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Clinical and Diagnostic Laboratory Immunology, January 1999, p. 133-136, Vol. 6, No. 1
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Streptococcal DNase B Is Immunologically Identical to Superantigen SpeF but Involves Separate Domains

Anna Eriksson,^1,* Björn Eriksson,^1,2 Stig E. Holm,¹ and Mari Norgren¹

Department of Clinical Bacteriology, Umeå University, S-901 85 Umeå,¹ and Division of Infectious Diseases, Departments of Immunology, Microbiology, Pathobiology, and Infectious Diseases, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge,² Sweden

Received 29 April 1998/Returned for modification 4 August 1998/Accepted 8 October 1998

	ABSTRACT

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The previous suggestion that streptococcal superantigen SpeF might be identical to DNase B was confirmed in this study. Polyclonal SpeF-specific antisera were able to inhibit depolymerization of methyl-green DNA by DNase B. However, T-cell mitogenicity and nuclease activity appear to involve separate immune epitopes on SpeF, since sera with the capacity to neutralize the mitogenic activity of SpeF did not always inhibit the DNase activity.

	TEXT

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Numerous bacterial proteins with superantigenic activity have been found in gram-positive bacteria (14). Several of the superantigens isolated from streptococci and staphylococci seem to have additional functions other than the activation of host T cells (1, 5, 7, 11, 13, 18, 21, 22). SpeF has been reported to have a heat-resistant nuclease activity resembling the properties of streptococcal DNase B (7, 8, 9). During streptococcal infection, DNases are produced and secreted, but very little is known about their involvement in pathogenesis. Among the four known streptococcal nucleases DNase A to DNase D, DNase B is the most common (19), and determination of levels of antibody to DNase B (ADNase B) is often used to confirm a clinical diagnosis of a previous group A streptococcal (GAS) infection. In this report, superantigen SpeF was shown to be immunologically identical to streptococcal DNase B. However, immune epitopes important for antibody-mediated neutralization of the mitogenic and nuclease activities of SpeF were found to be separate.

Immunological identity between SpeF and DNase B. Purified SpeF (16) was able to degrade a DNA PCR product (data not shown). Furthermore, it was shown that the nuclease activity of purified SpeF was comparable to that of streptococcal DNase B according to an assay system from BioSys Inova (Stockholm, Sweden). Briefly, DNase B or SpeF was added to methyl-green-conjugated DNA, and the depolymerization of DNA was determined optically (4). The hypothesis that SpeF and DNase B are identical was further tested by applying rabbit polyclonal antisera in this assay. Antisera against SpeA, SpeB, and SpeF were raised in rabbits, and SpeF-specific synthetic peptides conjugated to Keyhole limpet hemocyanin (Scandinavian Peptide Synthesis, Köping, Sweden) were used as described previously (2). In the ADNase B assay, a serum sample with inhibitory capacity at a dilution of 1:400 is regarded as positive (6). As a negative control, rabbit polyclonal antisera specific for SpeA and SpeB were used. A human antiserum known to inhibit DNase B could inhibit the nuclease activity of SpeF. The SpeF antisera could also inhibit streptococcal DNase B activity at a dilution of 1:800 (Table 1). No inhibitory activity could be detected with the SpeA and SpeB antisera, which confirmed that DNase B inhibition was specific for the rabbit anti-SpeF sera. None of the SpeF-specific peptide antisera could inhibit DNase B (data not shown); thus, the nuclease activity of SpeF might be dependent on conformational rather than linear epitopes.

View this table: [in this window] [in a new window]   TABLE 1.   DNase B activity of SpeF

Separate immune epitopes determine the mitogenic and nuclease activities of SpeF. In order to investigate whether the immune response patterns with regard to the two activities of SpeF differed among the patients, sera from individuals with ongoing GAS infections with various degrees of clinical severity were analyzed. Ninety human serum samples were tested for streptococcal DNase B titers as well as their ability to neutralize the mitogenic activity of SpeF. Acute-phase sera were drawn within 5 days of admission from patients with GAS bacteremia or GAS erysipelas at the Department of Infectious Diseases, Huddinge University Hospital, Huddinge, Sweden, during 1983 to 1995 (3, 15). Sera from patients with uncomplicated GAS tonsillitis were collected at Mariehems Health Center, Umeå, Sweden, in 1989, and 40 serum samples from healthy blood donors were collected at the Department of Serology, Umeå University Hospital, in 1994. The ability of human sera to neutralize SpeF-induced proliferation of human peripheral blood mononuclear cells (PBMCs) was determined as described previously (17). PBMCs were incubated in RPMI 1640 (GIBCO-BRL, Stockholm, Sweden) supplemented with 2 mM L-glutamine (GIBCO-BRL), 100 µg of gentamycin per ml (Sigma, St. Louis, Mo.), 7.5 ng of SpeF per ml, 2.5% human serum, and 7.5% fetal calf serum (FCS) (KEBO Lab AB, Stockholm, Sweden). As a negative control, SpeF-stimulated PBMCs incubated with 10% FCS were used. SpeF-stimulated PBMCs incubated with 250 µg of -globulin per ml (equivalent to antibody levels in sera) were used as a positive control. For determination of background cpm levels, PBMCs in RPMI medium supplemented with 10% FCS were used. All experiments were done in triplicate.

