOGY Vol. 88, No.4, p. 912-921 October, 1964 Copyright a1964 AmericanSocietyfor Microbiology Printedin

VOL. 88, 1964 SEPARATION OF STREPTOCOCCAL M PROTEIN COMPONENTS

preparations were not examined under theseconditions. In this experiment, 30 mg of S-2 weresubjected to electrophoresis as described. Elutedfractions were tested for serological activity bythe capillary precipitin method, and proteinconcentrations were determined. It can be seenthat a major peak of protein moved a short dis-tance toward the cathode. Smaller amounts ofprotein were found at the origin and near theanode end of the block. It was suspected that aportion of the antigen might have migrated intothe anodic buffer reservoir, but a shorter run (6hr) failed to reveal additional peaks. It can beseen that the 280:260 ultraviolet absorptionratios of the peak and of the material foundnearer the anode differed. Since initially the280:260 ratio of the antigen was 1.1, it can beassumed that one of the effects of starch-pasteelectrophoresis is to separate antigenic proteinfrom nucleic acids, the latter migrating towardthe anode. Pooled fractions -6 and -5 had a280:260 ratio of 1.25, which is similar to theratios reported by Lancefield and Perlmann(1952) and by Barkulis and Jones (1957).

TABLE 3. Hemagglutination tests on type 12(Bailey) fraction S-2 and pooled fractions

eluted from starch paste afterelectrophoresis*

Rabbit no.

437449762754774445437449762754774445437449762754774445

Antiserumtype

123614183612361418361236141836

* Protein concentrations used

HA titer reciprocal

40,9602,56010,24010,240

6402,560

40,9602,56010,24010,240

6402,56010,240

10<104010

<10

to sensitize

0fS-2.HP.

s-2

0

©FIG. 6. Ouchterlony plate. Center well, homolo-

gous unadsorbed antiserum (rabbit no. 8); well no. 1,type 12 (Bailey) fraction S-2; well no. 2, type 12(Bailey) fraction S-I; well no. 3, pooled type 12(Bailey) S-i electrophoretic fractions with low280:260 absorption ratios; well no. 4, pooled type 12(Bailey) S-i electrophoretic fractions with high280:260 absorption ratios; well no. 5, pooled type 12(Bailey) S-2 electrophoretic fractions with low280:260 absorption ratios; well no. 6, pooled type 12(Bailey) S-2 electrophoretic fractions with high280:260 absorption ratios.

The fractions eluted from a given starch blockwere pooled so that fractions -6 and -5, withhigh 280:260 ratios, were combined into one pool(HR); and fractions -4, -3, -2, -1, and 0,with low ratios, were combined into another(LR). When necessary, the pools were concen-trated with Carbowax 20-M (Giblett and Brooks,1962). Hemagglutination tests on the pooled frac-tions indicated that a relatively large amount ofthe LR antigen was required for sensitization oferythrocytes; however, it exhibited improvedspecificity (Table 3). The reactions of S-2 and itscorresponding HR antigen were identical in thisexperiment.The pooled material from several starch-paste

electrophoresis runs and the antigens used tocharge the starch blocks were compared in im-munodiffusion tests. Results of one such test areshown in Fig. 6. It can be seen that components 1and 2 of S-1 converged toward, and gave reactionsof identity with, component 1 of S-2. S-2 pro-

Antigen

S-2

HR

LR

erythrocytes: S-2, 4,g/ml; HR, 8 ,ug/ml; LR, 128,ug/ml. Meanings of the symbols are given in thetext.

