INFECTION AND IMMUNITY, Aug. 1976, p. 579-585Copyright © 1976 American Society for Microbiology

Vol. 14, No. 2Printed in U.S.A.

Two Previously Undescribed Extracellular StreptococcalAntigens Detectable with Naturally Occurring Human

AntibodiesD. KIEFER AND S. P. HALBERT*

Department ofPediatrics, University of Miami School of Medicine, Miami, Florida 33152

Received for publication 4 February 1976

Two streptococcal extracellular antigens (X and Y), which are detectable withnaturally occurring human antibodies, have been isolated and shown to repre-

sent entities distinct from previously described streptococcal enzymes, toxins,and antigens. They are each synthesized by both group C and group A strepto-cocci and appear to be proteins. Both antigens were found to be nonhemolyticand nontoxic for human leucocytes as well as for isolated beating newborn ratheart cells in tissue culture. In addition to being distinct from known streptococ-cal enzymes, they were also shown to be devoid of several other enzymaticactivities.

During naturally occurring human strepto-coccal infections, surprisingly large numbers ofextracellular antigens evoke antibody re-sponses that are readily detectable by variousimmunodiffusion methods (10, 11, 17, 19). Inearlier papers, many of the antigens thus visu-alized with human antibodies in group A strep-tococcal extracellular concentrates were iso-lated and studied (12-16). More recently, theantigens similarly detected in group C strepto-coccal concentrates were systematically puri-fied (21). Of the 12 group C antigens revealedwith human antibodies, 5 were obtained in arelatively high degree of purity. One of thelatter was identified as streptokinase. Two oth-ers were tentatively designated X and Y; thesewere also found in group A streptococcal con-centrates. Preliminary data suggested that theX and Y antigens were distinct from previouslydescribed group A or group C streptococcal anti-genic products. It is the purpose of the presentreport to elaborate on the evidence which indi-cates that the X and Y proteins are unrelated toknown streptococcal antigens, and to describean additional method for their separation.

MATERIALS AND METHODSThe group C streptococcal extracellular culture

concentrates were obtained from the H46A strain(we are deeply indebted to G. Schwick, ResearchDirector of the Behringwerke Co., for the very gen-erous donation of these preparations and the de-tailed information regarding their production). Thegrowth conditions and harvest of the extracellularproteins were described earlier (21). Also detailedthere were the purification methods used for isolat-ing the X and Y antigens and the various immuno-

diffusion and analytic techniques employed with hu-man gamma globulin concentrates as antibodysource.

For further chromatographic separation of the Xand Y antigens, carboxymethyl cellulose (micro-granular, CM52, preswollen; Whatman, Clifton,N.J.) was used according to the instructions of themanufacturer. The final equilibration of the adsorb-ent was carried out against 0.001 M sodium phos-phate, pH 6.8, and the columns were packed at 40Cunder flow conditions. The antigen mixtures appliedto the columns represented the 0.2 M sodium phos-phate eluate from the hydroxylapatite chromato-graphic step, described in the previous report (21).Lyophilized powders of this fraction (1,500 mg) weredissolved in 60 ml of the equilibrating buffer, al-lowed to stand overnight at 40C, and clarified byhigh-speed centrifugation. After the solution wasapplied to the column, it was submitted to a sodiumphosphate gradient (pH 6.8) between 0.001 and 0.15M. The eluate fractions were analyzed for proteinconcentration by absorption at 280 nm, and for rela-tive salt concentration by conductivity bridge meas-urements (Yellow Spring Instrument Co., Inc., Yel-low Springs, Ohio). The fractions comprising eachprotein peak were pooled and harvested by precipi-tation through dialysis against saturated am-monium sulfate. The sediments were dissolved inminimal volumes of 0.15 M NaCl-0.01 M sodiumphosphate, pH 7.4, and stored at -20°C until used.To determine whether the purified X and Y anti-

gens represented known streptococcal enzymes, thefollowing assays were performed with solutions con-taining at least 0.1 mg of the most purified prepara-tions per ml, using procedures described by the au-thors cited: adenosine triphosphatase, Ginsburg etal. (6); glucosaminidase, Ginsburg et al. (6); nicotin-amide adenine dinucleotidase (NADase), Carlson etal. (2); deoxyribonuclease, McCarty (25); ribonucle-ase, Kunitz (23); hyaluronidase, Dorfman (4); pro-

