Streptococcus pyogenes Strains - Infection and Immunity - American ...

INFECTION AND IMMUNITY, Dec. 1989, p. 3715-3719

Vol. 57, No. 12

0019-9567/89/123715-05$02.00/0 Copyright © 1989, American Society for Microbiology

Molecular Epidemiologic Analysis of the Type A Streptococcal Exotoxin (Erythrogenic Toxin) Gene (speA) in Clinical Streptococcus pyogenes Strains CHANG-EN YU AND JOSEPH J. FERRETTI* Department of Microbiology and Immunology, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73190 Received 9 June 1989/Accepted 17 August 1989

A molecular epidemiology analysis was performed with over 440 clinical isolates of Streptococcus pyogenes obtained from 11 different countries in order to determine the frequency of occurrence of the type A streptococcal exotoxin (erythrogenic toxin) gene (speA) among group A strains. The colony hybridization technique employing a specific internal fragment of the speA gene was used for initial screening, and all positive results were further confirmed by the Southern hybridization technique. Among over 300 general strains obtained from patients with a variety of diseases, except scarlet fever (such as tonsillitis, impetigo, cellulitis, pyoderma, abscess, rheumatic fever, and glomerulonephritis), 15% were found to contain the speA gene. Among a group of 146 strains obtained from individuals described as having scarlet fever, 45% were shown to contain the speA gene. Further analysis of the data indicated that strains with certain M- or T-type surface antigens showed a higher (such as M and T types 1 and 3/13) or lower (such as M2, M12, T4, T5, and T28) tendency to contain the speA gene. No correlation was found between speA content of a strain and the ability to cause a specific disease, although strains possessing the speA gene were more likely to be associated with scarlet fever and rheumatic fever than with other types of disease. to be associated with certain diseases, as well as with certain serotypes. In this investigation, we utilized a specific inter-

In recent years, molecular approaches to the study of Streptococcus pyogenes have been possible because of the ability to clone and express group A streptococcal genes in heterologous hosts. Among genes studied at the molecular level are those specifying streptokinase (14, 15), streptolysin O (20), hyaluronidase (16, 17), erythrogenic toxins (streptococcal exotoxins or pyrogenic exotoxins) type A (35, 36), type B (3), and type C (8, 9), several M proteins (24-26, 29, 30, 32), streptococcal acid glycoprotein (19), and immunoglobulin G Fc receptor protein (11). It is generally accepted that all group A strains possess the same complement of genes, and hybridization studies with specific probes have shown this to be true for the streptokinase gene (15) and the M protein gene (30). The factors responsible for the association of some strains with a particular disease, such as nephritogenic strains or rheumatogenic strains, is an active area of investigation. One of these factors, the type A erythrogenic toxin, has long been associated with the ability of group A streptococci to cause scarlet fever. Indeed, the type A toxin is thought to be the toxin primarily responsible for the skin rash of scarlet fever (6, 7). Streptococcal exotoxin A, also known as erythrogenic toxin or pyrogenic exotoxin A, is specified by the speA gene of group A streptococci. The speA gene has been cloned and shown to be a part of bacteriophage T12 (18, 35). The complete nucleotide sequence of this gene has been determined, and it specifies a mature protein with a molecular weight of 25,787 (36). The presence of a 30-amino-acid signal peptide, characteristic of secreted proteins, suggests that this gene is chromosomal in origin and not a functional part of the bacteriophage genome. In view of the presence of the speA gene on a mobile genetic element, such as a bacteriophage, we were curious about its distribution among clinical strains and its frequency of occurrence among strains known *

nal sequence of the speA gene as a probe in a molecular epidemiology analysis of clinical isolates of S. pyogenes obtained from 11 different countries. MATERIALS AND METHODS

Bacterial strains. Four hundred forty-eight clinical isolates of S. pyogenes (kindly provided by the streptococcal reference centers and World Health Organization laboratories) from around the world were used in this study. These strains were divided into two groups, general group A strains and scarlet fever-associated strains. The general group A strains were obtained from patients with a variety of group A streptococcal diseases except scarlet fever, while scarlet fever-associated strains were obtained from individuals described as having scarlet fever. Also, two standard laboratory strains NY5 (33) and K56 (22) were used as positive and negative controls, respectively, for the speA gene and SPEA toxin production. Media. The standard liquid medium used for bacterial growth was 3% (wt/vol) Todd-Hewitt (TH) broth (Difco Laboratories), with 1.5% (wt/vol) agar when required. Heatinactivated horse serum (GIBCO Laboratories), added to 3% (vol/vol), and 0.5% (wt/vol) sterile glycine were supplemented when colony hybridization and chromosomal DNA isolation were performed. Enzymes and chemicals. Restriction endonucleases, Klenow fragment, and the M13 17-base primer were purchased from Bethesda Research Laboratories, Inc. (Gaithersburg, Md.) and were used according to the specifications of the manufacturer. T4 DNA ligase was obtained from Amersham Corp.; [32P]dATP was from New England Nuclear Corp.; and mutanolysin, lysozyme, RNase, and protease were from Sigma Chemical Co. (St. Louis, Mo.). Construction of the pSF606 DNA probe. The speA gene

Corresponding author. 3715

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INFECT. IMMUN.