917

on October 2, 2017 by guest

http://jb.asm.org/

Dow

nloaded from

Page 7: JOURNAL OF BACTERIOLOGY Vol. No. American Printed U.S.A ... · JOURNAL OF BACTERIOLOGY Vol. 88, No.4, p. 912-921 October, 1964 Copyright a1964 AmericanSocietyfor Microbiology Printedin

J. BACTERIOL.

duced the same )attern as did the HR (peak)pooled material from an S-2 starch block. Datafrom other tests showed that S-1 likewise gave apattern identical to that of the corresponding HR(peak) pooled fraction from an S-1 starch block.Thus, in immunodiffusion tests, the antigen in thestarch-block peaks closely resembled the startingmaterial. The band of precipitate produced byS-2 (component 1) sep)arated into two zones inthe LR pool from an S-2 starch block, indicatingthat component 1 probably consisted of twoantigen-antibody reactions superimposed, aspreviously suggested. These formed separateprecipitates when the LR 1)ool was tested, prob-ably because of their changed relative concentra-tions in the pool. Component 2 of S-2 (LR), S-1(HR), S-1 (LR), and S-1 was apparently thetype-specific component (see Fig. 4). Component2 of S-2 and S-2 HR (component 3 of S-1 and S-1HR) was not seen in the reactions between theantiserum and either of the LR pools. It is tempt-ing to speculate that this component was the one

s-I

s-I

Ht.R

S-2

S-2

FIG. 7. Ouchterlony plate. Center well, homolo-gous typing antiserum; well no. 1, type 12 (Bailey)fraction S-i; well no. 2, pooled type 12 (Bailey) S-ielectrophoretic fractions with high 280:260 absorp-tion ratios; well no. 3, pooled type 12 (Bailey) S-2electrophoretic fractions with high 280:260 absorp-tion ratios; well no. 4, type 12 (Bailey) fraction S-2;well no. 5, pooled type 12 (Bailey) S-2 electrophoreticfractions with low 280:260 absorption ratios; wellno. 6, pooled type 12 (Bailey) S-i electrophoreticfractions with low 280:260 absorption ratios.

primarily responsible for the cross-reactions ob-served in hemagglutionation tests. As previouslyindicated, this component was found to cross-react with heterologous unadsorbed sera in im-munodiffusion tests.The absence of antigen 2 of S-2 and S-2 HR,

and antigen 3 of S-1 and S-1 HR from the LRpools, might be ascribed to its loss throughthe dialyzing membrane during concentrationwith Carbowax. Control experiments indicatedthat this was not the case.

In Fig. 7, the reactions of S-1, S-2, and theirrespective starch-block HR and LR pools withhomologous typing serum are shown. Type-specific antigen was present in all of the fractionsand pools, in highest concentration in S-2, inlower concentration in S-1, and in lowest con-centration in S-1 LR.

DISCUSSIONrThat partially l)urified MI protein is anti-

genically complex is evident from several earlierstudies (Lancefield, 1928b; Evans, 1946; Fleck,1956; Rantz, Randall, and Zuckerman, 1956;Schmidt, 1957; Kaplan, 1963). Wilson and Wiley(1963) demonstrated by immunoelectrophoresisin agar that M extracts of a type 17 strain of S.pyogenes, prepared according to the method ofLancefield (1928a), contain at least four anti-genic components, three of which appear to beserotype-specific or serotype-associated.Of the various methods that have been utilized

for the removal of 1I protein from the cell, therecently developed procedure involving the useof bacteriophage-associated lysin (Krause, 1958;Kantor and Cole, 1960) probably releases M pro-tein into solution from washed cell walls in itsmost nearly native configuration. However, afterfurther steps in purification, these preparationsstill contain other components, such as the Tprotein and the group-specific polysaccharide. Ifthe latter is linked in some fashion to M proteinin its native state, its presence in these prepara-tions could be an indication of the extraction in-tact of a larger, more complex molecule, possiblyequivalent to the "labile antigen" of Mudd et al.(1938). On the other hand, the rigorous conditionsof extraction in the procedure of Lancefield andPerlmann (1952) serve to eliminate the T protein,and further steps in the method usually removeall serologically detectable group-specific poly-saccharide. The protein obtained by this method

918 PIERCE

on October 2, 2017 by guest

http://jb.asm.org/

Dow

nloaded from

Page 8: JOURNAL OF BACTERIOLOGY Vol. No. American Printed U.S.A ... · JOURNAL OF BACTERIOLOGY Vol. 88, No.4, p. 912-921 October, 1964 Copyright a1964 AmericanSocietyfor Microbiology Printedin