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580 KIEFER AND HALBERT

TABLE 1. Tests ofpurified X and Y antigens for enzymes reported to be synthesized by group A orgroup C streptococci

Sp acta purified antigen/sp act crude Sp act purified antigen/sp act purifiedEnzyme concentrate enzyme

Antigen X Antigen Y Antigen X Antigen YDeoxyribonuclease 0.06b 0.5 0.0015 0.013Ribonuclease 1.4 1.2 0.000,02 0.000,02Hyaluronidase 0.02 0.02 NTc NTLipoproteinase Noned None NT NTAmylase 0.8 0.8 0.2e 0.2eProteinase None None NT NTStreptolysin 0 0.25 0.000,2 0.006 <0.000,02NADase 0.13 0.055 0.000,3 0.000,1Glucosaminidase None None NT NTAlkaline phosphatase None None NT NTGlucuronidase None None NT NTAcid phosphatase 0.4 None NT NTNeuraminidase None None NT NTStreptokinase 0.000,4 <0.000,4 0.000,03 <0.000,03

a Specific activity represents the number of units of enzyme activity per milligram of protein.b This ratio figure represents the equivalent of fold purification.c NT, Not tested; usually because of unavailability of purified enzyme.d None, No significant activity found.e In relation to a crude malt diastase preparation.

teinase, Liu et al. (24); lipoproteinase, Krumwiede(22); amylase, Crowley (3); esterase, Hardin et al.(18); glucuronidase, Fishman and Bernfeld (5); acidand alkaline phosphatase, Bessey et al. (1); andneuraminidase, Warren (29).

In addition, the immune precipitates represent-ing the isolated X and Y systems were analyzed forcertain enzyme activities by histochemical methods.In these tests, sodium azide was omitted during thedevelopment of the precipitin patterns. Tests forcatalase were performed by flooding the dried slideswith 0.03% H202 and observing the evolution of gasbubbles, as described by Howe et al. (20). Attemptsto detect glucuronidase, acid and alkaline phospha-tase, nonspecific esterase, glucosaminidase, leucineaminopeptidase, aryl sulfatase, and chymotrypsinactivities in the immune precipitates were carriedout according to the techniques of Uriel (28) andRaunio (26).

Leucocyte toxicity tests of the X and Y antigenswere performed by direct microscope observations.For this purpose, 10 ml of normal adult human bloodwas collected by venipuncture into heparin andcentrifuged at 500 x g for 10 min. After aspiratingthe plasma, the buffy coat was harvested and singledrops were spread on microscope slides. These wereincubated in a moist chamber at 37°C for 30 min toallow the leucocytes to attach to the glass surface,and then they were rinsed gently with warm (37°C)Hanks solution to remove the bulk of the remainingerythrocytes. Lyophilized purified X and Y antigenswere dissolved at 0.1 mg/ml in Hanks solution, and1 drop of each was added to the leucocyte prepara-tions. Cover slips were then placed over each dropand sealed with melted Vaseline. As controls, plainHanks solution was added to parallel leucocyte prep-arations. In addition, as positive control, purified

reduced streptolysin 0 was used at 0.1 mg/ml (8,000hemolytic units/ml). The slides were incubated at370C in a moist chamber and were observed atintervals over a 24-h period using phase microscopy.