YU AND FERRETTI

fragment from phage T12

I

0..

Aa

Aa Aa S S

S Ha

C

Av

Aa

Ac

J

I

A

ATG

I

A

Bg Be Bc

TAA

pSF606

I

pL

_w

_N _

S.

Ml3mpl8

_!

606 bp E Ba

P

FIG. 1. Restriction map of the phage T12 fragment containing the speA gene and the 606-bp fragment cloned in M13 mpl8. Restriction sites: A, AluI; Aa, AhaIII; Ac, AccI; Av, Avall; Ba, BamHI; Bc, BcIl; Be, BstEII; Bg, BgIIl; C, ClaI; E, EcoRI; H, HaeIII; P, PstI; S, Sau3AI. The positions of the initiation codon (ATG) and the termination codon (TAA) of speA are shown.

isolated from phage T12 was previously cloned in M13 phage by Weeks and Ferretti (35). One of the clones, which contained the whole gene except the last 29 base pairs (bp), was selected to construct a specific probe containing only internal coding sequences of the speA gene. To subclone this internal speA fragment, the recombinant DNA was simultaneously digested with BgIII and PstI. After electrophoresis through a low-melting-temperature agarose gel, a 600-bp DNA fragment was separated from the other DNA fragments, collected, and purified. The vector DNA was also double digested with BamHI and PstI, and the speA gene fragment was then subcloned into M13 phage mpl8 by the method described by Maniatis et al. (23). Colony hybridization. The colony hybridization technique employed in this study was that described by Maniatis et al. (23), with some modifications. First, the nitrocellulose filters were submerged in distilled water, sandwiched between two Whatman filters, and sterilized. Next, pure colonies of each bacterial strain were picked, inoculated onto a marked area on top of a glycine- and horse serum-supplemented TH agar plate, and then incubated at 37° C for 30 min. The sterile nitrocellulose filter was placed on top of the agar surface and incubated for up to 36 h. After incubation, the nitrocellulose filter was removed, placed on top of a Whatman 3MM filter, and treated successively with the following: 10 mM Tris buffer with mutanolysin (5 U/ml) and lysozyme (5 mg/ml) at 37° C for 2 h; 10 mM Tris-5 mM EDTA-0.5% sodium dodecyl sulfate with protease K (50 ,ug/ml) at 37° C for 1 h; 10% sodium dodecyl sulfate at room temperature for 5 min; 1.5 M NaCl-0.5 M NaOH for 10 min; 1.5 M NaCl-1 M Tris for 10 min; and 2x SSPE (360 mM NaCl, 20 mM Na2HPO4, 2 mM EDTA) for 10 min. Following this treatment, the nitrocellulose filter was baked at 80° C for 2 h and was ready for hybridization. Chromosomal DNA isolation. All the strains used in Southern hybridization experiments were grown in 10 ml of TH broth at 37° C for 8 h, transferred to 10 ml of fresh prewarmed TH broth with a final concentration of 0.5% (wt/vol) glycine, and allowed to grow for another 2 h. The bacterial cells were harvested by centrifugation at 15,000 rpm, using a Sorvall SS34 rotor at 4° C for 20 min. The cell pellets were suspended in 10 mM Tris buffer and transferred to microcentrifuge tubes. Again, the cells were pelleted and then suspended

a *a .

*

_

~i

FIG. 2. Autoradiography of colony hybridization. A negative control strain, K56, showed no hybridization signal.

with 400 ,ul of buffer containing 10 mM Tris and 50 mM EDTA. Next, 5 U of mutanolysin, 2 mg of lysozyme, and 5 ,u of DNase-free RNase (10 mg/ml) were added, and the mixtures were incubated at 37° C for 1 h with gentle shaking. Later, 20 ,ul of predigested (37° C for 30 min) protease (1 mg/ml) was added to the tube, which served to further weaken the cell walls and membranes, and incubated for another 30 min. Then, 20 ,ul of 20% sarcosyl was added to dissolve the lipid portion and lyse the cell. The DNA was extracted with phenol-chloroform and then precipitated with ethanol. Finally, the DNA pellets were suspended in 200 RId of TE buffer (10 mM Tris hydrochloride, 1 mM EDTA) and were ready for restriction enzyme digestion with HindIIl. Southern blot hybridizations. Transfer of DNA from gels to nitrocellulose filters was performed by the method of Southern (31), as described by Maniatis et al. (23). Radioactive labeling of M13 phage. The single-stranded M13 phage containing the 606-bp speA fragment was prepared as described by Maniatis et al. (23) and served as a template for making a radiolabeled probe by the procedure described by Hu and Messing (13). Hybridization and autoradiography. The nitrocellulose replica filters and nitrocellulose blots were hybridized with a 32P-labeled DNA probe as described by Maniatis et al. (23). RESULTS An internal sequence of the speA gene containing 606 bp was cloned into M13 mpl8 phage and used as a probe in all hybridization experiments. A physical map of this 606-bp probe is shown in Fig. 1. Initially, all hybridizations were performed by the colony hybridization technique. An example of the radioautography results from a typical filter is shown in Fig. 2. Results for all strains showing positive hybridization signals by the colony hybridization technique were subsequently confirmed by Southern hybridization experiments (data not shown). Interestingly, only single bands were observed, which indicated the presence of a single speA gene and no cross-hybridization with other

