Staphylococcal enterotoxin-like toxins U2 and V, two new staphylococcal superantigens arising from recombination within the enterotoxin gene cluster

Abstract

To test the hypothesis that the Staphylococcus aureus enterotoxin gene cluster (egc) can generate new enterotoxin genes by recombination, we analyzed the egc locus in a broad panel of 666 clinical isolates of S. aureus. egc was present in 63% of isolates, confirming its high prevalence. The archetypal organization of the egc locus, consisting of five enterotoxin genes plus two pseudogenes, was found in 409 of 421 egc-positive strains. The egc locus was incomplete in a few strains and occasionally harbored an insertion sequence and transposase genes. These strains may represent evolutionary intermediates of the egc locus. One strain with an atypical egc locus produced two new enterotoxins, designated SElV and SElU2, generated by (i) recombination between selm and sei, producing selv, and (ii) a limited deletion in the varphient1-varphient2 pseudogenes, producing selu2. Recombinant SElV and SElU2 had superantigen activity, as they specifically activated the T-cell families Vbeta 6, Vbeta 18, and Vbeta 21 (SElV) and Vbeta 13.2 and Vbeta 14 (SElU2). Immunoscope analysis showed a Gaussian CDR3 size distribution of T-cell receptor Vbeta chain junctional transcripts of expanded Vbeta subsets in toxin-stimulated cultures, reflecting a high level of polyclonality. These data show that egc is indeed capable of generating new superantigen genes through recombination.

FIG. 1.

Organization of the egc locus in reference and atypical strains. A900322 (GenBank accession number AFP285760) is the egc reference strain. A900624 harbors two new enterotoxin genes, selu2 and selv. LY19991287, A950227, LY19991222, A980341, and A940440 have an insertion sequence (Rve-like 1-transposase 8 or Rve-like 2-IstB-like). These sequences are shown schematically below the genes into which they are inserted.

FIG. 2.

Genetic organization of the strain A900624 egc locus in comparison with the archetypal egc locus of reference strain A900322. Black arrows indicate the locations of primers used for the SElV (R-SElV 1 and R-SElV 2) and SElU2 (R-SElU2 1 and R-SElU2 2) recombinant strategy.

FIG. 3.

Analysis of selv and selu2 transcripts by RT-PCR. cDNA was prepared from S. aureus A900624 total RNA using the RT primers shown (RT), followed by PCR using primer pairs A to E (boxed). Lanes A to E, results obtained using the corresponding primer pairs. Lane 1, 1-kb molecular size marker; lane 2, RT-PCR negative control (heat inactivation of reverse transcriptase); lanes A1 to E1, RT-PCR from extract of A900624; lanes A2 to E2, PCR positive control (A900624 DNA as template).

FIG. 4.

TCR Vβ repertoire analysis of superantigen-stimulated PBMC by the Immunoscope approach. (A) Quantitative Vβ repertoire determined by real-time PCR analysis on day 0 (D0) and day 14 (D14) following stimulation with SElV or SElU2. The x axis indicates Vβ families and the y axis their relative frequency of usage. Selective Vβ expansion was considered to occur when the D14/D0 Vβ frequency ratio was >3. (B) Immunoscope profiles of the fluorescent Vβ-Cβ runoff products obtained with 14-day-stimulated mononuclear cells. The x axis indicates the CDR3 length. Only profiles corresponding to Vβ expansion are shown.

FIG. 5.

Flow cytometric kinetic analysis of T cell stimulation by SElU2. Time courses of Vβ 13.2 (A) and Vβ 14 (B) expression in blast cells (closed circles) and small cells (open circles), identified with forward scatter/side scatter criteria, after stimulation of PBMC with 500 ng/ml SElU2 are shown. Vβ expansion was determined by flow cytometry. Data are percentages of CD3 T cells expressing the corresponding Vβ chains.

FIG. 6.

Immunoscope analysis of the time course of T-cell stimulation by SElU2. The quantitative Vβ repertoire determined with real-time PCR analysis on days 0, 1, 4, and 6 following stimulation with SElU2 is shown. The x axis indicates Vβ families and the y axis their relative frequency of usage.