Tests for cardiotoxicity were carried out usingisolated rhythmically beating newborn rat heartcells growing in tissue culture (we are grateful to R.Bruderer for preparations of beating heart cells).These were grown for 3 days in Rose chambers, aspreviously reported (27). To each culture (1.5 ml ofmedium) was added 0.15 ml of X and Y antigensolutions (1 mg/ml) in complete growth medium.Parallel negative control cultures received an addi-tional 0.15 ml of complete growth medium alone.The X and Y antigens were subjected to trypsin

digestion at final concentrations in 0.15 M NaCl-0.01M sodium phosphate (pH 7.4) of 0.5 mg of antigenper ml and 0.5 mg of crystalline trypsin per ml(Worthington Biochemicals Corp., Freehold, N.J.)for 60 min at 37°C. The reaction was stopped byaddition of an equal volume of soybean trypsin in-hibitor at 0.5 mg/ml. The treated samples and ap-propriate controls were then checked by immuno-diffusion.

RESULTSWhen solutions of the purified X and Y anti-

gens were tested for the enzymes known to besynthesized by group A and group C strepto-cocci, in no case was any activity found at po-tencies high enough to suggest that the X andY antigens represented these enzymes in ahighly purified state. In all instances, the as-says were performed in parallel with the crudegroup C streptococcal concentrate, and thespecific activity ratios between them repre-

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PREVIOUSLY UNDESCRIBED STREPTOCOCCAL ANTIGENS 581

sented the degree of "purification" achieved. Inaddition, where available, preparations of par-tially or highly purified enzymes were also as-sayed simultaneously as an indication of theabsolute specific activities present. These re-sults are summarized in Table 1. It may be seenthat both the X and Y antigen preparationscontained small amounts of ribonuclease and

XAg.5

SK.3

deoxyribonuclease activity, but these were ofvery low potency in relation to the purifiedenzymes. The nucleases were found in the puri-fied antigens at essentially the same levels asin the crude concentrates. Although traces ofhyaluronidase were present in both X and Yantigens, this enzyme was there in only 1/50 ofthe concentration found in the crude concen-

XAg-5

F LSLO 1f08

HGG

NAD.2

*/

iHGG

DNB AgEx..03 .1

CCCb.03

/PP.02

A X.Ag.5

J} JAgEx.1

D

HGGI

2 K~-Iqmmm~~~~...s

YAg-5

FIG. 1. Immunodiffusion reactions of"nonidentity" between purifiedX (XAg) and Y (YAg) antigens andpreviously purifled streptococcal antigens. In all cases, normal pooled human gamma globulin (HGG) servedas antibody source in the central wells. The numbers refer to the concentrations of the antigens tested, inmilligrams per milliliter. SLO, Streptolysin 0, group A; DNB, deoxyribonuclease B, group A; NAD,nicotinamide adenine dinucleotidase, group A; SK, streptokinase, group C; PP, proteinase precursor, groupA; CCb, group A streptococcal cellular "C" carbohydrate; FL, 'far left" group A streptococcal extracellularantigen (unidentified, see reference 13); AgEx, "antigen excess" group A streptococcal extracellular antigens,one of which appears to be related to erythrogenic toxin.

C XAg-5

YAg-5

FLI CCb

.03

PP.02

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582 KIEFER AND HALBERT

trate. Amylase was also present in both anti-gens, but in amounts slightly lower than seenin the starting material. Small quantities ofstreptolysin 0 and NADase were found in the Xantigen (one-fourth and one-eighth that of thecrude), but only traces of these were present inthe Y antigen. None of the other enzymes testedwas significantly detectable in these prepara-tions bv the methods used. On the basis of thesefinding; it is thus most probable that neither ofthese antigens, which were highly purified byimmunological criteria, represents any of theenzymes studied. Although some of the en-zymes were present in X and Y at low concen-trations, they were usually in considerablylower concentrations than that found in thecrude concentrates.

