Genomic analysis and identification of a novel superantigen, SargEY, in Staphylococcus argenteus isolated from atopic dermatitis lesions

Abstract

During surveillance of Staphylococcus aureus in lesions from patients with atopic dermatitis (AD), we isolated Staphylococcus argenteus, a species registered in 2011 as a new member of the genus Staphylococcus and previously considered a lineage of S. aureus. Genome sequence comparisons between S. argenteus isolates and representative S. aureus clinical isolates from various origins revealed that the S. argenteus genome from AD patients closely resembles that of S. aureus causing skin infections. We previously reported that 17%-22% of S. aureus isolated from skin infections produce staphylococcal enterotoxin Y (SEY), which predominantly induces T-cell proliferation via the T-cell receptor (TCR) Vα pathway. Complete genome sequencing of S. argenteus isolates revealed a gene encoding a protein similar to superantigen SEY, designated as SargEY, on its chromosome. Population structure analysis of S. argenteus revealed that these isolates are ST2250 lineage, which was the only lineage positive for the SEY-like gene among S. argenteus. Recombinant SargEY demonstrated immunological cross-reactivity with anti-SEY serum. SargEY could induce proliferation of human CD4⁺ and CD8⁺ T cells, as well as production of TNF-α and IFN-γ. SargEY showed emetic activity in a marmoset monkey model. S_argEY and SET (a phylogenetically close but uncharacterized SE) revealed their dependency on TCR Vα in inducing human T-cell proliferation. Additionally, TCR sequencing revealed other previously undescribed Vα repertoires induced by SEH. S_argEY and SEY may play roles in exacerbating the respective toxin-producing strains in AD.

Importance: Staphylococcus aureus is frequently isolated from active lesions of atopic dermatitis (AD) patients. We reported that 17%-22% of S. aureus isolated from AD patients produced a novel superantigen staphylococcal enterotoxin Y (SEY). Unlike many S. aureus superantigens that activate T cells via T-cell receptor (TCR) Vß, SEY activates T cells via TCR Vα and stimulates cytokine secretion. Staphylococcus argenteus was isolated from AD patients during the surveillance for S. aureus. Phylogenetic comparison of the genome indicated that the isolate was very similar to S. aureus causing skin infections. The isolate encoded a SEY-like protein, designated SargEY, which, like SEY, activated T cells via the TCR Vα. ST2250 is the only lineage positive for SargEY gene. ST2250 S. argenteus harboring a superantigen SargEY gene may be a novel staphylococcal clone that infects human skin and is involved in the exacerbation of AD.

Keywords: Staphylococcus; TCR Vα; argenteus; enterotoxins; human T-cell; superantigen.

Fig 1

Complete Genome Sequence of ST2250 s_(arg)ey-positive S. argenteus SARG0275. Circular representation of the S. argenteus chromosome and plasmid. Beginning with the outer region, the circles show nucleotide positions in base parts, predicted CDSs transcribed on the forward (clockwise) and reverse (counterclockwise) DNA strands, and percentage GC content (purple and green, respectively, represent the regions with higher and lower GC contents compared to the average value for the entire genome), and the GC skew curve. The position of s_(arg)ey on the chromosome is indicated by balloon shapes. Black up-pointing triangles indicate the inverted repeat. Arrows indicate coding sequences (CDS); mvaA gene encoding 3-hydroxy-3-methylglutaryl CoA reductase, mvaS gene encoding 3-hydroxy-3-methylglutaryl CoA synthase, ogt gene encoding methylated-DNA-[protein]-cysteine S-methyltransferase, clpL gene encoding ATP-dependent Clp protease ATP-binding subunit ClpL.

Fig 2

Phylogenetic analysis of sey-positive S. argenteus and staphylococcal enterotoxin gene profiles. A maximum likelihood phylogenetic tree of 244 S. argenteus strains with heatmaps indicating the metadata is shown. Only strains for which Illumina read data were available were analyzed. Metadata included AD patient isolates from Keio University in this study (Red), SCCmecIV cassette (Blue), CRISPR/Cas (Blue), coagulase type (coa-type), and staphylococcal enterotoxin (ent) genes. In the matrix of ent genes, blue indicates the presence of these genes and the presence of the sey-like gene is highlighted by red. The presence or absence of the toxin genes was determined using Abricate software v1.0.1. Parentheses in each cluster indicate the ST types of S. argenteus.

Fig 3

Phylogenetic tree of S_argEY and staphylococcal enterotoxin members in S. aureus with respective available accession numbers of the protein database. The tree was constructed by ClustalW alignment followed by the UPGMA method using Mega software.

Fig 4

T-cell stimulation activities of S_argEY. CFSE-labeled human PBMCs were stimulated with 10 ng/mL of S_argEY or PBS for 5 days. Flow cytometry analysis was performed to evaluate the percentage of dividing T cells in response to S_argEY stimulation by gating CD4^{+ high} CSFE^low and CD8^{+ high} CSFE^low (red box) (A). The percentages of proliferating CD4⁺ and CD8⁺ T cells were calculated and compared with PBS as negative control (B). Cytokine production was assessed by ELISA of culture supernatant from PBMCs stimulated with the toxin or PBS (C). The bars represent the mean and standard errors in three to four experiments.

Fig 5

Analysis of TCR Vα/β sequencing from total RNA samples after in vitro stimulation of human PBMCs with 10 ng/ml of SEB, SEH, and S_argEY or 100 ng/mL SET. Transcripts from TRAV (A) and TRBV (B) gene usage. Data represent the mean values and SEM of three healthy donors.