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Surface export of GAPDH/SDH, a glycolytic enzyme, is essential for Streptococcus pyogenes virulence.

Jin H, Agarwal S, Agarwal S, Pancholi V - MBio (2011)

Bottom Line: The complete attenuation of this mutant for virulence in the mouse model and the decreased and increased virulence of the wild-type and mutant strains postcomplementation with SDH(HBtail) and SDH, respectively, indicated that the SDH surface export indeed regulates GAS virulence.M1-SDH(HBtail) also displayed unaltered growth patterns, increased intracellular ATP concentration and Hpr double phosphorylation, and significantly reduced pH tolerance, streptolysin S, and SpeB activities.The ability of GAS as a successful pathogen to localize SDH in the cytoplasm as well as on the surface is physiologically relevant and dynamically obligatory to fine-tune the functions of many transcriptional regulators and also to exploit its virulence properties for infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Ohio State University College of Medicine, Columbus, Ohio, USA.

ABSTRACT

Unlabelled: Streptococcal surface dehydrogenase (SDH) (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) is an anchorless major multifunctional surface protein in group A Streptococcus (GAS) with the ability to bind important mammalian proteins, including plasmin(ogen). Although several biological properties of SDH are suggestive of its possible role in GAS virulence, its direct role in GAS pathogenesis has not been ascertained because it is essential for GAS survival. Thus, it has remained enigmatic as to "how and why" SDH/GAPDH is exported onto the bacterial surface. The present investigation highlights "why" SDH is exported onto the GAS surface. Differential microarray-based genome-wide transcript abundance analysis was carried out using a specific mutant, which was created by inserting a hydrophobic tail at the C-terminal end of SDH (M1-SDH(HBtail)) and thus preventing its exportation onto the GAS surface. This analysis revealed downregulation of the majority of genes involved in GAS virulence and genes belonging to carbohydrate and amino acid metabolism and upregulation of those related to lipid metabolism. The complete attenuation of this mutant for virulence in the mouse model and the decreased and increased virulence of the wild-type and mutant strains postcomplementation with SDH(HBtail) and SDH, respectively, indicated that the SDH surface export indeed regulates GAS virulence. M1-SDH(HBtail) also displayed unaltered growth patterns, increased intracellular ATP concentration and Hpr double phosphorylation, and significantly reduced pH tolerance, streptolysin S, and SpeB activities. These phenotypic and physiological changes observed in the mutant despite the unaltered expression levels of established transcriptional regulators further highlight the fact that SDH interfaces with many regulators and its surface exportation is essential for GAS virulence.

Importance: Streptococcal surface dehydrogenase (SDH), a classical anchorless cytoplasmically localized glycolytic enzyme, is exported onto the group A Streptococcus (GAS) surface through a hitherto unknown mechanism(s). It has not been known why GAS or other prokaryotes should export this protein onto the surface. By genetic manipulations, we created a novel GAS mutant strain expressing SDH with a 12-amino-acid hydrophobic tail at its C-terminal end and thus were able to prevent its surface exportation without altering its enzymatic activity or growth pattern. Interestingly, the mutant was completely attenuated for virulence in a mouse peritonitis model. The global gene expression profiles of this mutant reveal that the surface exportation of SDH is mandatory to maintain GAS virulence. The ability of GAS as a successful pathogen to localize SDH in the cytoplasm as well as on the surface is physiologically relevant and dynamically obligatory to fine-tune the functions of many transcriptional regulators and also to exploit its virulence properties for infection.

