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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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(A) Intracellular ATP concentrations in the wild-type M1-SF370 (M1-WT) and its isogenic mutant (M1-SDHHBtail) GAS strains. ATP concentrations in samples were determined using luciferin-luciferase bioluminescence assay based on the standard curve obtained with known concentrations of ATP. Error bars represent standard errors of the mean ATP concentrations obtained from three independent experiments (triplicate wells for each experiment). (B) Effect of pH on growth of the wild-type M1-SF370 (M1-WT) and mutant (M1-SDHHBtail) GAS strains. GAS strains were grown overnight in chemically defined medium (CDM), and culture density (600 nm) was determined as described in Materials and Methods. Error bars represent standard error of the mean OD600nm obtained from three independent experiments.
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f2: (A) Intracellular ATP concentrations in the wild-type M1-SF370 (M1-WT) and its isogenic mutant (M1-SDHHBtail) GAS strains. ATP concentrations in samples were determined using luciferin-luciferase bioluminescence assay based on the standard curve obtained with known concentrations of ATP. Error bars represent standard errors of the mean ATP concentrations obtained from three independent experiments (triplicate wells for each experiment). (B) Effect of pH on growth of the wild-type M1-SF370 (M1-WT) and mutant (M1-SDHHBtail) GAS strains. GAS strains were grown overnight in chemically defined medium (CDM), and culture density (600 nm) was determined as described in Materials and Methods. Error bars represent standard error of the mean OD600nm obtained from three independent experiments.

Mentions: Microarray analysis of the M1-SDHHBtail mutant revealed downregulation of 11 genes (SPy0148 to SPy0151, SPy0154, SPy0155, SPy0157, SPy0414, SPy0739, SPy1128, and SPy1849) and upregulation of three genes (SPy0755, SPy0757, and SPy0759) related to energy production and conversion (see Table S3 in the supplemental material). Since most of the downregulated genes belonged to V-type Na+ ATPase and upregulated genes belonged to proton-translocating ATPase, we hypothesized that the downregulation of ATPase may result in a surplus intracellular concentration of ATP. To validate the microarray data, we measured the intracellular concentration of ATP in the M1-SDHHBtail mutant and wild-type strains by luciferin-luciferase assay. An 8-fold increase in the intracellular ATP concentration in the mutant (Fig. 2A) indicated that the prevention of SDH export and its subsequent increase in the cytosol positively regulate the levels of ATP in the M1-SDHHBtail mutant.


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

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

(A) Intracellular ATP concentrations in the wild-type M1-SF370 (M1-WT) and its isogenic mutant (M1-SDHHBtail) GAS strains. ATP concentrations in samples were determined using luciferin-luciferase bioluminescence assay based on the standard curve obtained with known concentrations of ATP. Error bars represent standard errors of the mean ATP concentrations obtained from three independent experiments (triplicate wells for each experiment). (B) Effect of pH on growth of the wild-type M1-SF370 (M1-WT) and mutant (M1-SDHHBtail) GAS strains. GAS strains were grown overnight in chemically defined medium (CDM), and culture density (600 nm) was determined as described in Materials and Methods. Error bars represent standard error of the mean OD600nm obtained from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: (A) Intracellular ATP concentrations in the wild-type M1-SF370 (M1-WT) and its isogenic mutant (M1-SDHHBtail) GAS strains. ATP concentrations in samples were determined using luciferin-luciferase bioluminescence assay based on the standard curve obtained with known concentrations of ATP. Error bars represent standard errors of the mean ATP concentrations obtained from three independent experiments (triplicate wells for each experiment). (B) Effect of pH on growth of the wild-type M1-SF370 (M1-WT) and mutant (M1-SDHHBtail) GAS strains. GAS strains were grown overnight in chemically defined medium (CDM), and culture density (600 nm) was determined as described in Materials and Methods. Error bars represent standard error of the mean OD600nm obtained from three independent experiments.
Mentions: Microarray analysis of the M1-SDHHBtail mutant revealed downregulation of 11 genes (SPy0148 to SPy0151, SPy0154, SPy0155, SPy0157, SPy0414, SPy0739, SPy1128, and SPy1849) and upregulation of three genes (SPy0755, SPy0757, and SPy0759) related to energy production and conversion (see Table S3 in the supplemental material). Since most of the downregulated genes belonged to V-type Na+ ATPase and upregulated genes belonged to proton-translocating ATPase, we hypothesized that the downregulation of ATPase may result in a surplus intracellular concentration of ATP. To validate the microarray data, we measured the intracellular concentration of ATP in the M1-SDHHBtail mutant and wild-type strains by luciferin-luciferase assay. An 8-fold increase in the intracellular ATP concentration in the mutant (Fig. 2A) indicated that the prevention of SDH export and its subsequent increase in the cytosol positively regulate the levels of ATP in the M1-SDHHBtail mutant.

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