INFECTION AND IMMUNITY, Jan. 1991, p. 211-215 Vol. 59, No. 1 0019-9567/91/010211-05$02.00/0 Copyright © 1991, American Society for Microbiology Frequency of the Erythrogenic Toxin B and C Genes (speB and speC) among Clinical Isolates of Group A Streptococci CHANG-EN YU AND JOSEPH J. FERRETTI* Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190 Received 13 August 1990/Accepted 10 October 1990

DNA probes corresponding to the internal region of the erythrogenic toxin B and C genes, speB and speC, were used in hybridization studies with clinical isolates of Streptococcus pyogenes to determine the frequency of occurrence of these genes in a large population of group A streptococci. More than 500 strains from different geographical locations throughout the world were used in this study. The results from colony-lift hybridization experiments indicated that the frequency of occurrence of each toxin gene among all of these strains was 100% for speB and 50% for speC. Division of these strains into subgroups of general group A strains and strains associated with scarlet fever or rheumatic fever resulted in a frequency of occurrence of speC of about 50% for all subgroups. The speC gene was found to be more frequently associated with serotype M2, M4, and M6 strains and less frequently associated with serotype Ml, M3, and M49 strains. The results from a similar study with the speA gene have been previously reported (C.-E. Yu and J. J. Ferretti, Infect. Immun. 57:3715-3719, 1989).

The group A streptococci produce a number of extracel- genes among clinical isolates of Streptococcus pyogenes lular proteins, among which are included the erythrogenic obtained from 13 countries. toxins. Dick and Dick originally described erythrogenic toxins as all substances which cause a skin reaction, regard- less of their biological activity or biochemical nature (7, 8). MATERIALS AND METHODS Erythrogenic toxins (34) are also known as scarlet fever toxin (2), Dick toxin (1), streptococcal pyrogenic exotoxin Bacterial strains. A total of 512 clinical isolates of group A streptococcal strains were obtained from 13 countries (36), streptococcal exotoxin (22), blastogens (12), and mito- throughout the world (provided by streptococcal reference gens (30). Three antigenically distinct erythrogenic toxins have been centers and World Health Organization laboratories) and used in this study. These countries are the 12 included a described, including type A, B, and C toxins. The most in previous report (39) and the Republic of China. The majority extensively studied toxin is the type A toxin, which was of these strains were originally thought to be the toxin responsible for scarlet (90%) isolated between 1981 and 1988, and the rest were isolated from to fever (1, 8, 16). The type A toxin is encoded by the 1940 1980. These strains were divided into subgroups, general group A strains and bacteriophage speA gene (19, 37) and has a molecular weight scarlet fever strains, on the basis of the disease associated of 25,787 (38). The type B toxin is encoded by the speB gene with each strain as described previously (39). Furthermore, and has a molecular weight of 27,588 (15). The type C toxin strains isolated from patients with rheumatic fever were is encoded by the bacteriophage speC gene and has a molecular weight of 24,354 (11). separated from the general group A strains and regarded as an individual group. Two standard laboratory strains, NY5 The erythrogenic toxins have been associated with a variety of biological activities, including the following: (33) and K56 (23), were used as positive and negative for the gene. T18P erythematous skin reactions, probably due to enhancement controls, respectively, speA Strains (31) of delayed hypersensitivity (28a); pyrogenicity (4, 18, 36); and T19 (25) were used as positive and negative controls, specific and nonspecific T-cell mitogenicity (3); immunosup- respectively, for the speC gene. Strain NY5 was also used for speB gene amplification by the polymerase chain reaction pression by nonspecific activation of T-suppressor lympho- (PCR). cytes (6, 17); enhancement of susceptibility to endotoxin Media, chemicals, and enzymes. The standard liquid me- shock (21); cytotoxicity to splenic macrophages (21); car- (21, dium used for growth of group A streptococci was 3% diotoxicity 36); alteration ofthe blood-brain barrier (31); (wt/vol) Todd-Hewitt broth (Difco Laboratories) with 1.5% and alteration of antibody response to sheep erythrocytes in mice and rabbits (5, 14). (wt/vol) Bacto-Agar when required. Guanidium thiocyanate was purchased from Sigma Chemical Co. (St. Louis, Mo.). In view of the fact that at least two of