The Streptococcus pyogenes signaling peptide SpoV regulates streptolysin O and enhances survival in murine blood

Abstract

Streptococcus pyogenes (Group A Streptococcus, GAS) is a human pathogen that causes a wide range of diseases. For successful colonization within a variety of host niches, GAS must sense and respond to environmental changes. Intercellular communication mediated by peptides is one way GAS coordinates gene expression in response to diverse environmental stressors, which enhances bacterial survival and contributes to virulence. Using peptidomics we identified SpoV (Streptococcal peptide controlling virulence) in culture supernatant fluids. SpoV is a secreted peptide encoded near the gene encoding the extracellular cholesterol-dependent cytolysin streptolysin O (slo) The addition of synthetic SpoV peptide derivatives, but not control peptides, increased slo transcript abundance in an M49 isolate but not in an M3 isolate. Deletion of spoV decreased slo transcript abundance, extracellular SLO protein levels, and SLO-specific hemolytic activity. Complementation of the spoV mutant increased slo transcript abundance. Lastly, a spoV mutant was deficient in the ability to survive in murine blood compared to the parental strain. Moreover, pre-incubation of the spoV mutant with synthetic SpoV peptide derivatives increased GAS survival. Our findings show that slo expression is regulated, in part, by the GAS-specific signaling peptide SpoV.IMPORTANCEGAS secretes signaling peptides that can alter gene expression and impact virulence. We used peptidomics to identify a signaling peptide designated SpoV. Further, we showed that SpoV altered the expression of the cholesterol-dependent cytolysin SLO. Peptide signaling plays an important regulatory role during disease progression among several bacterial pathogens, including GAS. The therapeutic potential of manipulating peptide-controlled regulatory networks is an attractive option for the development of novel therapeutic strategies that disrupt virulence gene expression.

FIG 1

SpoV was identified in MGAS315 culture supernatant fluids using peptidomics. NZ131 and MGAS315 wild-type strains were grown with peptide-free medium overnight. The CSFs were filtered, and peptides were concentrated and analyzed with mass spectrometry. (A) Two peptides corresponding to SpyM3_0132 (designated SpoV) were identified in CSP obtained from MGAS315. (B) Alignment of full-length amino acid sequences of SpoV encoded by strains MGAS315 and NZ131. Asterisks indicate identical residues, colons indicate conserved differences, and periods indicate semiconserved differences. The predicted type II secretory system cleavage site is indicated with an arrow and the corresponding extracellular peptides are shown in red. The peptide identified by mass spectrometry is underlined. (C) Sequence of synthetic peptides containing the 21 C-terminal amino acids of MGAS315 SpoV (C21) or the 25 C-terminal amino acids of NZ131 SpoV (C25) as well as the associated scrambled peptides.

FIG 2

spoV transcripts were more abundant in MGAS315 than NZ131. MGAS315 and NZ131 were grown with THY or CDM until the mid-exponential phase (A₆₀₀ = 0.4 to 0.7) or stationary phase (A₆₀₀ = 0.9 to 1.0). RNA was extracted, and the relative quantity of spoV transcripts was determined by qRT-PCR. (A) In MGAS315, spoV transcripts were significantly higher in the exponential (EX) phase of growth than in the stationary (S) phase when grown with THY but not when grown with CDM. (B) In NZ131, spoV transcripts were significantly higher in the exponential phase than in the stationary phase when grown with either THY or CDM. For both strains, GAS was grown on 4 different occasions, and each RNA sample was analyzed by qRT-PCR in duplicate. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test. ns, not significant (P > 0.05); ****, P < 0.0001; **, P < 0.01; *, P < 0.05.