Analysis of the X and Y immune precipitatesfor the enzymes assayed by histochemicalmethods were all negative: esterase, catalase,glucuronidase, acid and alkaline phosphatase,glucosaminidase, leucine-aminopeptidase, aryl-sulfatase and chymotrypsin. Except the non-specific esterase, which was found previouslyto be unrelated immunologically to X and Y,none of these enzymes was seen in the 12 im-mune precipitin systems revealed with thecrude group C streptococcal concentrate. In allcases, suitable positive controls were run inparallel, with several human tissue antigen-antibody patterns, e.g., liver, pancreas, sub-maxillary gland, etc. (7-9).Immunodiffusion comparisons of the X and Y

antigens were carried out against variousgroup A streptococcal extracellular antigensthat had been similarly detected and purifiedearlier (12, 13). These results are illustrated inFig. 1, where it may be seen that both X and Yantigens were immunologically unrelated tothe following group A products: proteinaseprecursor, deoxyribonuclease B (DNase B),NADase, streptolysin 0, C carbohydrate, "farleft" component (13), and the two "antigen ex-cess" antigens. It was shown previously thatone of the latter is probably related to erythro-genic toxin (13). In addition, the immunologicalnonidentity of X, Y, and the purified group Cstreptokinase described previously is also seen.The nonidentity of streptokinase with all of thepurified group A streptococcal antigens was ob-served in these and other plates (i.e., the noni-dentity of streptokinase and NADase is seen inFig. 1A).Both X and Y antigens proved to be nontoxic

to human leucocytes under the assay conditionsused. After exposure of such cells to these prep-arations at 0.1 mg/ml for 24 h, no cytotoxiceffects were observed, which distinguished theexposed cells from the negative controls that

PBS

XAgT.5

XAg.5

HGG

YAgT.5

YAg*5

PBS

FIG. 2. Destruction ofimmunological reactivity ofpurifiedX and Y antigens by treatment with trypsin.Numbers refer to antigen concentration in milli-grams per milliliter. HGG, Human gamma globulinas antibody source; X Ag, X antigen, untreated; YAg, Y antigen, untreated; X Ag T, X antigen, treatedwith trypsin; Y Ag T, Y antigen, treated with trypsin;PBS, phosphate-buffered saline, pH 7.2.

were treated only with medium. Since it wastheoretically possible that both X and Y anti-gens together might possess synergistic effects,as is the case for staphylococcal leucocidin (30),additional leucocytoxicity tests were carriedout in which mixtures of the X and Y antigenswere exposed to the leucocyte cultures. Notoxic effects were observed with the mixtures aswell.

In the case of toxicity for isolated beating ratheart cells in tissue culture, the Y antigenshowed no detectable effects. After addition ofthis antigen to the culture, the cells continuedto beat for 24 h at the normal rate of 85/minseen in the parallel negative control culturesthat received only complete growth medium.However, addition of the X antigen to the heartcultures did result in some beating irregulari-ties, which persisted for 24 h. Replacement ofthe culture fluid with fresh medium free ofantigen resulted in a normal beating ratewithin 1 day. It has been shown that similarcultures treated with purified streptolysin 0showed rapid killing of 80% of the cells within 5min at concentrations of 250 hemolytic units/ml(27). Since the antigen X fraction was contami-nated with a small amount of streptolysin 0(1,200 hemolytic units/mg), it seems reasonable

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PREVIOUSLY UNDESCRIBED STREPTOCOCCAL ANTIGENS 583

to assume that the minimal amount of cardi-otoxicity observed was due to this contaminant.Previous studies had indicated that streptoly-sin 0 is definitely toxic for the heart cells at 50to 100 hemolytic units/ml, and that only strep-tolysin 0 in the crude group C streptococcalconcentrate possesses such cardiotoxic proper-ties in this system (27). The complete lack oftoxicity of the purified Y antigen for the heartcells supports this conclusion, since the Y prep-aration contained only minute traces of strepto-lysin 0.That both X and Y antigens were proteins

was indicated by the destruction of their immu-nological reactivity on exposure to trypsin. Thisis clearly seen in Fig. 2, where pretreatment ofpurified X and Y antigens with trypsin abol-ished development of the precipitin bands inimmunodiffusion against the antibodies in hu-man gamma globulin.The purified X and Y antigens studied above

had been isolated by a sequence of steps involv-ing ammonium sulfate salting out, hydroxylap-atite chromatography, Sephadex G-100 gel fil-tration, and isoelectric focusing. It was subse-quently found that carboxymethyl cellulosechromatography was a more simple procedurefor separating these two proteins, using the