VOL. 57, 1989

speA FREQUENCY IN S. PYOGENES

TABLE 1. Hybridization of the speA gene to clinical isolates from 11 different countries Country of origin

Canada Denmark England France German Democratic Republic Federal Republic of Germany India Japan New Zealand Thailand United States

USSR % Positive

No. of No. of general No. speA scarlet No. speA group A positive fever positive strains strains

45 26 44 33 0 0 22 4 22 49 33 24

3 8 9 5 0 0 2 1 5 4 6 2 14.9

17 24 0 13 20 11 0 0 18 1 23 19

5 13 0 5 2 7 0 0 16 1 6 12 45.2

Additionally, several band sizes were observed different isolates, indicating heterogeneity of sequences adjacent to the speA gene. Among the 448 strains examined, there were 302 that were general group A strains, i.e., strains obtained from individuals with diseases such as tonsillitis, impetigo, cellulitis, pyoderma, abscess, rheumatic fever, and glomerulonephritis, but not scarlet fever. The other group of strains included 146 isolates obtained from individuals described as having had scarlet fever. The results of these experiments along with the country of origin of each of the strains are given in Table 1. The speA gene was found to be present in 15% of general group A strains and 45% of scarlet fever-associated strains. The occurrence of the speA gene among group A streptococcal M- and T-serotype strains is presented in Table 2. Although M-type or T-type information was not available for all strains employed in this study, there appeared to be a correlation between the presence or absence of the speA gene among certain serotype strains. For example, strains such as Ml, M3, M49, Ti, and T3/13 showed a high frequency of occurrence of the speA gene, whereas strains such as M4, M12, M22, T2/22, and T4 had a low or no frequency of occurrence of the speA gene. For the most part, there was no particular pattern of association of the speA gene with a particular disease. However, in addition to the scarlet fever-associated strains, of which 45% contained the speA gene, it was also found in a study of strains documented to be associated with rheumatic fever that 48% of the strains (19 positive of 39 tested) also contained the speA

genes. among

streptococcal strains and more specifically, the frequency of the speA gene in scarlet fever-associated strains. It seems reasonable that only 15% of general group A strains contain the speA gene since not all group A infections result in scarlet fever. However, it was surprising that only 45% of the strains isolated from patients with scarlet fever contained the speA gene. A similar frequency of occurrence (48%) in strains associated with rheumatic fever was also observed. Thus, it appears that possession of the speA gene by a strain makes it more likely to be associated with a severe form of streptococcal disease. The product of the speA gene, erythrogenic toxin A, has long been implicated as the exotoxin responsible for the rash of scarlet fever (6, 7); thus, it was expected that the speA gene would be found in almost all of the scarlet feverassociated strains. There are two possible explanations for the finding that only 45% of these strains contained the speA gene. First, there may have been variations among individual physicians in the criteria and clinical symptoms used to diagnose scarlet fever. Second, the type B and C erythrogenic toxins are also known to cause a skin rash (12) and in the absence of the type A toxin produce identical clinical symptoms. Since these three antigenically distinct streptococcal exotoxins have similar biological activities (1, 2, 4, 5, 10, 21, 27, 28, 34), it is possible that some regions among the type A, B, and C exotoxins are similar. Indeed, the nucleotide sequence of the speC gene has recently been determined, and the gene was shown to have a high degree of similarity to speA (9). The presence of the speA gene on a mobile genetic element, aside from plasmid-carrying antibiotic resistance genes, is one of the few examples in which a genetic determinant is able to naturally transfer among the group A streptococci. Most other genes and their products are found uniformly among all strains, although differences in the ability of some strains to express a particular product have been noted with continued laboratory subculturing. Such variable expression was also true of speA-carrying strains in this study, in which only a small percentage of strains produced increased levels of exotoxin under laboratory conditions. The reason for the variable exotoxin production is unclear and was most likely due to regulatory rather than gene dosage effects. The 448 S. pyogenes strains employed in this study were obtained from 11 countries in different geographical locations. Most of the strains were isolated from different regions TABLE 2. Occurrence of the speA gene among group A streptococcal M and T serotypes

gene.

