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. 2021 Jun 4:12:685343.
doi: 10.3389/fmicb.2021.685343. eCollection 2021.

Incidence and Effects of Acquisition of the Phage-Encoded ssa Superantigen Gene in Invasive Group A Streptococcus

Chuan Chiang-Ni  1   2   3   4 , Yen-Shan Liu  2 , Chieh-Yu Lin  2 , Chih-Yun Hsu  1 , Yong-An Shi  2 , Yi-Ywan M Chen  1   2   3 , Chih-Ho Lai  1   2   3 , Cheng-Hsun Chiu  2   3   5
Affiliations

Incidence and Effects of Acquisition of the Phage-Encoded ssa Superantigen Gene in Invasive Group A Streptococcus

Chuan Chiang-Ni et al. Front Microbiol. .

Abstract

The acquisition of the phage-encoded superantigen ssa by scarlet fever-associated group A Streptococcus (Streptococcus pyogenes, GAS) is found in North Asia. Nonetheless, the impact of acquiring ssa by GAS in invasive infections is unclear. This study initially analyzed the prevalence of ssa+ GAS among isolates from sterile tissues and blood. Among 220 isolates in northern Taiwan, the prevalence of ssa+ isolates increased from 1.5% in 2008-2010 to 40% in 2017-2019. Spontaneous mutations in covR/covS, which result in the functional loss of capacity to phosphorylate CovR, are frequently recovered from GAS invasive infection cases. Consistent with this, Phostag western blot results indicated that among the invasive infection isolates studied, 10% of the ssa+ isolates lacked detectable phosphorylated CovR. Transcription of ssa is upregulated in the covS mutant. Furthermore, in emm1 and emm12 covS mutants, ssa deletion significantly reduced their capacity to grow in human whole blood. Finally, this study showed that the ssa gene could be transferred from emm12-type isolates to the emm1-type wild-type strain and covS mutants through phage infection and lysogenic conversion. As the prevalence of ssa+ isolates increased significantly, the role of streptococcal superantigen in GAS pathogenesis, particularly in invasive covR/covS mutants, should be further analyzed.

Keywords: CovR/CovS; SSA; group A Streptococcus; invasive GAS infection; streptococcal superantigen; superantigen.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Prevalence of emm type, ssa+, and erythromycin-resistant (ErmR) isolates in 2008–2019. (A) The emm type of ssa+ isolates in 2008–2019. (B) Prevalence of emm1-type, emm12-type, ssa+, and ErmR isolates in 2008–2019. (C) Prevalence of emm1 and emm12 ssa+ isolates in 2008–2019. (D) Prevalence of emm1 and emm12 ErmR isolates in 2008–2019.
Figure 2
Figure 2
Phosphorylated CovR and streptolysin O (SLO) expression and ssa transcription in selected clinical isolates, covS isogenic mutants, vector-control strains, and covR/covS trans-complementary strains. (A) Detection of the phosphorylated CovR in the selected clinical isolates using Phostag western blot assay. A20 and its covS (∆covS) and covR (∆covR) mutants were utilized as experimental controls. (B) SLO secretion in selected ssa-positive isolates. SLO was detected in the culture supernatants using western blot analysis. (C) ssa transcription in the SPY131 (emm1) and SPY128 (emm12; Wt) and their covS mutants (∆covS). (D) Phosphorylated CovR expression and ssa transcription in vector-control (∆covS+Vec) and the covR/covS trans-complementary (∆covS+PcovR/S) strains. RNA was extracted for reverse transcription-PCR (RT-qPCR) analysis. *p < 0.05. CovR~P, phosphorylated CovR; CovR, nonphosphorylated CovR. Total protein is served as the internal loading control.
Figure 3
Figure 3
Growth activity of SPY131 (emm1), SPY128 (emm12), their ssa mutants (∆ssa), covS mutants (∆covS), and covS and ssa double mutants (∆covS∆ssa) in human whole blood. Group A Streptococcus (GAS) strains were incubated with whole blood from donors for 1.5 h. The number of surviving bacteria in human blood was determined by plating and enumerating the colony forming units (CFUs), which determined growth relative to the initial inoculum. *p < 0.05.
Figure 4
Figure 4
PCR and Southern blot analyses of the ssa mutant and lysogenic convertants. (A) Detection of the chloramphenicol cassette (cm) and the chromosomal speB gene in the chromosomal DNA and filtrated supernatant after mitomycin C treatment using PCR. SCN279, the phage donor. A20 (emm1-type), the recipient. (B) Southern blot hybridization of lysogenic convertants. The upper panel shows the HindIII site and the predicted sizes for cm probe hybridization according to ΦHKU.vir (HKU360, NCBI accession no. CP009612) and A20 (NCBI accession no. CP003901.1) sequence. The recipient strains (A20, CovSH280A, and ∆covS) showed no signal for cm probe hybridization and used as the experimental negative control. M, DNA marker.
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