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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

Growth curves of the wild-type M1-SF370 (M1-WT) and its isogenic mutant (M1-SDHHBtail) GAS strains in chemically defined medium supplemented with glucose, sucrose, fructose, maltose, maltodextrin, and mannose. Error bars represent standard error of the mean OD600nm obtained from three independent experiments.
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f1: Growth curves of the wild-type M1-SF370 (M1-WT) and its isogenic mutant (M1-SDHHBtail) GAS strains in chemically defined medium supplemented with glucose, sucrose, fructose, maltose, maltodextrin, and mannose. Error bars represent standard error of the mean OD600nm obtained from three independent experiments.

Mentions: Since about half of the differentially regulated genes in the M1-SDHHBtail mutant were related to metabolism, most of which were downregulated and essentially belonged to carbohydrate transporters, we investigated the growth properties of the M1-SDHHBtail mutant and the wild-type GAS strains in chemically defined medium (CDM) supplemented with different sugars as the sole carbon source (Fig. 1). Our results revealed that the lack of surface exportation of SDH did not change the growth characteristics of the mutant in complex nutritionally enriched THY medium (Todd-Hewitt broth supplemented with 0.5% yeast extract). However, when the mutant was grown in CDM with different sugars as the sole carbon source, different growth patterns of M1-SDHHBtail were observed compared to M1-WT. More importantly, the growth rates of the M1-SDHHBtail mutant were not affected when grown in CDM with glucose, sucrose, fructose, and mannose (Fig. 1). On the other hand, when maltose or maltodextrin was used as the sole carbon source, the growth rate of the M1-SDHHBtail mutant was reduced (Fig. 1), indicating that the inhibition of SDH exportation or increased intracellular concentration of SDH directly influences the ability of GAS to metabolize and transport specific carbon sources.


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

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

Growth curves of the wild-type M1-SF370 (M1-WT) and its isogenic mutant (M1-SDHHBtail) GAS strains in chemically defined medium supplemented with glucose, sucrose, fructose, maltose, maltodextrin, and mannose. 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

f1: Growth curves of the wild-type M1-SF370 (M1-WT) and its isogenic mutant (M1-SDHHBtail) GAS strains in chemically defined medium supplemented with glucose, sucrose, fructose, maltose, maltodextrin, and mannose. Error bars represent standard error of the mean OD600nm obtained from three independent experiments.
Mentions: Since about half of the differentially regulated genes in the M1-SDHHBtail mutant were related to metabolism, most of which were downregulated and essentially belonged to carbohydrate transporters, we investigated the growth properties of the M1-SDHHBtail mutant and the wild-type GAS strains in chemically defined medium (CDM) supplemented with different sugars as the sole carbon source (Fig. 1). Our results revealed that the lack of surface exportation of SDH did not change the growth characteristics of the mutant in complex nutritionally enriched THY medium (Todd-Hewitt broth supplemented with 0.5% yeast extract). However, when the mutant was grown in CDM with different sugars as the sole carbon source, different growth patterns of M1-SDHHBtail were observed compared to M1-WT. More importantly, the growth rates of the M1-SDHHBtail mutant were not affected when grown in CDM with glucose, sucrose, fructose, and mannose (Fig. 1). On the other hand, when maltose or maltodextrin was used as the sole carbon source, the growth rate of the M1-SDHHBtail mutant was reduced (Fig. 1), indicating that the inhibition of SDH exportation or increased intracellular concentration of SDH directly influences the ability of GAS to metabolize and transport specific carbon sources.

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