All of the data presented in Tables 1 and 2 were subjected to statistical analysis by the chi-square test with the following results (data not shown): (i) the samples from the different countries are highly heterogeneous; (ii) there is a

significant difference in the distribution of the speA gene among general and scarlet fever-associated strains; (iii) no significant correlation was observed between the presence of the speA gene and a particular disease, except scarlet fever; and (iv) there was a high level of significance for the presence of the speA gene in certain serotypes and not in others, as described above. DISCUSSION The results of this study provide data about the frequency of the speA gene in the general population of group A

3717

Strain

% speA positive (no. positive/no. tested) Scarlet fever General group strains A strains

Ml M3 M49 M4 M12 M22

67 72 0 0 0 0

(14/21) (8/11) (0/6) (0/16) (0/14) (0/8)

100 (11/11) 88 (7/8) 100 (13/13)

Ti

49 (18/37) 24 (15/63) 0 (0/9)

100 (13/13) 81 (17/21) 88 (7/8) 0 (0/26) 0 (0/1) 0 (0/1)

T3/13 T14/49 T4 T5 T28

0 (0/35) 0 (0/12) 0 (0/14)

0 (0/14) 17 (1/6) 0 (0/5)

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of each country and in many instances in different years. The strain collection is therefore not a regional sampling from a single outbreak but rather samples which represent the general population of S. pyogenes. Several potential technical problems could have influenced the results of this study, the first of which concerns the difficulty in lysing group A streptococcal cells, especially in the colony hybridization assays in which unlysed colonies would not have shown positive hybridization results even if the speA gene was present. To prove that negative hybridizations were indeed negative, 50 negative strains were randomly selected, and the DNA was isolated by conventional techniques and used in Southern blot hybridizations with the speA gene probe. In no instance did previously negative speA strains prove to be positive in these assays, whereas positive control strains always remained positive. A second potential problem that may have influenced the results of this study was the conditions used for hybridizations. However, these experiments were performed under high stringency conditions to eliminate short-region bindings and assure that only the long and continuously complementary bindings remained on the nitrocellulose filters, preventing any nonspecific hybridizations. The speA internal gene fragment used as a probe has a G+C content of 29% and a Tm of 81.6° C. Under stringent conditions such as those employed in these experiments, i.e., 1 M Na+ and 68° C overnight, there is an allowance of only 10% base-pair mismatch. Thus, it seems highly unlikely that technical problems influenced the results of this study. The finding that the speA gene was present with high frequency in certain serotype strains (Ml, M3, M49, Ti, and T3/13) and not others (M4, M12, M22, T2/22, and T4) may be indicative of the host range of the T12 bacteriophage rather than of immediate clinical significance. Indeed, we observed no correlation of the speA gene with strains associated with a particular disease. On the other hand, the fact that strains from individuals with scarlet fever or rheumatic fever were more likely to contain the speA gene may indicate that the type A erythrogenic toxin contributes to the armamentarium of pathogenic factors in the group A streptococci. ACKNOWLEDGMENTS This research was supported by Public Health Service grant Al 9304 from the National Institutes of Health. We thank the following individuals for providing strains used in this study: Geoffrey Colman, Central Public Health Laboratory, London, England; Richard Facklam, Centers for Disease Control, Atlanta, Ga.; Jorgen Henrichsen, Statens Seruminstitut, Copenhagen, Denmark; Thea Horaud, Institute Pasteur, Paris, France; Dwight Johnson, University of Minnesota, Minneapolis, Minn.; Werner Kohler, Zentralinstitut fur Mikrobiologie und Experimentelle Therapie, Jena, German Democratic Republic; A. Eugenia Lipinski, Provincial Laboratory of Public Health, Edmonton, Alberta, Canada; Rudolf Lutticken, Der Universitat Koln, Koln, Federal Republic of Germany; Diana Martin, National Health Institute, Wellington, New Zealand; Teiko Murai, Toho University, Tokyo, Japan; K. Prakash, Lady Hardinge Medical College, New Dehli, India; Amporn Sukonthaman, Chulalongkorn Hospital, Bangkok, Thailand; and Artem A. Totolian, Institute of Experimental Medicine, Leningrad, USSR. LITERATURE CITED 1. Barsumian, E. L., C. M. Cunningham, P. M. Schlievert, and D. W. Watson. 1978. Heterogeneity of group A streptococcal pyrogenic exotoxin type B. Infect. Immun. 20:512-518. 2. Barsumian, E. L., P. M. Schlievert, and D. W. Watson. 1978. Non-specific and specific immunological mitogenicity by group

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