Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens

Abstract

Previous studies have demonstrated that a proportion of Staphylococcus aureus isolates from bovine mastitis coproduce toxic shock syndrome toxin (TSST) and staphylococcal enterotoxin C (SEC). In this study, molecular genetic analysis of one such strain, RF122, revealed the presence of a 15,891-bp putative pathogenicity island (SaPIbov) encoding the genes for TSST (tst), the SEC bovine variant (sec-bovine), and a gene (sel) which encodes an enterotoxin-like protein. The island contains 21 open reading frames specifying hypothetical proteins longer than 60 amino acids including an integrase-like gene. The element is bordered by 74-bp direct repeats at the left and right junctions, and the integration site lies adjacent to the 3' end of the GMP synthase gene (gmps) in the S. aureus chromosome. SaPIbov contains a central region of sequence identity with the previously characterized tst pathogenicity island SaPI1 (J. A. Lindsay et al., Mol. Microbiol. 29:527-543, 1998). A closely related strain, RF120, of the same multilocus enzyme electrophoretic type, random amplified polymorphic DNA type, and ribotype, does not contain the island, implying that the element is mobile and that a recent insertion/deletion event has taken place. TSST and TSST/SEC-deficient mutants of S. aureus strain RF122 were constructed by allele replacement. In vitro bovine Vbeta-specific lymphocyte expansion analysis by culture supernatants of wild-type strains and of tst and sec-bovine allele replacement mutants revealed that TSST stimulates BTB13-specific T cells whereas SEC-bovine stimulates BTB93-specific T cells. This suggests that the presence of SaPIbov may contribute to modulation of the bovine immune response.

FIG. 1

Mapping SaPIbov by PCR and Southern hybridization. Vectorette PCR was performed on DNA flanking the 6.5-kb HindIII fragment harboring tst and sec. DNA was cleaved with BclI, ligated with the Vectorette cassette, and subjected to PCR with outward-directed primer VL or VR and a primer specific for the Vectorette unit attached to the end of each fragment. These PCR products provided probes A and B, which were used to analyze genomic DNA of strain RF122 tst sec (lanes 1 and 3) and wild-type strain RF120 (lanes 2 and 4). Primers VR and JR1 were used to amplify the 9-kb right junction fragment. The left and right junctions of SaPIbov are denoted by open boxes.

FIG. 2

Southern blot analysis of HindIII-digested genomic DNA from strain RF120 (lane 1) and strain RF122 tst sec (lane 2) using pJRF101 containing the 6.5-kb HindIII fragment as a probe. The probe hybridized to a 6.5-kbp fragment of RF122 and also, unexpectedly, to a second 3-kb fragment of both strains, suggesting that the 6.5-kb tst/sec fragment contains homologous sequences elsewhere in the genome.

FIG. 3

The 15,891-bp SaPIbov. Black arrows indicate ORFs of unknown function. White arrows indicate genes referred to in the text: sec, sel, int, tst, and gmps. The hatched boxes indicate positions of the direct repeats (DR), the sequence of which is given. Open boxes represent the erythromycin (erm) and tetracycline (tet) cassettes used to construct the TSST⁻ and TSST⁻ SEC⁻ mutants. Primer juncf3 was used in Vectorette PCR to amplify DNA containing the SaPIbov integration site in strain RF120 after digestion with AluI.

FIG. 4

Southern blot hybridization analysis of tst and tst sec mutants. Genomic DNA from wild-type strain RF122 (lane 1), mutant RF122-1 tst (lane 2), and double mutant RF122-2 tst sec (lane 3) was cleaved with HindIII, separated by agarose electrophoresis, and transferred to a nylon membrane. It was hybridized with a probe specific for tst covering the site of insertion of the tet marker.

FIG. 5

Representative analysis of boVβ expansion in bovine lymphocyte cultures. Bovine lymphocyte cultures were treated with protein preparations recovered from cultures of S. aureus RF122 (wt [wild-type]), S. aureus RF122-1 (Δtst), and S. aureus RF122-2 (Δtst/sec). Fold increase reflects the fold difference in measured PDU for each boVβ. This value was determined by quantifying reaction products (in PDU) generated from treated lymphocytes to those from an identical but untreated cell culture. All boVβ PDU values were normalized by quantifying bovine lymphocyte TCR Cα levels.