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Related in: MedlinePlus

Relationship of the surface export of SDH and SpeB secretion. (A) Determination of the relative cysteine protease activities of SpeB present in the culture supernatants of M1-WT and M1-SDHHBtail GAS using FITC-labeled casein. The released fluorescence activity by the culture supernatant of the wild-type GAS strain was set at 100% activity, and its specificity was determined in the presence of cysteine protease inhibitor E-64. Values are means plus standard errors of the means (error bars) of three to six independent experiments. (B) Determination of SpeB-specific cysteine protease activity (hydrolysis of milk casein) present in the culture supernatants of M1-WT, M1-SDHHBtail, M1-SDHHBtail::sdh, and M1-WT::sdhHBtail GAS strains using milk agar. (C) Western blot analysis showing the effect on the secretion of SpeB in the culture supernatants of M1-WT, M1-SDHHBtail, M1-SDHHBtail::sdh, and M1-WT::sdhHBtail strains and the ability of SDH to bind secreted SpeB as determined by the blot overlay method.
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f5: Relationship of the surface export of SDH and SpeB secretion. (A) Determination of the relative cysteine protease activities of SpeB present in the culture supernatants of M1-WT and M1-SDHHBtail GAS using FITC-labeled casein. The released fluorescence activity by the culture supernatant of the wild-type GAS strain was set at 100% activity, and its specificity was determined in the presence of cysteine protease inhibitor E-64. Values are means plus standard errors of the means (error bars) of three to six independent experiments. (B) Determination of SpeB-specific cysteine protease activity (hydrolysis of milk casein) present in the culture supernatants of M1-WT, M1-SDHHBtail, M1-SDHHBtail::sdh, and M1-WT::sdhHBtail GAS strains using milk agar. (C) Western blot analysis showing the effect on the secretion of SpeB in the culture supernatants of M1-WT, M1-SDHHBtail, M1-SDHHBtail::sdh, and M1-WT::sdhHBtail strains and the ability of SDH to bind secreted SpeB as determined by the blot overlay method.

Mentions: Streptococcal pyogenic exotoxin B (SpeB/SPy0274) is another virulence factor which degrades host serum proteins, such as human extracellular matrix, immunoglobulins, complement components, and even S. pyogenes surface and secreted proteins (34). Downregulation of speB (7.01-fold downregulation found by microarray and 3.22-fold downregulation by qRT-PCR [P < 0.0001]; see Table S4 in the supplemental material) in the M1-SDHHBtail mutant prompted us to compare the SpeB-specific cysteine protease activity in the culture supernatants of M1-SDHHBtail and M1-WT strains. This was accomplished by measuring the fluorescence released upon casein hydrolysis in the reaction mixture containing casein-fluorescein isothiocyanate (FITC) (Fig. 5A). About 94% of the cysteine protease activity observed in the culture supernatants of M1-WT was inhibited by cysteine protease inhibitor E-64, indicating that most of the detected hydrolytic activity in the supernatant was contributed by cysteine protease (Fig. 5A). Compared to the wild type, the M1-SDHHBtail mutant displayed significantly reduced cysteine protease activity (22% for M1-SDHHBtail and 100% for M1-WT) (Fig. 5A).


Surface export of GAPDH/SDH, a glycolytic enzyme, is essential for Streptococcus pyogenes virulence.

Jin H, Agarwal S, Agarwal S, Pancholi V - MBio (2011)

Relationship of the surface export of SDH and SpeB secretion. (A) Determination of the relative cysteine protease activities of SpeB present in the culture supernatants of M1-WT and M1-SDHHBtail GAS using FITC-labeled casein. The released fluorescence activity by the culture supernatant of the wild-type GAS strain was set at 100% activity, and its specificity was determined in the presence of cysteine protease inhibitor E-64. Values are means plus standard errors of the means (error bars) of three to six independent experiments. (B) Determination of SpeB-specific cysteine protease activity (hydrolysis of milk casein) present in the culture supernatants of M1-WT, M1-SDHHBtail, M1-SDHHBtail::sdh, and M1-WT::sdhHBtail GAS strains using milk agar. (C) Western blot analysis showing the effect on the secretion of SpeB in the culture supernatants of M1-WT, M1-SDHHBtail, M1-SDHHBtail::sdh, and M1-WT::sdhHBtail strains and the ability of SDH to bind secreted SpeB as determined by the blot overlay method.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104492&req=5