\tOL. 88, 1964 SEPARATION OF STREPTOCOCCAL M PROTEIN COMPONENTS

retains many of its original properties, precipitat-ing with antibody and functioning as a completeantigen (Hayashi and Walsh, 1961; Wolfe et al.,1963). Evidence has been obtained in this lab-oratory (Domingue and Pierce, 1964) that Mprotein extracted and purified by the Lancefieldand Perlmann (1952) method, when adsorbed toglossy homologous cells, does not increase the re-sistance of these cells to phagocytosis by rabbitperitoneal exudative leukocytes. However, lackof resistance in this instance could be ascribed toone or more other, unknown deficiencies in theglossy cells which develop at the time of mutationto the glossy phase. Even though M protein ad-sorbed to such cells might retain its biologicalactivity, this could be obscured by a lack of theother essential factor(s) in such cells.

Passive hemagglutination is a convenient tech-nique which is said to be able to detect as littleas 0.001 pg of antibody to egg albumen or diph-theria toxoid (Stavitsky, 1954b). Its verysensitivity, however, is a liability if the antigenemployed is not of the highest purity, since anti-bodies specific for contaminants in the antigenwill also be readily detected. If a completelytype-specific streptococcal antigen could beobtained, it would greatly facilitate the perform-ance of this test. In the present study, an antigenof improved specificity has been demonstrated.However, practical considerations render theoutlook unfavorable for its useful employment inhemagglutination tests for type-specific antibody.The chemical and physical procedures requiredfor its purification are sormewhat complex and theyields are small. In a typical starch electropho-retic run, the yield of specific antigen was 0.67 mg,where the block had been loaded initially with 11mg of partially purified antigen, representing theyield from 7.2 g (wet weight) of cells obtainedfrom approximately 5.7 liters of culture. Consid-ering that as much as 128 mg of protein per mlmay be required in solutions to be used to sensi-tize erythrocytes, this yield is not satisfactory.Furthermore, in surveys designed to detect anti-body responses to several streptococcal types, allof these difficulties would be compounded. It ispossible that a less highly purified antigen couldbe used as the starting material in starch-pasteelectrophoresis, and that different conditions ofpH and ionic strength, or other modifications inthe electrophoretic procedure, might afford higheryields.

ACKNOWLEDGMENTS

This investigation was supported in part byPublic Health Service grant E-1263 from theNational Institute of Allergy and Infectious Dis-eases.The author wishes to express his appreciation

for the helpful technical assistance of John N.Theall and Don R. Saucier.

LITERATURE CITED

BARKULIS, S. S., AND M. F. JONES. 1957. Studiesof streptococcal cell walls. I. Isolation, chemi-cal composition, and preparation of M pro-teins. J. Bacteriol. 74:207-216.

BONE, M., A. I. BRAUDE, AND H. KLEINMAN. 1957.Complement-fixing antibody response toM-protein of nephritogenic streptococci inglomerulonephritis. J. Lab. Clin. Med. 50:705-711.

BOYDEN, S. V. 1951. The adsorption of proteins onerythrocytes treated with tannic acid andsubsequent hemagglutination by antiproteinsera. J. Exptl. Med. 93:107-120.

CROWNrLE, A. J. 1961. Immunodiffusion, p. 304.Academic Press, Inc., New York.

DENNY, F. W., JR., AND L. THOMAS. 1953. Thedemonstration of type specific streptococcalantibody by a hemagglutination techniqueemploying tannic acid. J. Clin. Invest. 32:1085-1093.

DOMINGUE, G. J., AND W. A. PIERCE, JR. 1964.Effect of partially purified streptococcal Mprotein on in vitro phagocytosis of glossystreptococci. Bacteriol. Proc., p. 50.

EVANS, A. C. 1946. Protection between strains ofheterologous agglutinogenic type of betahemolytic streptococci of group A. J. Im-munol. 52:1-15.

