INFECTION AND IMMUNITY, Dec. 1981, p. 915-9190019-9567/81/120915-05$02.00/0

Vol. 34, No. 3

Phage-Host Interactions and the Production of Type AStreptococcal Exotoxin in Group A Streptococci

LARRY McKANEt AND JOSEPH J. FERRETTI*

Department ofMicrobiology and Immunology, The University of Oklahoma Health Sciences Center,Oklahoma City, Oklahoma 73190

Received 13 April 1981/Accepted 10 August 81

The infection of Streptococcus pyogenes nontoxigenic strain T253 with bacte-riophage T12 to form lysogen T253 (T12) resulted in the production of type Astreptococcal exotoxin (erythrogenic toxin or streptococcal pyrogenic exotoxin).Two lines of evidence indicated that lysogeny per se was not sufficient to promotetoxigenic conversion of strain T253. First, a virulent mutant of phage T12, unableto form stable lysogens, was able to affect type A exotoxin production by strainT253. An unrelated virulent phage A25 did not affect type A exotoxin productionafter infection of strain T253. Second, the temperate phage H4489A, whichestablished stable lysogens with strain T253, did not promote type A exotoxinproduction. These results suggest that there is a strain specificity to the phage-host interaction which affects type A exotoxin synthesis. Additional evidence ispresented which indicates that type A streptococcal exotoxin was not a structuralcomponent of phage T12.

Type A streptococcal exotoxin (11), alsoknown as erythrogenic toxin, Dick toxin, andstreptococcal pyrogenic exotoxin, is elaboratedby certain strains ofStreptococcuspyogenes andis responsible for the rash that accompaniesscarlet fever. In 1964, Zabriskie (21) demon-strated that bacteriophages had a role in theproduction of type A streptococcal exotoxin; i.e.,the infection of a nonlysogenic strain T253 witha temperate bacteriophage, T12, obtained froma strain of streptococci known to be associatedwith scarlet fever, resulted in the formation of alysogen that produced type A streptococcal ex-otoxin. This observation has been confinred byNida et al. (17) and Johnson et al. (9). Prelimi-nary evidence has also been presented by Nidaet al. (17) that toxigenic conversion is not limitedto the T12 phage-T253 host system but can beaccomplished with a number of other phage-host strains.

In recent years, type A streptococcal exotoxinhas been purified and characterized (3, 7, 8, 10,15, 16); however, little work has been reportedconcerning the mechanism by which bacterio-phages affect the production of type A exotoxinby lysogenic streptococci. In this report, we pres-ent evidence that lysogeny itself was not suffi-cient to promote toxigenic conversion, and fur-thermore, we suggest that there is a specificityto the phage-host interaction which affects type

t Present address: Department of Biological Sciences, Cal-ifornia State Polytechnic University, Pomona CA 91768.

A exotoxin synthesis. Data are also presentedwhich indicate that type A streptococcal exo-toxin was not a structural component of bacte-riophage T12.

MATERIALS AND METHODSBacteria and bacteriophages. The primary

strains of S. pyogenes known to produce type A strep-tococcal exotoxin which were used in this study wereNY5 type 10 (6) and T253(T12) (21). Two strainsknown to produce no extraceilular type A exotoxinwere T253 and K56. Bacteriophage strains includedthe temperate phage T12, isolated from its originaltoxigenic donor (21), and phage H4489A, isolated froma nephritogenic strain of S. pyogenes. Virulent phagesincluded strain A25 (14) and T12cpl, a spontaneousmutant of phage T12 which formed clear plaques ona bacterial lawn of strain T253. Bacterial strain T253was relysogenized in this laboratory by phage T12 toconstruct strain T253(T12)B and by phage H4489A toproduce strain T253 (H4489A).

Media. Supplemented proteose peptone broth (19)was the standard liquid medium used for bacterialgrowth and phage propagation. The medium em-ployed for growing toxigenic strains when detection oftype A exotoxin was desired was a Todd-Hewitt dialy-sate medium prepared by dialyzing 100 ml of 10-foldconcentrated Todd-Hewitt broth (30 g/100 ml of dis-tilled water) in 500 ml of distilled water for 16 h at4.0°C. The dialysate was then autoclaved and was

completed with the addition of sterile glucose (0.05%).Bacteriophage assays were performed by using themethod of Wannamaker et al. (20) on serum Todd-Hewitt agar plates. Serum Todd-Hewitt plates con-

sisted of Todd-Hewitt broth (Difco Laboratories) ad-justed to pH 6.9 before addition of 1% bacteriological

915

916 McKANE AND FERRETTI

agar and 0.03% Na2HPO4 and subsequent autoclaving.The sterile mixture was supplemented with 5% normalhorse serum and 0.02% CaCl2.