13.5

X 13.0z0

C 1.0

wz

0Cl)5

'-4

-C)., t.

fraction eluting with 0.2 M sodium phosphatefrom hydroxylapatite as the starting material(see reference 21). A typical elution patternusing a sodium phosphate gradient is shown inFig. 3. It may be seen that the antigen inhighest concentration in fraction H.2-CMV isdistinct from that eluting in fractions H.2-CMIIand H.2-CMIII. Other immunodiffusion testsdemonstrated that the single line in fractions IIand III represented antigen X, and that fractionV represented antigen Y in relatively purifiedstates. Based on previous data (21), it is proba-ble that an additional isoelectric focusing stepof each would result in preparations of consider-able purity.The absorption spectra of the most highly

purified X and Y antigens were studied be-tween 250 and 450 nm. Both showed spectratypical of simple proteins, with absorption max-ima at about 280 nm, and no evidence of otherabsorption peaks within this range. The 280/260(nm) ratios for the X and Y antigens were 1.06and 1.2, respectively.

DISCUSSION

Although the two streptococcal extracellularantigens studied here were derived from a

I-

-J0

vJI I I I I '

0 20 40 60 80TUBE

FIG. 3. Chromatographic separation of X and Y antigens on carboxymethyl cellulose, using a sodiumphosphate concentration gradient, pH 6.8. The single antigen seen with peaks HI and III was identified as X,whereas the considerably purified antigen ofpeak V was shown to be Y. The material applied to the columnwas the 0.2 M peak obtained from hydroxylapatite chromatography (see reference 21).

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584 KIEFER AND HALBERT

group C streptococcal strain, a previous reportdemonstrated that apparently identical anti-gens were also synthesized by group A strepto-cocci. It may be emphasized once again that theantigens studied here were all detected withhuman antibodies that had been evoked as re-sponses to naturally occurring infections. Sincethe majority of such infections are caused bygroup A streptococci, it is probable that the Xand Y antigens released by organisms of thislatter group had been primarily responsible forthe human antibodies present.The data presented in this paper document

the previous indications that the X and Y anti-gens are unrelated to other reported streptococ-cal antigens, enzymes, or toxins. Both X and Yappear to be proteins, as evidenced by the de-struction of their immunological reactivitiesupon exposure to trypsin and their ultravioletabsorption spectra, as well as their general be-havior during purification, i.e., with regard tosalting out, column chromatography on variousadsorbents, etc. In addition to being unrelatedto known streptococcal products, the X and Yantigens do not appear to represent certainother enzymes such as aryl-sulfatase, leucine-amino peptidase, etc. Although insufficientquantities were available to test their generaltoxicity for experimental animals, both anti-gens were devoid of appreciable cellular toxicityfor human leucocytes, or for rhythmically beat-ing newborn rat heart cells in tissue culture.Although the X antigen preparation used re-

vealed slight effects on the pulsations of theheart cell cultures, this could be entirely ac-

counted for by the small amount of contaminat-ing streptolysin 0 present. The latter toxin hasbeen clearly shown to have profound cardio-toxicity, which is evident at low concentrationsin this tissue culture system. Both X and Yantigens were found to be nonhemolytic forhuman erythrocytes.Although the functions of the X and Y anti-

gens have not yet been determined, numerousadditional tests aimed at clarifying their poten-tial role in the infectious process are feasible.For example, studies on their generalized toxic-ity, on the possible protection induced againstexperimental infection by active immunizationwith each, analysis of their skin toxicity, moreextensive study of their possible enzymaticproperties, quantitative assays of the antibodyresponses to X and Y after different poststrepto-coccal illnesses, etc., could be carried out whenmore adequate supplies of the purified antigensare isolated. It is hoped that such a systematicanalysis of the streptococcal products shown tobe released in vivo during human infectionsmay ultimately permit precise identification of

those factors that initiate the various nonsup-purative poststreptococcal complications.

ACKNOWLEDGMENT

These investigations were supported by a Public HealthService grant from the National Institute of Allergy andInfectious Diseases (no. AI10081).

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