Differential secretomics of Streptococcus pyogenes reveals a novel peroxide regulator (PerR)-regulated extracellular virulence factor mitogen factor 3 (MF3)

Abstract

Streptococcus pyogenes is a human pathogen that causes various diseases. Numerous virulence factors secreted by S. pyogenes are involved in pathogenesis. The peroxide regulator (PerR) is associated with the peroxide resistance response and pathogenesis, but little is known about the regulation of the secretome involved in virulence. To investigate how PerR regulates the expression of the S. pyogenes secretome involved in virulence, a perR deficient mutant was used for comparative secretomic analysis with a wild-type strain. The conditioned medium containing secreted proteins of a wild-type strain and a perR deficient mutant at the stationary phase were collected for two-dimensional gel electrophoresis analysis, where protease inhibitors were applied to avoid the degradation of extracellular proteins. Differentially expressed protein spots were identified by liquid chromatography electrospray ionization tandem MS. More than 330 protein spots were detected on each gel. We identified 25 unique up-regulated proteins and 13 unique down-regulated proteins that were directly or indirectly controlled by the PerR regulator. Among these identified proteins, mitogen factor 3 (MF3), was selected to verify virulence and the expression of gene products. The data showed that MF3 protein levels in conditioned medium, as measured by immunoblot analysis, correlated well with protein levels determined by two-dimensional gel electrophoresis analysis. We also demonstrated that PerR bound to the promoter region of the mf3 gene. The result of an infection model showed that virulence was attenuated in the mf3 deficient mutant. Additional growth data of the wild-type strain and the mf3 deficient mutant suggested that MF3 played a role in digestion of exogenous DNA for promoting growth. To summarize, we conclude that PerR can positively regulate the expression of the secreted protein MF3 that contributes to the virulence in S. pyogenes. The analysis of the PerR-regulated secretome provided key information for the elucidation of the host-pathogen interactions and might assist in the development of potential chemotherapeutic strategies to prevent or treat streptococcal diseases.

Fig. 1.

Validation of characteristics of the perR deficient mutant. A, RT-PCR analysis of perR gene in the wild-type strain (A-20) and the perR deficient mutant (SW-612). Electrophoresis of the RT-PCR products using RNA extracted from cultures of the strain A-20 and the strain SW-612 grown under the same conditions. B, Western blotting analysis of PerR protein in the strain A-20 and the strain SW-612. Total protein (100 μg/lane) from the lysates was separated on a 4–12% NuPAGE BisTris gel and probed by anti-PerR mouse antibody. C, Virulence of S. pyogenes wild-type and perR deficient mutant strains. Skin air sacs of BALB/c mice (n = 9 per group) were injected with 2 × 10⁸ CFU of the strain A-20 or the strain SW-612. Animals were monitored regular intervals four times per day. The results are expressed as percentages of survival over time.

Fig. 2.

Growth curve of the wild-type strain (A-20) and the perR deficient mutant (SW-612). The strain A-20 (●) and the strain SW-612 (■) of S. pyogenes were cultured in TSBYE. The strain A-20 (▵) and the strain SW-612 (×) of S. pyogenes were cultured in TSBYE supplemented with protease inhibitor mixture (PIC). The error bars indicate the standard deviations.

Fig. 3.

Comparison of secretomes of the wild-type strain (A-20) and the perR deficient mutant (SW-612). Each gel of the secretomes of the A-20 strain and the SW-612 strain were grown for 12 h under the same conditions are shown. Proteins were separated by 2DGE using 18-cm wide range IPG strips (pH 4–7) and 12% SDS-PAGE and stained by a silver staining method. The designation “A” is used for those spots overexpressed in the A-20 strain, and “P” is used for those spots over-expressed in the strain SW-612. The spots differentially expressed in each strain are numbered and correspond to the proteins listed in Table I.

Fig. 4.

Validation of expression level of mf3 gene product in the wild-type strain (A-20) and the perR deficient mutant (SW-612). A, Analysis of MF3 expression by Western blotting. Total proteins (10 μg/lane) from the CM of S. pyogenes strains were extracted and probed by anti-MF3 mouse antibody. Silver staining of the gel is shown in the supplemental Fig. 5 as a protein loading control. B, Analysis of MF3 expression by Western blotting. Total proteins (10 μg/lane) from the lysates of the A-20 strain and the SW-612 strain were extracted and probed by an anti-MF3 mouse antibody. C, Relative gene expression of mf3 in the wild-type strain (A-20) and the perR deficient mutant (SW-612) measured by real time RT-PCR analysis. The fold change of mf3 gene expression was calculated by dividing the expression value of the SW-612 strain by the expression value of the A-20 strain. Bars represent mean values (three biological replicates) of fold changes ± standard deviation (S.D.).

Fig. 5.

Confirmation of PerR binding to the mf3 promoter region. The mf3 promoter and positive control dpr promoter were incubated with different concentrations of r-PerR (lanes1–5 and lanes 6–7) for 15 min at room temperature. The mixtures were separated by native PAGE, and the mobility shift of the promoter was visualized by ethidium bromide staining.

Fig. 6.

Virulence of S. pyogenes wild-type and mf3 deficient mutant strains. Skin air sacs of BALB/c mice (n = 10 per group) were injected with 2 × 10⁸ CFU of the strain A-20 and the strain SW-917. Animals were monitored four times per day. The results are expressed as percentages of survival over time.

Fig. 7.

Growth curve of S. pyogenes strains using DNA as the sole source of carbon, nitrogen, and phosphate. Symbols: (●) is indicated the wild-type strain A-20, (■) is indicated the perR deficient mutant SW-917. (▴) is indicated the complementary strain SW- 930.