Bacteriophage propagation and phage lysatepreparation. Phage propagation was accomplishedby simultaneous inoculation of 1 drop of an overnightculture of the sensitive bacterial strain and 1 drop ofa phage preparation containing 108 plaque-formingunits per ml to 5 ml of 6% proteose peptone broth #3(Difco). To the sterile proteose peptone broth wereadded (in final concentrations) 0.038% Na2HPO4,0.02% CaCl2, 0.05% glucose, 5% horse serum, and 0.1mg of hyaluronidase per ml. Incubation was carriedout at 37°C for 4 h for most phages and for 6 to 10 hfor phage T12. Virulent phages, such as A25 and themutant T12cpl, resulted in predominantly clear ly-sates, whereas temperate phages, such as H4489A andT12, resulted in predominantly turbid culture lysates.After the incubation period, the culture was centri-fuged at 10,000 x g for 15 min, and the phage lysatewas filtered through a Millipore membrane (0.45 ,im).Phage titers of approximately 109 plaque-forming unitsper ml were obtained for most phages. Concentratedphage preparations were obtained by centrifugation at101,000 x g for 90 min. Disruption of phage T12 wasaccomplished by resuspension of the phage pellet indistilled water.

Broken-cell extracts. Bacterial cells (50 ml) weregrown to the stationary phase, washed in phosphate-buffered saline, and suspended in 10 ml of phosphate-buffered saline. The cells were broken in a Braun cellhomogenizer (3 min), and the particulate matter wasremoved from the extract by centrifugation for 30 minat 6,450 x g. Protein concentrations of the broken-cellextract were determined; they ranged between 1 to 2mg/ml, depending on the extent of cell breakage.

Extracellular culture filtrates. All strains wereindividually incubated at 37°C in 500 ml of Todd-Hewitt dialysate for 16 h, and growth kinetics werefollowed turbidimetrically in a Klett colorimeter (660filter) to insure that all cultures were in the stationaryphase of growth. The cells were removed by centrifu-gation, and the supernatant fluids were filteredthrough Millipore membranes (0.45 im). The culturefiltrates contained approximately 2.5 mg of protein perml, about 0.3% of which was type A exotoxin (8).Concentrated samples of culture filtrate were obtainedby polyethylene glycol (20,000 MJ) absorption ofwaterfrom dialysis membranes. Protein concentrations, asdetermined by the method of Lowry et al. (12), of theconcentrated culture filtrates were in the range of 250mg/ml.Type A streptococcal exotoxin. The purification

of type A streptococcal exotoxin was accomplished bythe procedure described by Cunningham et al. (3) withthe modification described by us in a previous paper(8).Preparation of antisera. Hyperimmune rabbit

antisera were prepared with culture filtrates of thefollowing streptococcal strains: T253, T253 (T12), andNY5 type 10. Immunization of rabbits with culturefiltrates was accomplished by biweekly intramuscularand intraperitoneal injection of 1.5 ml of filtrate emul-sified in an equal volume of Freund incomplete adju-vant (Difco) for the first 3 weeks. The same procedure

was employed for the next 21 days of immunizationexcept that the samples were first suspended in 7.0%polyacrylamide and then polymerized with riboflavin(0.02%) for 30 min in the presence of direct visiblelight. The gel suspensions were then emulsified withequal volumes (3.0 ml) of Freund incomplete adjuvant.The animals were bled by arterial puncture every 10days, and precipitating antibody assays were per-formed by double immunodiffusion (Ouchterlonymethod). Serum titers were subsequently determinedby the tube dilution of antigen in constant serumconcentrations.

Polyacrylamide gel electrophoresis. Analyticaldisk gel electrophoresis was performed on purifiedtype A streptococcal exotoxin and phage T12. Thephage preparation was previously sedimented by ul-tracentrifugation (100,000 x g, 90 min) and disruptedby resuspension in distilled water. Electrophoresis ofsamples in 7.5% polyacrylamide was performed by themethod of Ornstein (18) and Davis (4). Protein wasdetected in the gels by using the Coomassie brilliantblue staining method of Diezel et al. (5).