the toxin genes, The GeneAmp DNA amplification reagent kit was purchased speA and speC, are located on mobile genetic elements, it from Perkin Elmer Cetus. Terminal transferase and the DNA was of interest to determine the frequency of occurrence of labeling and detection kit (nonradioactive) were from Boehr- the erythrogenic toxin genes in a population of clinical inger Mannheim Biochemicals. The random primer DNA isolates. In a previous study, we reported that the speA gene labeling kit was from Bethesda was found in 15% of general strains and 45% of strains Research Laboratories, Inc. The T7 kit was obtained from patients with scarlet fever (39). We report sequencing from Pharmacia, and the Gene- clean kit was from Bio 101. All were used to here on the frequency occurrence according the of of the speB and speC specifications of the manufacturers. [ct-32P]dCTP, [,y-32p] ATP, and [a-35S]dATP were obtained from DuPont New England Nuclear Corp. * Corresponding author. Construction of toxin gene-specific probes. The specific 211 212 YU AND FERRETTI INFECT. IMMUN. probe for the speA gene, pSF606, containing only the internal sequences of speA, was constructed as previously reported (39). An oligonucleotide was synthesized and used as the specific speC gene probe. It contained 16 nucleotides (5'-GGAATTACGCCTGCTC-3') and was selected from bp 345 to 360 of the published speC DNA sequence data (11). The specific speB gene probe was obtained by using the PCR with degenerate primers. The primers used for speB gene amplification were synthetic oligonucleotides derived from the amino acid sequences of mature streptococcal proteinase ''W YSi~~~~~~~ii;0- as described by Tai et al. (35). Primer 1 (5'-ATG AAA[G] TAT[C] CAT[C] AAT[C] TATECI CC-3') was a 20-mer oligo- nucleotide derived from amino acids 56 to 62 of the strepto- ..'.''.L'~~~~~~~~~~~~~~~~~~~~~~...... ';.e: coccal proteinase sequence, starting with an ATG start codon. Primer 2 (5'-GAA TTC CCC CAA[G] TCA[CTGJ ACA[GI TGA[GI TAA[G] AA-3') was from amino acids 206 to 212 of the streptococcal proteinase sequence. The amplified PCR product was used as the speB-specific probe. PCR. Amplification of speB by PCR was performed as follows. Chromosomal DNA from strain NY5 was first melted at 94°C for 1 min, and primers were annealed at 37°C (for the first 5 cycles) and 48°C (for the following 30 cycles) FIG. 1. Example of colony-lift hybridization with the speC-gene- for 2 min each. Then the polymerization and extension were specific probe, using a nonradioactive color detection system on a completed at 72°C for 3 min. The amplified products were Nytran membrane. The bottom row contains strain T18p, a strain purified and recovered by gel electrophoresis and Geneclean positive for type C erythrogenic toxin production, and strain T19 treatment. and a group G strain, both of which are known not to produce type DNA sequencing. The PCR-amplified DNA fragment, used C toxin. The remaining strains were clinical isolates and showed as the speB gene-specific probe, was confirmed for the some variation in signal intensity due to differences in growth of correct sequence specifying streptococcal proteinase (35) by individual colonies. the dideoxy sequencing method (28) with the double- stranded DNA and degenerate primers. Colony-lift hybridization. Colony-lift hybridizations were RESULTS accomplished as described previously (39), with some mod- ifications. First, each strain was inoculated by dropping 2 ,ul speB- and speC-specific probes. The observation by of a sedimented overnight culture from the bottom of a tube Gerlach et al. (10) that the proteinase precursor was identical onto the top of the agar surface. After incubation at 37°C for to erythrogenic toxin B and the recent sequence information 30 min, the agar surface was covered with a piece of sterile showing their identity (15) allowed us to obtain a speB- Nytran membrane (Schleicher & Schuell, Inc., Keene, specific probe. The primers derived from the proteinase N.H.). Next, instead of 10% sodium dodecyl sulfate, the amino acid sequence described in Materials and Methods GES solution (5 M guanidium thiocyanate, 100 mM EDTA, were used to amplify chromosomal DNA of strain NY5 by 0.5% [vol/vol] sarkosyl) (26) was employed at room temper- the PCR. A single DNA fragment, estimated to be 470 bp in ature for ensure cell All size by gel electrophoresis, was obtained following amplifi- 10 min to complete lysis. hybridiza- cation. This DNA fragment was further purified by gel tion experiments were performed under high-stringency con- electrophoresis and Geneclean treatment before being sub- ditions, i.e., hybridization and washing at 68°C