Characterization of a novel covS SNP identified in Australian group A Streptococcus isolates derived from the M1UK lineage

Abstract

Group A Streptococcus (GAS) is a human-adapted pathogen responsible for a variety of diseases. The GAS M1_UK lineage has contributed significantly to the recently reported increases in scarlet fever and invasive infections. However, the basis for its evolutionary success is not yet fully understood. During the transition to systemic disease, the M1 serotype is known to give rise to spontaneous mutations in the control of virulence two-component regulatory system (CovRS) that confer a fitness advantage during invasive infections. Mutations that inactivate CovS function result in the de-repression of key GAS virulence factors such as streptolysin O (SLO), a pore-forming toxin and major trigger of inflammasome/interleukin-1β-dependent inflammation. Conversely, expression of the streptococcal cysteine protease SpeB, which is required during initial stages of colonization and onset of invasive disease, is typically lost in such mutants. In this study, we identified and characterized a novel covS single nucleotide polymorphism detected in three separate invasive M1_UK isolates. The resulting CovS^Ala318Val mutation caused a significant upregulation of SLO resulting in increased inflammasome activation in human THP-1 macrophages, indicating an enhanced inflammatory potential. Surprisingly, SpeB production was unaffected. Site-directed mutagenesis was performed to assess the impact of this mutation on virulence and global gene expression. We found that the CovS^Ala318Val mutation led to subtle, virulence-specific changes of the CovRS regulon compared to previously characterized covS mutations, highlighting an unappreciated level of complexity in CovRS-dependent gene regulation. Continued longitudinal surveillance is warranted to determine whether this novel covS mutation will expand in the M1_UK lineage.IMPORTANCEThe M1_UK lineage of GAS has contributed to a recent global upsurge in scarlet fever and invasive infections. Understanding how GAS can become more virulent is critical for infection control and identifying new treatment approaches. The two-component CovRS system, comprising the sensor kinase CovS and transcription factor CovR, is a central regulator of GAS virulence genes. In the M1 serotype, covRS mutations are associated with an invasive phenotype. Such mutations have not been fully characterized in the M1_UK lineage. This study identified a novel covS mutation in invasive Australian M1_UK isolates that resulted in a more nuanced virulence gene regulation compared to previously characterized covS mutations. A representative isolate displayed upregulated SLO production and triggered amplified interleukin-1β secretion in infected human macrophages, indicating an enhanced inflammatory potential. These findings underscore the need for comprehensive analyses of covRS mutants to fully elucidate their contribution to M1_UK virulence and persistence.

Keywords: CovRS; SLO; SNP; SpeB; Streptococcus pyogenes; inflammation; interleukin-1β; transcriptional regulation; two-component system.

Fig 1

Protein expression of SLO and SpeB in M1 GAS strains and SP1450 isogenic mutants. All strains were grown in Todd-Hewitt broth supplemented with 1% yeast extract to the late exponential growth phase (optical density at 600 nm = 0.8). Supernatant protein was purified using trichloroacetic acid precipitation. Expression of SLO and SpeB was visualized via immunoblotting. (A) Representative Western blot of n = 3 biological repeats; upper panel shows SLO; lower panel shows SpeB. Gaps between lanes indicate that additional samples have been cropped out of the full blot image (see Fig. S2). (B) Quantification of SLO expression of n = 3 immunoblots, which was performed using ImageJ. Error bars represent SEM. Significance was analyzed by Tukey one-way analysis of variance. ****P < 0.0001. ns, not significant.

Fig 2

CovS^Ala318Val increases slo expression in M1 GAS. To account for possible RNA degradation at later growth stages, strains were grown in Todd-Hewitt broth supplemented with 1% yeast liquid culture to the mid-exponential growth phase (optical density at 600 nm = 0.4). Quantified RNA transcripts of slo (A) and speB (B), relative to the gyrA gene, were normalized to 5448. Precipitated preparations of total culture supernatant protein were probed for SLO and SpeB by immunoblotting. (C) Representative Western blot of n = 3 biological repeats; upper panel shows SLO, and lower panel shows SpeB. (D) SLO protein expression in n = 3 replicates was quantified with ImageJ. All error bars represent SEM. Significance was analyzed by unpaired Welch t-test comparing wild-type strains and respective isogenic mutants. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3

RNA sequencing analysis investigating the impact of CovS^Ala318Val on global gene transcription in 5448 and SP1380. (A and B) Volcano plots highlighting upregulated genes in red and downregulated genes in blue (P  <  0.05, ≥1 log₂ fold change; n  =  3). (C) Venn-diagram showing overlapping and individual genes that are differentially expressed in both comparison pairs (upregulated in red, downregulated in blue).

Fig 4

SP1450 triggers enhanced IL-1β secretion from differentiated THP-1 macrophages. Cells were infected with M1 GAS strains at MOI = 25. Nigericin, a potent bacterial ionophore, was used as a positive control for inflammasome activation. At the indicated timepoints, assay supernatants were collected and analyzed for (A) IL-1β and (B) LDH release. Data are presented as the mean percentage of the 2 h nigericin control for IL-1β release (A) or as mean percentage of complete cell lysis induced by Triton X-100 for LDH release (B) ± SEM of n = 3 independent replicate experiments. Significance was calculated using Brown-Forsythe and Welch analysis of variance tests. *P < 0.05.