Immunodiffusion. Ouchterlony double diffusionwas performed in gels of 0.8% agarose and 0.3% Nobleagar in glycine-saline buffer (0.1 M glycine, 0.15 MNaCl), pH 8.2.

Kinetics of type A exotoxin production. StrainT253 was grown in Todd-Hewitt dialysate, and phageT12 or phage T12cpl was added at the early log phaseof growth. A sample was removed at each 2-h interval,the cells were removed by centrifugation, and thesupernatant fluid was passed through a 0.45-,um Mil-lipore filter. A portion of each sample (0.1 ml) wasused to determine the phage titer and to assay type Aexotoxin by the erythematous skin test. The backs ofNew Zealand white rabbits were carefully shaven be-fore injection, and 0.1 ml of the Todd-Hewitt dialysatefiltrate was injected intradermally. A positive skinreaction was demonstrated by an erythematous re-sponse, which formed the basis of the test. The rabbitskins were observed at 24 to 48 h after injection. Thesame rabbits were used repeatedly for skin testing.

RESULTSPhage conversion and production of type

A exotoxin. The Ouchterlony immunodiffusionprocedure was used to assay for the presence oftype A streptococcal exotoxin. An antiserumsample to a culture filtrate of strain NY5 extra-cellular products was reacted with a sample ofthe corresponding filtrate and a sample of puri-fied type A exotoxin (Fig. 1). A line of identitybetween purified type A exotoxin and NY5 ex-tracellular products confirmed the presence ofantibody specific to type A exotoxin. Type Aexotoxin was also detected in the extracellularfiltrate of the lysogenic strain T253(T12) but nbtin the nonlysogenic strain T253 (Fig. 2). Theseresults are consistent with the observations ofZabriskie (21), who first reported lysogenic con-version in these strains of group A streptococci.To determine whether type A exotoxin was

INFECT. IMMUN.

TYPE A STREPTOCOCCAL EXOTOXIN PRODUCTION 917

A NY5Toxin Filtrate

Ant

FIG. 1. Immunodiffusion of purified type A exo-toxin and extracellular culture filtrate with antibodyto the extracellular culture filtrate of strain NY5(anti-NY5).

FIG. 2. Immunodiffusion of antibody to the extra-cellular culture filtrate ofstrain NY5 (anti-NY5) with(top well, clockwise direction) purified type A exo-toxin, culture filtrate of strain T253(T12), broken-cellextract of strain T253, purified type A exotoxin, bro-ken-cell extract of strain T253(T12), and culture fil-trate of strain T253.

present intracellularly, we washed bacterialcells, brokethem in a Braun cell homogenizer,and assayed the broken-cell extracts for the pres-ence oftype A exotoxin. The broken-cell extractsof neithxr the T253 (T12) lysogen nor the T253nonlysogen contained a level of type A exotoxinthat could be detected, whereas toxin was easilydetected in the extracellular filtrate of strainT253(T12) (Fig. 2).Role of bacteriophage T12 in type A ex-

otoxin production. The mechanism by whichphage affects production of type A exotoxin bystreptococci is unknown; however, the resultsobtained in polyacrylamide gel electrophoresisexperiments with phage T12 components (Fig.3) indicate that phage T12 did not have a struc-tural component capable of migrating to the

same position as that to which purified type Aexotoxin migrated. The lack of immunologicalreactivity of the phage components to anti-NY5serum indicated further that type A exotoxinwas probably unrelated to any structural com-ponent of bacteriophage T12.The isolation of a clear plaque mutant of

phage T12, designated T12cpl, provided thenecessary strain for determining whether lyso-geny of strain T253 by phage T12 was essentialfor type A exotoxin production. After infectionof strain T253 by phage T12cpl or by the virulentphage A25, the lysates were obtained and wereused in the Ouchterlony immunodiffusion assay.Neither the lysate obtained after infection byphage A25 nor the filtrate of the T253 nonlyso-gen contained detectable type A exotoxin (Fig.4). However, the T253 lysate obtained after in-fection by the virulent mutant T12cpl was con-verted to the toxigenic state, as evidenced by apositive reaction in the Ouchterlony immunodif-fusion assay. The possibility that a small per-centage of the cells were lysogenized by T12cpland were responsible for the detectable type Aexotoxin seems remote, because a study of thekinetics of type A exotoxin synthesis revealedthat maximum type A exotoxin levels obtainedafter infection of T253 by T12cpl correspondedto the major lytic event, whereas a 6-h lag oc-curred before maximum extracellular toxin

A B

Now

FIG. 3. Polyacrylamide gel electrophoresis (micro-grams ofprotein) of (A) purified type A exotoxin (30pg; arrow) and of (B) disrupted phage T12 prepara-tion (200 pJ. Gels were aligned by determining Rfvalues of tracking dye and bands with a Gilfordspectrophotometric gel scanning attachment.

