Differential Secretomics of Streptococcus pyogenes Reveals a Novel Peroxide Regulator (PerR)-regulated Extracellular Virulence Factor Mitogen Factor3 (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 downregulated 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

Peroxide‐Sensing Transcriptional Regulators in Bacteria

“…In Bacillus subtilis, PerR regulates itself (30) and other genes, including those involved in oxidative stress defense (katA, ahpCF, mrgA) (10,14), metal homeostasis (zosA, fur, hemAXCDBL) (14,30,31), and surfactant production (srfA) (41), in response to peroxide exposure. As with OxyR, PerR homologs in other organisms tend to regulate similar classes of genes (7,8,36,48,79,91,117).…”

Peroxide-Sensing Transcriptional Regulators in Bacteria

“…In Bacillus subtilis, PerR regulates itself (30) and other genes, including those involved in oxidative stress defense (katA, ahpCF, mrgA) (10,14), metal homeostasis (zosA, fur, hemAXCDBL) (14,30,31), and surfactant production (srfA) (41), in response to peroxide exposure. As with OxyR, PerR homologs in other organisms tend to regulate similar classes of genes (7,8,36,48,79,91,117 (46,52) (Fig. 2A).…”

Automated Mass Spectrometry–Based Functional Assay for the Routine Analysis of the Secretome

“…4,5,8,16,17,19 The differences between two different secretomes are identified either before (e.g., in SDS-PAGE or 2D SDS-PAGE) or after protein digestion (e.g., in LC-MS/MS or during data processing and/or analysis). Since the posttranslationally modified proteins are digested into many peptides, parts of these proteins that are not modified can be analyzed by LC-MS/MS and the sequence of the peptides determined and, implicitly, the protein identified and then quantified.…”

“…[1][2][3][4][5][6]8 Secretomes may be classified according to various criteria, such as the type of the cell or the type of the tissue producing the secretome, or the location of the secreted proteins such as extracellular but nonsystemic, intraorgan (e.g., intrahepatic) or extracellular but systemic, extraorgan (e.g., blood, saliva, urine, or lymphatic fluid). [16][17][18][19][20][21][22][23][24][25][26][27][28][29] Depending on the type of secretome analyzed, specific information may be obtained.…”

“…Therefore, characterization of the proteins that form a secretome (secretomics) can help us not only in the identification, quantification, and characterization of proteins but also in the identification or prediction of the physiological or pathological state of a cell type or a cell population. [3][4][5][6][7][8][9][10] Many methods can be used to analyze secreted proteins, and historically, perhaps the most frequently used ones have been Western blotting and enzyme-linked immunosorbent assay. [11][12][13] However, recent advances in mass spectrometrybased proteomics approaches allow one not only to identify all proteins in a particular secretome but also to automatize these proteomics approaches.…”