FEINBERG, J. G. 1957. Identification, discrimina-tion and quantification in Ouchterlony gelplates. Intern. Arch. Allergy Appl. Immunol.11:129-152.

FLECK, D. G. 1956. Mouse protection and enhance-ment of phagocytosis by antisera to Strepto-coccus pyogenes. Brit. J. Exptl. Pathol. 37:406-422.

GIBLETT, E. R., AND L. E. BROOKS. 1962. A simpleand practical method for concentrating bloodgroup antibodies. Transfusion 2:261-264.

HAYASHI, J. A., AND G. WALSH. 1961. Studies ofstreptococcal cell walls. VI. Effects of adju-vants on the production of type-specific anti-bodies to cell walls and isolated M proteins.J. Bacteriol. 82:736-742.

HOLMES, M. C. 1958. Type specific antibodies to

919

on October 2, 2017 by guest

http://jb.asm.org/

Dow

nloaded from

Page 9: JOURNAL OF BACTERIOLOGY Vol. No. American Printed U.S.A ... · JOURNAL OF BACTERIOLOGY Vol. 88, No.4, p. 912-921 October, 1964 Copyright a1964 AmericanSocietyfor Microbiology Printedin

J. BACTERIOL.

Streptococcus pyogenes. Australian J. Exptl.Biol. 36:395-404.

KANTOR, F. S., AND R. M. COLE. 1960. Preparationand antigenicity of M protein released fromgroup A, type 1 streptococcal cell walls byphage-associated lysin. J. Exptl. Med. 112:77-96.

KAPLAN, M. H. 1963. Immunologic relation ofstreptococcal and tissue antigens. I. Proper-ties of an antigen in certain strains of group Astreptococci exhibiting an immunologic cross-reaction with human heart tissue. J. Immunol.90:595-606.

KRAUSE, R. M. 1958. Studies on the bacteriophagesof hemolytic streptococci. II. Antigens re-leased from the streptococcal cell wall by aphage associated lysin. J. Exptl. Med. 108:803-821.

KUNKEL, H. G., AND R. G. SLATER. 1952. Zoneelectrophoresis in a starch supporting me-dium. Proc. Soc. Exptl. Biol. Med. 80:42-47.

LANCEFIELD, R. C. 1928a. The antigenic complexof Streptococcus haemolyticus. I. Demonstra-tion of a type-specific substance in extracts ofStreptococcus haemolyticus. J. Exptl. Med.47:91-103.

LANCEFIELD, R. C. 1928b. The antigenic complexof Streptococcus haemolyticus. III. Chemicaland immunological properties of the proteinfractions. J. Exptl. Med. 47:469-480.

LANCEFIELD, R. C., AND G. E. PERLMANN. 1952.Preparation and properties of type-specificM antigen isolated from a group A, type 1hemolytic streptococcus. J. Exptl. Med. 96:71-82.

LANCEFJELD, R. C., AND E. W. TODD. 1928. Anti-genic differences between matt hemolyticstreptococci and their glossy variants. J.Exptl. Med. 48:769-790.

LAYNE, E. 1957. Spectrophotometric and turbidi-metric methods for measuring proteins, p.447. In S. P. Colowick and N. 0. Kaplan [ed.],Methods in enzymology, vol. 3. AcademicPress, Inc., New York.

LIAN, T. S., AND W. A. PIERCE, JR. 1961. A studyof bentonite flocculation test for measurementof type-specific streptococcal antibodies.Proc. Soc. Exptl. Biol. Med. 106:207-211.

MUDD, S., E. J. CZARNETSKY, D. LACKMAN, ANDH. PETTIT. 1938. The antigenic structure ofhemolytic streptococci of Lancefield group A.I. The preparation of a labile, type-specificantigen; its identification as the Griffith type-specific agglutinogen and as a substance fromwhich a group-specific and a type-specifichapten are derivable. J. Immunol. 34:117-153.

