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 |
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 |
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.
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
|
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Top
Abstract
Text
(IL-1
