Molecular Pathogenesis

Two Novel Superantigens Found in Both Group A and Group C Streptococcus

Thomas Proft, Phillip D. Webb, Vanessa Handley, John D. Fraser
DOI: 10.1128/IAI.71.3.1361-1369.2003

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Family tree of streptococcal and staphylococcal SAgs. The tree was created using ClustalW (39) and the TreeView computer program (30). The novel SAgs belong to group A, together with SPE-C, SPE-G, SPE-J, and SMEZ, but build a separate branch within this group.

  • FIG. 2.
    FIG. 2.

    Multiple alignment of streptococcal SAg protein sequences. The protein sequences of mature proteins were aligned using the ClustalX computer program. The clear box near the C terminus represents a primary zinc binding motif (H-X-D), a feature of all streptococcal toxins except SPE-A and SSA. Structural elements of SMEZ-2 and SPE-C, respectively, are shown below.

  • FIG. 3.
    FIG. 3.

    Stimulation of human T cells with recombinant toxins. PBLs were isolated from human blood samples and incubated with various concentrations of recombinant toxin (in duplicates). After 3 days, 0.1 μCi of [3H]thymidine was added, and cells were incubated for another 24 h before being harvested and counted on a scintillation counter.

  • FIG. 4.
    FIG. 4.

    Competition binding studies with rSPE-L and rSPE-M. LG-2 cells were incubated in duplicate with 1 ng of 125I-labeled recombinant toxin and increasing amounts of unlabeled toxins. After 1 h cells were washed and counted. (A) Competition assay with labeled rSPE-L; (B) competition assay with labeled rSPE-M.

Tables

  • TABLE 1.

    Genotyping of S. pyogenes isolates from New Zealanda

    S. pyogenes isolateM/emmSiteDisease or conditionPCR result with primer for:
    23SspeAspeCspeGbspeHbspeIspeJssaspeLspeMsmezb
    FP 1943M53TSST+−++−−+−−−+
    FP 2658M59TSST+−−+−−+−+−+
    FP 4223M80TSST+−−+−−+−−++
    FP 5417M41TSST++−+−−+−+−+
    FP 5971M57TSST+++++−+−−−+
    1/5045M4TSST++++−−++−−+
    82/20M4SKUlcer+−−+−−++−−+
    85/167M12TSST+−−++++−−−+
    85/314M89WSWound+−−+−−+−+−+
    85/437M81WSInfections eczema+−++−−++−−+
    85/723M22EarOtitis+−++−−++−−+
    86/435M4TSRF+−++−−++−−+
    87/19M12TSST+++++++−−−+
    89/26M1TSAGN++−+−−+−−−+
    90/424M4TSST++++−−++−−+
    94/229M49HVSEndometritis++−++++−−−−c
    94/712M89WSCellulitis+−++−−+−−−+
    95/31(2)M89WSAbscess+−−+−−+−−−+
    96/1M4HVSEndometritis+−++−−++−−+
    9779emm56TSST+−−+−−+−+−+
    9893M82TSST+−+++++−−−+
    10019emm44TSST+−+++++−−−+
    10303emm59TSST+−−+−−+−+−+
    10438ST3018TSST++−+−−+−−−+
    10463emm49TSST++−+−−++−−+
    10649ST2267TSST+−−+−−+−−−+
    10763M88TSST+−++−−++−−+
    10791MNTTSST+−++++++−−+
    10989M87TSST+−−+−−+−−−+
    11070emm65TSST+−−++−+−−−−c
    11152M85TSST+−−++−+−−−−c
    11222M92TSST+++++−+−−++
    11227emm14TSST+−−+−−+−−−+
    11276MNTTSST++−+−−++−−+
    11574ST809TSST+−−+−−+−−−+
    11646ST4547TSST+−−+−−++−−+
    11686M91TSST+−−+−−++−−+
    11789MNTTSST++++−−+−−−+
    ATCCM1WoundND+−+++++−−−+
    33117T28R28NDSTSS+−−+−−+−+−+
        % With positive PCR result100314210026161003215592
    Strain M89
    85/314M89WSWound+−−+−−+−+−+
    95/361M89PSAbscess+−−++−+−+−+
    82/675M89WSWound+−++−−+−+−+
    95/31M89WSAbscess+−−+−−+−−−+
    94/712M89WSCellulitis+−++−−+−+−+
    10846M89TSST++−+−−+−+−+
    89/54M89TSST++−+−−+−+−+
    94/11M89PSAbscess+−−+−−+−+−+
    95/127M89BCCellulitis+−−+−−+−+−+
    84/781M89TSST++−+−−+−−−+
    96/364M89BCBurns+−−+−−+−−−+
        % With positive PCR result1002718100901000730100

    • ↵ a Fifty-one S. pyogenes isolates (38 from New Zealand, 1 from France, 1 ATCC reference strain, and 11 S. pyogenes M89 isolates collected in New Zealand) were genotyped. The results are based on PCR analysis using purified genomic DNA and specific primers for each sag gene. The primers for speL and speM were designed using the DNA sequences of the orthologous speLSe and speMSe genes from S. equi. Site and disease abbreviations are as follows: TS, throat site; WS, wound site; SK, skin; PS, pus site; HVS, high vaginal site; BC, blood culture; ST, sore throat; RF, rheumatic fever; AGN, acute glumerulonephritis; STSS, streptococcal toxic shock; ND, not determined.

    • ↵ b Reported previously (34).

    • ↵ c Positive by Southern blotting.

  • TABLE 2.

    Vβ specificity of recombinant toxins on human PBLsa

    Vβ cDNA% of Vβ enrichment after stimulation with:
    ConASPE-LSPE-M
    1.10.1 8.44 6.75
    2.13.944.464.2
    3.119.576.539.83
    4.10.120.981.41
    5.11.822.783.69
    5.312.0610.213.96
    5.83.314.494.43
    6.31.031.572.15
    6.40.631.411.47
    6.95.964.854.92
    7.30.840.941.1
    7.42.311.711.47
    8.10.30.680.79
    9.11.341.782.05
    12.315.367.768.93
    12.51.171.381.94
    14.10.30.790.95
    15.12.263.924.25
    17.1−0.071.051.12
    18.11.512.543.13
    22a1.12.092.27
    23.10.451.391.41
        Total71.67882.2

    • ↵ a Human PBLs were incubated with recombinant toxin (20 pg/ml) for 4 days. Relative enrichment of Vβ cDNAs was analyzed from RNA of toxin-stimulated and ConA-stimulated PBLs by anchored-primer PCR and reverse dot blot to a panel of 22 different Vβ cDNAs. The figures represent the percentage of each Vβ with respect to total Cβ. Significant responses are underlined.

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KEYWORDS

Bacterial Proteins
Exotoxins
membrane proteins
Streptococcus
Streptococcus pyogenes
superantigens

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