f5: Relationship of the surface export of SDH and SpeB secretion. (A) Determination of the relative cysteine protease activities of SpeB present in the culture supernatants of M1-WT and M1-SDHHBtail GAS using FITC-labeled casein. The released fluorescence activity by the culture supernatant of the wild-type GAS strain was set at 100% activity, and its specificity was determined in the presence of cysteine protease inhibitor E-64. Values are means plus standard errors of the means (error bars) of three to six independent experiments. (B) Determination of SpeB-specific cysteine protease activity (hydrolysis of milk casein) present in the culture supernatants of M1-WT, M1-SDHHBtail, M1-SDHHBtail::sdh, and M1-WT::sdhHBtail GAS strains using milk agar. (C) Western blot analysis showing the effect on the secretion of SpeB in the culture supernatants of M1-WT, M1-SDHHBtail, M1-SDHHBtail::sdh, and M1-WT::sdhHBtail strains and the ability of SDH to bind secreted SpeB as determined by the blot overlay method.
Mentions: Streptococcal pyogenic exotoxin B (SpeB/SPy0274) is another virulence factor which degrades host serum proteins, such as human extracellular matrix, immunoglobulins, complement components, and even S. pyogenes surface and secreted proteins (34). Downregulation of speB (7.01-fold downregulation found by microarray and 3.22-fold downregulation by qRT-PCR [P < 0.0001]; see Table S4 in the supplemental material) in the M1-SDHHBtail mutant prompted us to compare the SpeB-specific cysteine protease activity in the culture supernatants of M1-SDHHBtail and M1-WT strains. This was accomplished by measuring the fluorescence released upon casein hydrolysis in the reaction mixture containing casein-fluorescein isothiocyanate (FITC) (Fig. 5A). About 94% of the cysteine protease activity observed in the culture supernatants of M1-WT was inhibited by cysteine protease inhibitor E-64, indicating that most of the detected hydrolytic activity in the supernatant was contributed by cysteine protease (Fig. 5A). Compared to the wild type, the M1-SDHHBtail mutant displayed significantly reduced cysteine protease activity (22% for M1-SDHHBtail and 100% for M1-WT) (Fig. 5A).

Bottom Line: The complete attenuation of this mutant for virulence in the mouse model and the decreased and increased virulence of the wild-type and mutant strains postcomplementation with SDH(HBtail) and SDH, respectively, indicated that the SDH surface export indeed regulates GAS virulence.M1-SDH(HBtail) also displayed unaltered growth patterns, increased intracellular ATP concentration and Hpr double phosphorylation, and significantly reduced pH tolerance, streptolysin S, and SpeB activities.The ability of GAS as a successful pathogen to localize SDH in the cytoplasm as well as on the surface is physiologically relevant and dynamically obligatory to fine-tune the functions of many transcriptional regulators and also to exploit its virulence properties for infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Ohio State University College of Medicine, Columbus, Ohio, USA.

ABSTRACT

Unlabelled: Streptococcal surface dehydrogenase (SDH) (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) is an anchorless major multifunctional surface protein in group A Streptococcus (GAS) with the ability to bind important mammalian proteins, including plasmin(ogen). Although several biological properties of SDH are suggestive of its possible role in GAS virulence, its direct role in GAS pathogenesis has not been ascertained because it is essential for GAS survival. Thus, it has remained enigmatic as to "how and why" SDH/GAPDH is exported onto the bacterial surface. The present investigation highlights "why" SDH is exported onto the GAS surface. Differential microarray-based genome-wide transcript abundance analysis was carried out using a specific mutant, which was created by inserting a hydrophobic tail at the C-terminal end of SDH (M1-SDH(HBtail)) and thus preventing its exportation onto the GAS surface. This analysis revealed downregulation of the majority of genes involved in GAS virulence and genes belonging to carbohydrate and amino acid metabolism and upregulation of those related to lipid metabolism. The complete attenuation of this mutant for virulence in the mouse model and the decreased and increased virulence of the wild-type and mutant strains postcomplementation with SDH(HBtail) and SDH, respectively, indicated that the SDH surface export indeed regulates GAS virulence. M1-SDH(HBtail) also displayed unaltered growth patterns, increased intracellular ATP concentration and Hpr double phosphorylation, and significantly reduced pH tolerance, streptolysin S, and SpeB activities. These phenotypic and physiological changes observed in the mutant despite the unaltered expression levels of established transcriptional regulators further highlight the fact that SDH interfaces with many regulators and its surface exportation is essential for GAS virulence.

Importance: Streptococcal surface dehydrogenase (SDH), a classical anchorless cytoplasmically localized glycolytic enzyme, is exported onto the group A Streptococcus (GAS) surface through a hitherto unknown mechanism(s). It has not been known why GAS or other prokaryotes should export this protein onto the surface. By genetic manipulations, we created a novel GAS mutant strain expressing SDH with a 12-amino-acid hydrophobic tail at its C-terminal end and thus were able to prevent its surface exportation without altering its enzymatic activity or growth pattern. Interestingly, the mutant was completely attenuated for virulence in a mouse peritonitis model. The global gene expression profiles of this mutant reveal that the surface exportation of SDH is mandatory to maintain GAS virulence. The ability of GAS as a successful pathogen to localize SDH in the cytoplasm as well as on the surface is physiologically relevant and dynamically obligatory to fine-tune the functions of many transcriptional regulators and also to exploit its virulence properties for infection.

Show MeSH
Related in: MedlinePlus