for both the jected to DNA sequencing. The deduced amino acid se- speA and speB probes, which allowed for only 10% mis- quence of the protein specified by the 470-bp DNA fragment match. The speC oligonucleotide probe, with a melting was identical to the amino acid sequence of streptococcal temperature of 50°C, was hybridized and washed at 42°C, proteinase described by Tai et al. (35), and this purified which also allowed for a 10% mismatch. All results were fragment was used as the speB-specific gene probe. confirmed by at least two separate experiments with each The speC gene probe was a 16-mer synthetic oligonucle- strain. otide derived from the published DNA sequence (11). This DNA probe labeling and reprobing. The speA gene probe, sequence was specifically selected because of its lack of derived from EcoRI-PstI doubly digested fragments of homology to speA as well as to sequences of other genes. pSF606, and the speB gene probe, obtained from PCR The speC-specific probe hybridized strongly with chromo- amplification, were purified from agarose gels and subjected somal DNA from strain T18P, a strain known to produce to Geneclean treatment. They were then labeled either type C erythrogenic toxin, but did not hybridize with strain radioactively ([a-32P]dCTP) or nonradioactively (digoxige- T19, a strain known not to produce type C erythrogenic nin-dUTP) by the random priming method. The speC gene toxin. probe, a 16-mer oligonucleotide, was labeled radioactively Hybridization results. All hybridizations with the spe gene ([y-32P]ATP) or nonradioactively (digoxigenin-dUTP) by the probes were performed by the colony-lift technique under end-labeling method as described by Maniatis et al. (27). high-stringency conditions. Both radioautography and non- Reprobing was achieved by removing the probes from radioactive color detection methods were used in this study Nytran membranes with 0.2% NaOH-0.1% sodium dodecyl (Fig. 1). Of the 512 clinical strains, 315 were general group A sulfate and incubating at 37°C for 30 min, and the membranes strains isolated from patients with diseases such as tonsilli- were subjected to the next hybridization reaction. tis, impetigo, cellulitis, pyoderma, abscess, and glomerulo- VOL. 59, 1991 FREQUENCY OF speB AND speC AMONG GROUP A STREPTOCOCCI 213

TABLE 1. Frequency of occurrence of erythrogenic toxin genes Table 2. As noted previously (39), the speA gene is found among clinical isolates of group A streptococci with a high frequency of occurrence in serotype Ml, M3, less % of strains (no. positive/no. tested) M49, Ti, and T3/13 strains. The speC gene is found frequently in these strains. The speC gene is found with a Gene(s) Rheumatic General group A Scarlet fever fever high frequency of occurrence in serotype M2, M4, M6, M12, T2, T4, T6, and T28 strains. The speA gene is found less speA 14.9 (47/315) 52.6 (80/152) 51.1 (23/45) frequently in these strains. The only serotype containing speB 100 (315/315) 100 (152/152) 100 (45/45) both the speA and speC genes in a large number was M3. speC 50.8 (160/315) 48.7 (74/152) 46.7 (21/45) speA and 5.0 (16/315) 14.5 (22/152) 24.2 (11/45) speC DISCUSSION Neither speA 39.4 (124/315) 13.2 (20/152) 26.7 (12/45) nor speC The availability of specific speB and speC probes has allowed the completion of the molecular epidemiological analysis of the erythrogenic toxin genes among clinical S. pyogenes strains. In our previous study (39), only the speA nephritis but not scarlet fever or rheumatic fever. The other gene was analyzed in terms of its frequency of occurrence two groups included 152 strains obtained from patients with among clinical isolates. In the present study, additional scarlet fever and 45 strains obtained from patients with strains were analyzed that were not previously reported, rheumatic fever. The overall results of the frequency of particularly those obtained from patients with documented occurrence of speA, speB, and speC genes among these cases of rheumatic fever. clinical isolates are presented in Table 1. The speA gene The 512 S. pyogenes strains used in this study represent a hybridizations were performed again with essentially the geographical distribution of strains throughout the world, same strains as presented previously (39), with the deletion and most of the strains were isolated from different regions of several strains which were not recovered following stor- of each country and, in many instances, in different years. age and with the addition of several new strains. More than 90% of the strains were obtained in the 1980s, and The speB gene was found in all strains of group A there was no time dimension to the collection of