A Novel CovS Variant Harbored by a Colonization Strain Reduces Streptococcus pyogenes Virulence

Abstract

Streptococcus pyogenes, also known as group A Streptococcus, causes a wide variety of diseases ranging from mild noninvasive to severe invasive infections. To identify possible causes of colonization-to-invasive switches, we determined the genomic sequences of 10 isolates from five pairs each composed of an invasive strain and a carriage strain originating from five infectious clusters. Among them, one pair displayed a single-nucleotide difference in covS, encoding the sensor histidine kinase of the two-component CovRS system that controls the expression of 15% of the genome. In contrast to previously described cases where the invasive strains harbor nonfunctional CovS proteins, the carriage strain possessed the mutation covST115C, leading to the replacement of the tyrosine at position 39 by a histidine. The CovSY39H mutation affected the expression of the genes from the CovR regulon in a unique fashion. Genes usually overexpressed in covS mutant strains were underexpressed and vice versa. Furthermore, the covS mutant strain barely responded to the addition of the CovS-signaling compounds Mg²⁺ and LL-37. The variations in the accumulation of two virulence factors paralleled the transcription modifications. In addition, the covST115C mutant strain showed less survival than its wild-type counterpart in murine macrophages. Finally, in two murine models of infection, the covS mutant strain was less virulent than the wild-type strain. Our study suggests that the CovSY39H protein compromises CovS phosphatase activity and that this yields a noninvasive strain. IMPORTANCE Streptococcus pyogenes, also known as group A Streptococcus, causes a wide variety of diseases, leading to 517,000 deaths yearly. The two-component CovRS system, which responds to MgCl₂ and the antimicrobial peptide LL-37, controls the expression of 15% of the genome. Invasive strains may harbor nonfunctional CovS sensor proteins that lead to the derepression of most virulence genes. We isolated a colonization strain that harbors a novel covS mutation. This mutant strain harbored a transcriptome profile opposite that of other covS mutant strains, barely responded to environmental signals, and was less virulent than the wild-type strain. This supports the importance of the derepression of the expression of most virulence genes, via mutations that impact the phosphorylation of the regulator CovR, for favoring S. pyogenes invasive infections.

Keywords: adaptation; covRS; environmental signal; group A Streptococcus; infection; two-component regulatory system.

FIG 1

Localization of the Y39H mutation. (A) Amino acid sequences of the first 50 amino acid residues in the M1-CovS-WT and M1-CovS-Y39H strains. The 39th amino acid residue is in boldface type. (B) Localization of the mutation in a schematic representation of CovS. Black arrow, 39th residue. (Modified from reference .)

FIG 2

The M1-CovS-Y39H strain displays an atypical transcriptomic profile and responds poorly to an environmental cue. Shown are log₂ fold transcript differences in selected virulence genes between the M1-CovS-Y39H and M1-CovS-WT strains grown in THY broth (A), M1-CovS-WT grown in THY broth supplemented with MgCl₂ and THY broth (B), and M1-CovS-Y39H grown in THY broth supplemented with MgCl₂ and THY broth (C), as determined by RNA-seq analysis (see Table S2 in the supplemental material). Genes are in black; those that are overexpressed and underexpressed are highlighted with a blue diamond above the zero bar and a red diamond below the zero bar, respectively. *, statistically significant after a multiple-testing-adjustment procedure.

FIG 3

The CovSY39H variant responds poorly to environmental cues. Log₂ fold transcript differences in selected genes between the M1-CovS-Y39H and M1-CovS-WT strains grown in THY broth (A), the M1-CovS-WT and M1-CovS-Y39H strains grown in THY broth supplemented or not with MgCl₂ (B), and the M1-CovS-WT and M1-CovS-Y39H strains grown in THY broth supplemented or not with LL-37 (C) were determined by qRT-PCR. Genes above or below the zero bar are those that are overexpressed or underexpressed, respectively. Mean values and standard deviations (SD) are indicated (n = 4 [A and C] and n = 3 [B]). P values were determined by an unequal-variance t test on ΔC_T (*, P < 0.05; **, P < 0.01; ****, P < 0.001).

FIG 4

Effect of the CovSY39H variant on SpeB and SLO production. Shown are the relative abundances of the accumulated SpeB (A and C) or SLO (B and D) in the supernatants of M1-CovS-WT (closed circles) and M1-CovS-Y39H (open squares) grown in THY broth or THY broth with MgCl₂ (A and B) or in THY broth or THY broth with LL-37 (C and D) compared to those in the supernatants of the wild-type strain grown in THY broth. Strains were grown to the late (OD₆₀₀ = 0.6 to 0.7) and early (OD₆₀₀ = 0.3 to 0.4) exponential phases for SpeB and SLO quantifications, respectively. The experiments were carried out for the SpeB and the SLO quantifications 10 and 4 times, respectively. Statistical analysis was performed by a Kruskal-Wallis test with a Dunn posttest and by two-way ANOVA with a Bonferroni posttest for SpeB and SLO analyses, respectively (Prism 9). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 5

The M1-CovS-WT strain survives better than the M1-CovS-Y39H mutant strain inside macrophages. A macrophage survival assay was performed with mouse RAW264.7 macrophages infected with M1-CovS-WT (closed circles) and M1-CovS-Y39H (open squares) at an MOI of 10. GAS survival is expressed as a percentage of intracellular bacteria at T₂, T₄, and T₆ relative to those at T₀. The results are representative of data from 7 independent experiments performed in triplicate. Error bars represent the means and standard errors of the means (**, P < 0.01).

FIG 6

The M1-CovS-WT strain is more virulent than the M1-CovS-Y39H mutant strain. Mice were infected with the M1-CovS-WT and M1-CovS-Y39H strains intravenously (n = 10) at 4 × 10⁷ and 5 × 10⁷ CFU, respectively (A), and intranasally (n = 6) at 5 × 10⁸ and 1.2 × 10⁹ CFU, respectively (B). The survival of the animals was scored for a period of 14 days. Dashed lines, M1-CovS-WT; dotted lines, M1-CovS-Y39H. **, P < 0.001 (by log rank and Gehan-Breslow-Wilcoxon tests).