Identification of single nucleotide polymorphisms associated with hyperproduction of alpha-toxin in Staphylococcus aureus

Abstract

The virulence factor α-toxin (hla) is needed by Staphylococcus aureus in order to cause infections in both animals and humans. Although the complicated regulation of hla expression has been well studied in human S. aureus isolates, the mechanisms of of hla regulation in bovine S. aureus isolates remain undefined. In this study, we found that many bovine S. aureus isolates, including the RF122 strain, generate dramatic amounts of α-toxin in vitro compared with human clinical S. aureus isolates, including MRSA WCUH29 and MRSA USA300. To elucidate potential regulatory mechanisms, we analyzed the hla promoter regions and identified predominant single nucleotide polymorphisms (SNPs) at positions -376, -483, and -484 from the start codon in α-toxin hyper-producing isolates. Using site-directed mutagenesis and hla promoter-gfp-luxABCDE dual reporter approaches, we demonstrated that the SNPs contribute to the differential control of hla expression among bovine and human S. aureus isolates. Using a DNA affinity assay, gel-shift assays and a null mutant, we identified and revealed that an hla positive regulator, SarZ, contributes to the involvement of the SNPs in mediating hla expression. In addition, we found that the bovine S. aureus isolate RF122 exhibits higher transcription levels of hla positive regulators, including agrA, saeR, arlR and sarZ, but a lower expression level of hla repressor rot compared to the human S. aureus isolate WCUH29. Our results indicate α-toxin hyperproduction in bovine S. aureus is a multifactorial process, influenced at both the genomic and transcriptional levels. Moreover, the identification of predominant SNPs in the hla promoter region may provide a novel method for genotyping the S. aureus isolates.

Figure 1. Hemolytic analysis and SDS-PAGE analysis of the expression profiles of exported proteins.

(A) Hemolytic analysis on Sheep Blood Agar. SDS-PAGE analysis of the expression profiles of the exported proteins. (B) Human S. aureus isolates, Lane 1, WCUH29; Lane 2, NRS105; Lane 3, NRS194; Lane 4, NRS 237; Lane 5, NRS243; Lane 6, NRS248; Lane 7, NRS384; Lane 8, MW2(956); Lane 9, USA300(1371); M, Precision Plus Protein Standard. (C) Alpha-toxin hyper-producing bovine mastitis S. aureus isolates. Lane 1, RF122; Lane 2, BSa39; Lane 3, BSa55; Lane 4, BSa60; Lane 5, BSa67; Lane 6, BSa68; Lane 7, BSa74; Lane 8, BSa97. M, Precision Plus Protein Standard. (D) Bovine mastitis S. aureus isolates. Lane 1, RF122; Lane 2, BSa12; Lane 3, BSa22; Lane 4, BSa28; Lane 5, BSa83; Lane 6, BSa110. M, Precision Plus Protein Standard. Arrow indicates α-toxin.

Figure 2. Structural alignments of Bovine and Human S. aureus isolates.

Structural alignments with homologues of hla promoter region from sequenced bovine and published human S. aureus genomes. The symbol “–” represents the upstream region from the start codon of hla. The boxed nucleotide represents the major difference between bovine S. aureus isolates and the human S. aureus isolate. (A) Human S. aureus isolates (B) Bovine mastitis S. aureus isolates.

Figure 3. Influence of SNPs hla promoter-luxABCDE reporters on bioluminescence intensity of S. aureus WCUH29.

The maximal light intensity values are given as relative light units (RLU). The symbol “*” indicates a significant difference (P≤0.05) between SaWH1207 and all other strains.

Figure 4. Impact of hla promoter and nucleotide mutations on hla expression in RF122 strain.

(A) SDS-PAGE analysis of hla expression of the S. aureus RF122 strain carrying different hla promoter-gfp fusions. Lane 1, RF122 control; Lane 2, SaRF1207 (RF122 hla promoter-gfp fusion); Lane 3, SaRF1307 (G-376 mutated RF122 hla promoter-gfp fusion); Lane 4, SaRF1407 (AT-484-483 mutated RF122 hla promoter-gfp fusion); Lane 5, SaRF1507 (G-376 AT-484-483 mutated RF122 hla promoter-gfp fusion); Lane 6, SaRF1107 (WCUH29 hla promoter-gfp fusion); Lane 7, WCUH29 control. M, Precision Plus Protein Standard. (B) Western blot analysis of Gfp expression from different hla promoter-gfp fusions in RF122. Lane 1, SaRF1507; Lane 2, SaRF1307; Lane 3, SaRF1407; Lane 4, SaRF1207; Lane 5, SaRF1107. Black triangle indicates the increase of intensity of reaction band. (C) SDS-PAGE analysis of hla expression of the S. aureus RF122 sarZ null mutant (BsasarZ). Lane 1, RF122 control, Lane 2, BsasarZ control, Lane3, BSasarZ1107 (WCUH29 hla promoter-gfp fusion); Lane 4, BSasarZ1207 (RF122 hla promoter-gfp fusion); Lane 5, BSasarZ1307 (G-376 mutated RF122 hla promoter-gfp fusion); Lane 5, BSasarZ1407 (AT-484-483 mutated RF122 hla promoter-gfp fusion); Lane 7, BSasarZ1507 (G-376 AT-484-483 mutated RF122 hla promoter-gfp fusion); M, Precision Plus Protein Standard. (D) Western blot analysis of Gfp expression from different hla promoter-gfp fusions in BSasarZ. Lane 1, RF122 wild-type control; Lane 2, BSasarZ1107; Lane 3, BSasarZ1207; Lane 4, BSasarZ1307; Lane 5, BSasarZ1407; Lane 6, BSasarZ1507. (E) Hemolytic assay on Sheep Blood Agar.