strains. The streptococci, a result that may have been expected since overall results represent a baseline of information concern- there is no evidence of speB phage association as there is ing the frequency of occurrence of spe genes in the general with the speA and speC genes. The speB probe did not population of group A streptococci. hybridize with a group G streptococcal strain. The speC The selection of the speB gene probe was based on the gene was found uniformly among the three groups of strains: previous finding of Gerlach et al. (10) that erythrogenic toxin 50.8% in general group A strains, 48.7% in scarlet fever- B is identical to the proteinase precursor. Further evidence associated strains, and 46.7% in rheumatic fever-associated that these two proteins are identical comes from the fact that strains. the deduced amino acid sequence of the protein encoded by The number of strains containing both the speA and speC speB shows a high degree of identity to the streptococcal genes is low, only 5% ofthe general strains and 9% of the 512 proteinase sequence, and they are considered the same (15). strains employed in the study. In contrast, the number of In view of the fact that the majority of strains produce an strains containing neither the speA nor the speC gene was extracellular proteinase, it was not surprising to find speB in 30% of the total number of strains employed in the study. 100% of the strains tested. On the other hand, the speC gene The occurrence of the speA and speC genes among group was expected to be found with a lower frequency of occur- A streptococcal M- and T-serotype strains is presented in rence because of its presence on a bacteriophage and indeed

TABLE 2. Occurrence of the speA or speC gene among group A streptococcal M and T serotypes % of strains (no. positive/no. tested) Serotype General group A Scarlet fever Rheumatic fever speA speC speA speC speA speC Ml 62.5 (10/16) 12.5 (2/16) 94.4 (17/18) 11.1 (2/18) 33.3 (1/3) 0 (0/3) M2 20.0 (1/5) 100 (5/5) 0 (0/2) 100 (2/2) a M3 81.8 (9/11) 27.3 (3/11) 100 (8/8) 50.0 (4/8) 100 (4/4) 0 (0/4) M4 0 (0/13) 92.3 (12/13) 0 (0/15) 93.3 (14/15) M6 0 (0/3) 100 (3/3) 0 (0/1) 100 (1/1) 100 (2/2) 100 (2/2) M12 7.1 (1/14) 64.3 (9/14) 16.7 (1/6) 66.7 (4/6) M22 0 (0/7) 14.3 (1/) 20.0 (1/5) 20.0 (1/5) M49 0 (0/6) 33.3 (2/6) 100 (7/7) 0 (0/7) Ti 47.8 (11/23) 30.4 (7/23) 94.1 (16/17) 11.8 (2/17) - T2 10.0 (1/10) 70.0 (7/10) 0 (0/3) 100 (3/3) T3/13 30.0 (18/60) 43.3 (26/60) 90.0 (27/30) 33.3 (10/30) T4 0 (0/20) 80.0 (16/20) 0 (0/8) 87.5 (7/8) T6 0 (0/4) 75.0 (3/4) 0 (0/1) 100 (1/1) T12 3.0 (1/33) 48.5 (16/33) 15.4 (2/13) 38.5 (5/13) T14/49 0 (0/8) 37.5 (3/8) 100 (6/6) 0 (0/6) T28 0 (0/16) 68.8 (11/16) 0 (0/1) 100 (1/1) a -, Data not available. 214 YU AND FERRETTI INFECT. IMMUN. was found in approximately 50% of the strains tested. There the speA gene. The availability of specific spe probes to was little variation of the speC gene frequency of occurrence monitor strains will be important for future epidemiological among the general, scarlet fever-associated, and rheumatic studies. fever-associated strains. Thus, the presence of either speC Whereas this study focuses on the detection of the indi- or speB does not appear to be correlated with the ability of vidual erythrogenic toxin genes, it says nothing about their a strain to cause either scarlet fever or rheumatic fever. expression. It is clear from previous studies that toxin In contrast, the speA gene was found in 15% of general production and identification among individual strains has strains, 52% of scarlet fever-associated strains, and 51% of been variable (20, 24, 25, 29). The key to understanding the rheumatic fever-associated strains. Thus, speA appears to be role of each toxin in disease may lie in the regulatory factors the only gene among the three erythrogenic toxin genes that involved in the expression of each erythrogenic toxin gene. has a significant correlation with disease. These results support predictions from the earliest studies that erythro- ACKNOWLEDGMENT genic toxin A was the major toxin responsible for the development of scarlet fever (1, 8, 16). Whether there is a This research was supported by Public Health Service grant correlation between development of scarlet fever and rheu- A119304 from the National Institutes of Health. matic fever is difficult to ascertain, since neither scarlet fever REFERENCES nor rheumatic fever is a presently reportable disease. Nev- 1. Ando, K., K. Kurauchi, and H. Nishimura. 1930. Studies on the ertheless, a high proportion of rheumatic fever-associated "toxins" of hemolytic streptococci. III. 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