POUL1K, M. D. 1957. Starch gel electrophoresis ina discontinuous system of buffers. Nature180:1477-1479.

POULIK, M. D. 1960. In L. A. Lewis [ed.], Electro-phoresis in physiology. Charles C Thomas,Publisher, Springfield, Ill.

QUINN, R. W., AND N. P. LOWRY. 1963. Strepto-coccal M protein antibodies. J. Infect. Diseases 113:33-38.

RANTZ, L. A., E. RANDALL, AND A. ZUCKERMAN.1956. Hemolysis and hemagglutination bynormal and immune serums of erythrocytestreated with a nonspecies specific bacterialsubstance. J. Infect. Diseases 98:211-222.

ROTHBARD, S. 1945. Bacteriostatic effect of humansera on group A streptococci. I. Type-specificantibodies in sera of patients convalescentfrom group A streptococcal pharyngitis. J.Exptl. Med. 82:93-106.

ROTHBARD, S., AND R. F. WATSON. 1948. Variationoccurring in group A streptococci during hu-man infection. Progressive loss of M sub-stance correlated with increasing suscepti-bility to bacteriostasis. J. Exptl. Med. 87:521-533.

SCHMIDT, W. C. 1957. The quantitative precipitinreaction of type 19M protein antigen of groupA streptococci and antistreptococcal rabbitsera. J. Immunol. 78:178-184.

SMITHIES, 0. 1955. Zone electrophoresis in starchgels: variations in the serum proteins of nor-mal human adults. Biochem. J. 61:629-641.

SMITHIES, 0. 1959. An improved procedure forstarch gel electrophoresis: further variationsin the serum proteins of normal individuals.Biochem. J. 71:585-587.

STAVITSKY, A. B. 1954a. Micromethods for thestudy of proteins and antibodies. I. Procedureand general applications of hemagglutinationand hemagglutination-inhibition reactionswith tannic acid and protein-treated red bloodcells. J. Immunol. 72:360-375.

STAVITSKY, A. B. 1954b. Micromethods for thestudy of proteins and antibodies. II. Specificapplications of hemagglutination and hemag-glutination-inhibition reactions with tannicacid and protein-treated red blood cells. J.Immunol. 72:368-375.

STOLLERMAN, G. H., AND R. EKSTEDT. 1957. Longchain formation by strains of group A strepto-cocci in the presence of homologous anti-serum: a type-specific reaction. J. Exptl. Med.106 :345-356.

SWIFT, H. F., A. T. WILSON, AND R. C. LANCE-FIELD. 1943. Typing group A hemolytic strep-tococci by M precipitin reactions in capillarypipettes. J. Exptl. Med. 78:127-133.

920 PIERCE

on October 2, 2017 by guest

http://jb.asm.org/

Dow

nloaded from

Page 10: JOURNAL OF BACTERIOLOGY Vol. No. American Printed U.S.A ... · JOURNAL OF BACTERIOLOGY Vol. 88, No.4, p. 912-921 October, 1964 Copyright a1964 AmericanSocietyfor Microbiology Printedin

VOL. 88, 1964 SEPARATION OF STREPTOCOCCAL M PROTEIN COMPONENTS

WADSWORTH, A. B. 1947. Standard methods of thedivision of laboratories and research of theNew York State Department of Health, 3rded. The Williams & Wilkins Co., Baltimore.

WILSON, A. T., AND G. G. WILEY. 1963. The cellu-lar antigens of group A streptococci. Immuno-electrophoretic studies of the C, M, T, PGP,

E4 , F, and E antigens of serotype 17 strepto-cocci. J. Exptl. Med. 118:527-556.

WOLFE, C. K., JR., J. A. HAYASHI, G. WALSH, ANDS. S. BARKULIS. 1963. Type-specific antibodyresponse in man to injection of cell walls andM protein from group A, type 14 streptococci.J. Lab. Clin. Med. 61:459-468.

921

on October 2, 2017 by guest

http://jb.asm.org/

Dow

nloaded from