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918 McKANE AND FERRETTI

levels were obtained with wild-type T12 (Fig. 5).Lysogeny per se, therefore, was not essential fortoxin production. Additionally, phage-mediatedlysis alone was not sufficient to account for theappearance of type A exotoxin.To determine whether a temperate phage un-

related to phage T12 could affect the conversionof strain T253 to the toxigenic state, we con-structed a T253 lysogen containing temperatephage H4489A as well as a relysogenized strainof T253 containing phage T12, which was desig-nated T253 (T12)B. After growth ofthese strains,the culture filtrates were obtained and wereutilized in the Ouchterlony immunodiffusion as-

FIG. 4. Immunodiffusion of antibody to the extra-cellular culture filtrate ofstrain NY5 (anti-NY5) with(top well clockwise direction) purified typeA exotoxin,T253 lysate obtained after infection with virulentphage A25, T25 lysate obtained after infection withphage T12, purified type A exotoxin, extracellularculture filtrate from an uninfected T253 culture, andT253 lysate obtained after infection with phageT12cpl.

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INFECT. IMMUN.

say. Whereas the relysogenized strain producedexotoxin identical to purified type A exotoxin,the other lysogen, T253 (H4489A), was incapableof producing type A exotoxin (Fig. 6). Appar-ently, there is a specificity to the interactionbetween phage and bacterial cell to affect aconversion to the toxigenic state.

DISCUSSIONThe results of this study provide several lines

of new information concerning the role of phageT12 in the conversion of group A strain T253 toproduction of type A streptococcal exotoxin. Ofspecial interest is the observation that stablelysogeny and presumptive integration of thephage genome into the host chromosome are notessential for expression of the toxigenic genes.This conclusion was reached when it was ob-served that virulent phage mutant T12cpl wascapable of producing type A exotoxin after in-

FIG. 6. Immunodiffusion of antibody to the extra-cellular culture filtrate ofstrain NY5 (anti-NY5) withculture filtrates ofthe lysogen T253 (H4489A), purifiedtype A exotoxin, and the reconstructed lysogen T253(T12)B.

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FIG. 5. Kinetics of type A exotoxin production during infection by phages T12 (A) and T12cpl (B). Type Aexotoxin was assayed by the erythematous skin reaction.

TYPE A STREPTOCOCCAL EXOTOXIN PRODUCTION 919

fection into host bacterial strain T253. The pos-

sibility that a structural component of phageT12 was related to type A streptococcal exotoxinwas considered; however, this possibility was

reduced by the observation of the absence ofprotein bands similar to those of type A exotoxinin polyacrylamide gel electrophoresis experi-ments and the lack of immunological reactionbetween phage components and antibody spe-

cific for type A exotoxin.One of the most pertinent questions concern-

ing toxigenic conversion in group A streptococciis whether the host bacterial strain or the in-fecting phage possesses the specificity for typeA exotoxin production. Our evidence indicatesthat phage T12 has a definite role in affectingtoxigenic conversion, since two unrelatedphages, virulent phage A25 and temperate phageH4489A, were unable to influence strain T253 toproduce type A exotoxin. In another communi-cation, we will present evidence that a numberof phages isolated from clinical strains of strep-tococci known to cause scarlet fever not onlydetermine the type of toxin the host synthesizesbut also affect toxigenic conversion in other hostbacterial strains.Whether the gene(s) specifying toxin synthesis

is located in the phage genome or in the bacterialchromosome is still unclear. We hypothesizethat bacterial strain T253 possesses the ability toproduce at least low levels of intracellular typeA toxin and that once introduced into the hoststrain, phage T12 affects the host to excrete or

possibly modify (or both) the intracellular toxinto an extracellular form. Further genetic analysiswill be necessary to determine the location ofthe gene(s) specifying type A exotoxin produc-tion and to elucidate the role of phage T12 intoxin production. The development of strepto-coccal cloning vehicles (1) and recombinant de-oxyribonucleic acid techniques in streptococci(2, 13) should facilitate such an analysis.