FIG 3

Synthesized C21 and C25 peptides increased slo transcript abundance. MGAS315 wild type (A), NZ131 wild type (B), MGAS315 ΔspoV (C), and NZ131 ΔspoV (D) were grown with peptide-free medium to the mid-exponential phase of growth (A₆₀₀, 0.4 to 0.7). Then, a 10 μM concentration of the indicated peptides (C21, C21 Scr, C25, and C25 Scr) or diluent (DMSO) was added to cultures. After 3 h, RNA was extracted, and slo transcript abundance was measured by qRT-PCR. RNA was extracted from GAS cultures grown on 3 to 12 different occasions. Each RNA sample was analyzed by qRT-PCR in duplicate. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test. P values were determined by comparison among the indicated strains. ns, not significant (P > 0.05); ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 4

Deletion of spoV decreased slo transcript abundance. To determine if spoV affects slo transcript levels, MGAS315 wild type (WT), MGAS315 ΔspoV, MGAS315 ΔspoV (pAH32), MGAS315 ΔspoV (pAH-VC), MGAS315 ΔspoV (pAH5), MGAS315 ΔspoV (pAM401), MGAS315 ΔspoV (pAH49) (A) and NZ131 WT, NZ131 ΔspoV, NZ131 ΔspoV (pAH49), NZ131 ΔspoV (pAH-VC), NZ131 WT (pAH49), NZ131 WT (pAH-VC), and NZ131 ΔspoV (pAH32) (B) strains were grown with THY until mid-exponential phase (A₆₀₀, 0.4 to 0.7). RNA was extracted from GAS cultures, which were grown on 3 to 13 different occasions. From each RNA sample, slo transcripts were measured by qRT-PCR analysis in duplicate. slo transcripts were significantly decreased in both MGAS315 ΔspoV and NZ131 ΔspoV compared to the parental strains. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test. P values were determined by comparison among the indicated strains. ns, not significant (P > 0.05); ****, P < 0.0001; **, P < 0.01; *, P < 0.05.

FIG 5

Deletion of spoV decreased SLO protein abundance and hemolytic activity. MGAS315 wild-type (WT), MGAS315 ΔspoV, MGAS315 ΔspoV (pAH32), MGAS315 ΔspoV (pAH-VC), MGAS315 ΔspoV (pAH5), and MGAS315 ΔspoV (pAM401) strains were grown with THY to exponential phase. (A) The amount of SLO present in CSPs was determined by Western blotting using an anti-SLO antibody. Densitometry analysis was used to quantify the results obtained from two independent experiments. A representative image of a blot is shown. (B) SLO-specific hemolysis was determined by measuring the amount of hemoglobin released from erythrocytes. Controls containing 5 % erythrocytes and sterile water, which was considered 100 % hemolysis, were used to determine the percentage of total hemolysis. Results are presented as means and standard errors of the means (SEM). Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test. P values were determined by comparison among the indicated strains. ns, not significant (P > 0.05); ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 6

SpoV enhanced GAS survival in whole blood. Approximately 10⁴ CFU of each bacterial strain were mixed with 200 μl of whole murine blood and incubated at 37°C. At specific time points, diluted samples were plated onto agar plates for enumeration. Percent survival is calculated as number of CFU at a given time point divided by the initial number of CFU. (A) Survival of MGAS315 wild type (WT), MGAS315 ΔspoV, MGAS315 ΔspoV (pAH32), MGAS315 ΔspoV (pAH-VC), MGAS315 ΔspoV (pAH5), and MGAS315 ΔspoV (pAM401) in whole blood. (B) Survival of MGAS315 ΔspoV following incubation with 10 μM C21, C21 Scr, C25, and C25 Scr peptides or diluent (DMSO) for 15 min at room temperature. After exposure to the peptides or diluent, the bacteria were mixed with 200 μl whole murine blood and incubated at 37°C. After 30 min or 2 h, diluted samples were plated onto agar plates for enumeration of viable GAS. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test. P values were determined by comparison among the indicated strains. ns, not significant (P > 0.05); ***, P < 0.001; **, P < 0.01; *, P < 0.05.