The history of GAS infections among blood donors was undocumented, and 12 of 40 (30%) had no reactivity in either the mitogen assay or nuclease neutralization test. These sera were not included in the comparisons made. The most striking difference between the sera from healthy donors and those from patients with documented GAS infections was noted in sera with a small capacity to neutralize SpeF mitogenicity (50% or less) and with DNase B inhibition titers at or below 200. Seven of twenty-eight (25%) of the serum samples with these immune reactivities were identified in the group of blood donors, while only 2 of 56 (3%) serum samples from the patient groups had the same immune pattern. Sera from patients suffering from bacteremias with various types of clinical focus included the largest group of double reactives: 11 of 20 (55%), compared to only 3 of 11 (27%) and 3 of 8 (37%) serum samples from patients with streptococcal toxic shock syndrome (STSS) and patients with bacteremia with erysipelas, respectively. In sera from patients with uncomplicated tonsillitis, only 1 of 7 (14%) serum samples shared the same pattern (Tables 2 and 3). However, only 4 of 17 serum samples from patients with uncomplicated GAS infections had ADNase B titers above 1:400.

View this table: [in this window] [in a new window]   TABLE 2.   Antistreptococcal DNase B activity of human sera in relation to the ability to neutralize SpeF-induced lymphocyte proliferation

View this table: [in this window] [in a new window]   TABLE 3.   Antistreptococcal DNase B activity in sera from different patient categories in relation to the ability to neutralize SpeF-induced mitogenicity

Fifty percent (28 of 56) of the serum samples with a large capacity to neutralize SpeF mitogenicity were not able to inhibit streptococcal DNase B activity, irrespective of the patient category (Table 2). The observed immune reactivities in patient sera indicated that antibody epitopes involved in neutralization of the two activities of SpeF were located on separate domains of the protein. Age is among several factors that may influence antibody levels in sera after a GAS infection (10, 20). In this study, age did not influence the immune response: 9 of 17 (56%) serum samples with ADNase B titers below 1:400 were found in patients over 70 years of age, compared to 23 of 36 (64%) serum samples in the group 15 to 70 years of age (data not shown). In acute-phase sera from patients with documented GAS infections, only 2 of 56 serum samples had low SpeF mitogen neutralization and positive DNase B titers, while 22 of 56 samples with at least 50% neutralization of the mitogenic activity and with DNase B titers at or equal to 200 were found. Thus, the antibody response against the SpeF mitogen-related epitopes seemed to be more long lasting. High antibody titers in antiserum to DNase B are generally regarded as an indication of a recent history of GAS infection (4, 6), while antibodies to the mitogen-related epitopes, according to the results in the present report, persist at higher levels over time. Thus, at least in adults, the presence of DNase B titers in human serum may be a better indicator of recent GAS infections than the SpeF neutralization levels.

Conclusions. In this study, antiserum raised against SpeF was shown to inhibit the nuclease activity of DNase B, and a DNase B-neutralizing antiserum could inhibit SpeF's nuclease activity, which showed that the two enzymes were immunologically identical. The fact that none of the antipeptide sera could inhibit the DNase activity indicated that the active site may be conformational. It was equally common that either mitogen neutralization activity or DNase B inhibition, both of these activities, or none of these activities were detected in individual sera. The results indicated that the T-cell mitogenicity of SpeF was independent of its nuclease activity.

	ACKNOWLEDGMENTS

This work was supported by grants from the Swedish Medical Research Council (10844) and the Västerbottens Läns Landsting to S.E.H. and M.N. M.N. was also supported by Umeå University Medical Faculty, the Wiberg Foundation, and the Magnus Bergvall Foundation. A.E. was supported by a grant from the Kempe Foundation.

We thank H. Edebro for technical assistance.

	FOOTNOTES

^* Corresponding author. Mailing address: Department of Clinical Bacteriology, Umeå University, S-901 85 Umeå, Sweden. Phone: 46 90 785-1121. Fax: 46 90 785-2225. E-mail: anna.eriksson{at}climi.umu.se.

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Clinical and Diagnostic Laboratory Immunology, January 1999, p. 133-136, Vol. 6, No. 1
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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