ACKNOWLEDGMENT

This work was supported by a grant from the AmericanHeart Association, Oklahoma Affiliate.

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3. Cunningham, C. M., E. L. Barsumian, and D. W.Watson. 1976. Further purification of group A strep-

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4. Davis, B. J. 1964. Disc electrophoresis. II. Method andapplication to human serum proteins. Ann. N.Y. Acad.Sci. 121:404-427.

5. Diezel, W., G. Kopperachlager, and E. Hoffman. 1972.An improved procedure for protein staining in poly-acrylamide gels with a new Coomassie brilliant blue.Anal. Biochem. 48:617-620.

6. Dochez, A. R., and F. A. Stevens. 1927. Studies on thebiology of streptococcus. VI. Allergic reactions withstrains from erysipelas. J. Exp. Med. 46:487-493.

7. Gerlach, D., H. Knoll, and W. Kohler. 1980. Purifica-tion and characterization of erythrogenic toxins. 1. In-vestigation of erythrogenic toxin-A produced by Strep-tococcus pyogenes NY-5. Zentralbl. Bakteriol. Parasi-tenkd. Infektionskr. Hyg. Abt. 1. Orig. Reihe A 247:177-185.

8. Houston, C. W., and J. J. Ferretti. 1981. Enzyme-linkedimmunosorbent assay for detection of type A strepto-coccal exotoxin: kinetics and regulation during growthof Streptococcus pyogenes. Infect. Immun. 33:862-869.

9. Johnson, L. P., P. M. Schlievert, and D. W. Watson.1980. Transfer of group A streptococcal pyrogenic exo-toxin production to nontoxigenic strains by lysogenicconversion. Infect. Immun. 28:254-257.

10. Kim, Y. B., and D. W. Watson. 1970. A purified groupA streptococcal pyrogenic exotoxin. Physicochemicaland biological properties including the enhancement ofsusceptibility to endotoxin lethal shock. J. Exp. Med.131:611-628.

11. Kim, Y. B., and D. W. Watson. 1972. Streptococcalexotoxins: biological and pathological properties, p. 33-50. In L. W. Wannaker and J. M. Matsen (ed.), Strep-tococci and Streptococcal Diseases. Academic Press,Inc., New York.

12. Lowry, O., N. Rosebrough, A. Farr, and R. Randall.1951. Protein measurement with the Folin phenol re-agent. J. Biol. Chem. 193:265-275.

13. Macrina, F. L., K. R. Jones, and P. H. Wood. 1980.Chimeric streptococcal plasmids and their use as mo-lecular cloning vehicles in Streptococcus sanguis (Chal-lis). J. Bacteriol. 143:1425-1435.

14. Maxted, W. 1964. Streptococcal bacteriophages, p. 25-52.In J. Uhr (ed.), Streptocci, rheumatic fever, and glo-merulonephritis. WilLiams & Wilkins Co., Baltimore.

15. Nauciel, C., J. Blass, R. Mangalo, and M. Raynaud.1969. Evidence for two molecular forms of streptococcalerythrogenic toxin. Eur. J. Biochem. 11:160-164.

16. Nauciel, C., M. Raynaud, and B. Bizzini. 1968. Purifi-cation et properties de la toxine erythrogene du strep-tocoque. Ann. Inst. Pasteur 114:796-811.

17. Nida, S. K., C. W. Houston, and J. J. Ferretti. 1978.Erythrogenic toxin production by group A streptococci,p. 66. In M. T. Parker (ed.), Pathogenic streptococci.Reedbooks Ltd., City, England.

18. Ornstein, L. 1964. Disc electrophoresis. I. Backgroundand theory. Ann. N.Y. Acad. Sci. 121:321-349.

19. Wannamaker, L., S. Almquist, and S. Skjold. 1973.Intergroup phage reactions and transduction betweengroup C and group A streptococci. J. Exp. Med. 137:1338.

20. Wannamaker, L., S. Skjoid, and W. Maxted. 1970.Characterization of bacteriophages from nephritogenicgroup A streptococci. J. Infect. Dis. 121:407418.

21. Zabriskie, J. 1964. The role of temperate bacteriophagein the production of erythrogenic toxin by group Astreptococci. J. Exp. Med. 119:761-779.

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