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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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Retention of SDH in the cytoplasm attenuates GAS virulence in an experimental mouse intraperitoneal infection model. (A) Survival/mortality curves for M1-WT and M1-SDHHBtail (20 mice per group) and their corresponding complemented strains (10 mice per group), M1-SDHHBtail::sdh and M1-WT::sdhHBTail (complemented strains created with pDC123 plasmid containing genes encoding SDHHBtail and SDH, respectively). Mice infected with GAS strains were monitored for 10 days postinfection (P.I.) and statistically evaluated by the log rank test, and the results were plotted using GraphPad Prism 4 software. All the mock-infected mice survived throughout the observation period (not shown). (B) qRT-PCR-based expression analysis of the indicated genes in the complemented strains. The relative fold change in the expression level was calculated with respect to the expression levels in the corresponding parent strains (M1-SDHHBtail::sdh versus M1-SDHHBtail and M1-WT::sdhHBTail versus M1-WT) after normalization with the housekeeping gene.
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f6: Retention of SDH in the cytoplasm attenuates GAS virulence in an experimental mouse intraperitoneal infection model. (A) Survival/mortality curves for M1-WT and M1-SDHHBtail (20 mice per group) and their corresponding complemented strains (10 mice per group), M1-SDHHBtail::sdh and M1-WT::sdhHBTail (complemented strains created with pDC123 plasmid containing genes encoding SDHHBtail and SDH, respectively). Mice infected with GAS strains were monitored for 10 days postinfection (P.I.) and statistically evaluated by the log rank test, and the results were plotted using GraphPad Prism 4 software. All the mock-infected mice survived throughout the observation period (not shown). (B) qRT-PCR-based expression analysis of the indicated genes in the complemented strains. The relative fold change in the expression level was calculated with respect to the expression levels in the corresponding parent strains (M1-SDHHBtail::sdh versus M1-SDHHBtail and M1-WT::sdhHBTail versus M1-WT) after normalization with the housekeeping gene.

Mentions: Microarray analysis in conjunction with other functional analyses collectively indicated that the prevention of SDH export onto the cell surface results in downregulation of several virulence factors (Table 3). We therefore hypothesized that the inhibition of SDH export might adversely affects GAS virulence in an experimental mouse intraperitoneal infection model. A group of 20 mice injected with the M1-SDHHBtail strain displayed 100% survival, while all 20 mice injected with the wild-type strain died by day 3 postinfection (P < 0.0001) (Fig. 6A). While live bacteria were recovered from the spleen (106 CFU/mg of tissue), lung, liver, and kidneys (5 × 104 to 8 × 104 CFU/mg of tissue) of mice infected with M1-WT, no bacteria were recovered from the mice infected with the M1-SDHHBtail mutant, indicating its attenuation for virulence. Together, these results indicate that the mere retention of SDH in the cytoplasm by preventing its exportation onto the GAS surface adversely affect both GAS metabolism and virulence. Hence, the accumulation of SDH/GAPDH in the cytoplasm beyond its physiological concentration results in the attenuation of GAS virulence.


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

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

Retention of SDH in the cytoplasm attenuates GAS virulence in an experimental mouse intraperitoneal infection model. (A) Survival/mortality curves for M1-WT and M1-SDHHBtail (20 mice per group) and their corresponding complemented strains (10 mice per group), M1-SDHHBtail::sdh and M1-WT::sdhHBTail (complemented strains created with pDC123 plasmid containing genes encoding SDHHBtail and SDH, respectively). Mice infected with GAS strains were monitored for 10 days postinfection (P.I.) and statistically evaluated by the log rank test, and the results were plotted using GraphPad Prism 4 software. All the mock-infected mice survived throughout the observation period (not shown). (B) qRT-PCR-based expression analysis of the indicated genes in the complemented strains. The relative fold change in the expression level was calculated with respect to the expression levels in the corresponding parent strains (M1-SDHHBtail::sdh versus M1-SDHHBtail and M1-WT::sdhHBTail versus M1-WT) after normalization with the housekeeping gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Retention of SDH in the cytoplasm attenuates GAS virulence in an experimental mouse intraperitoneal infection model. (A) Survival/mortality curves for M1-WT and M1-SDHHBtail (20 mice per group) and their corresponding complemented strains (10 mice per group), M1-SDHHBtail::sdh and M1-WT::sdhHBTail (complemented strains created with pDC123 plasmid containing genes encoding SDHHBtail and SDH, respectively). Mice infected with GAS strains were monitored for 10 days postinfection (P.I.) and statistically evaluated by the log rank test, and the results were plotted using GraphPad Prism 4 software. All the mock-infected mice survived throughout the observation period (not shown). (B) qRT-PCR-based expression analysis of the indicated genes in the complemented strains. The relative fold change in the expression level was calculated with respect to the expression levels in the corresponding parent strains (M1-SDHHBtail::sdh versus M1-SDHHBtail and M1-WT::sdhHBTail versus M1-WT) after normalization with the housekeeping gene.
Mentions: Microarray analysis in conjunction with other functional analyses collectively indicated that the prevention of SDH export onto the cell surface results in downregulation of several virulence factors (Table 3). We therefore hypothesized that the inhibition of SDH export might adversely affects GAS virulence in an experimental mouse intraperitoneal infection model. A group of 20 mice injected with the M1-SDHHBtail strain displayed 100% survival, while all 20 mice injected with the wild-type strain died by day 3 postinfection (P < 0.0001) (Fig. 6A). While live bacteria were recovered from the spleen (106 CFU/mg of tissue), lung, liver, and kidneys (5 × 104 to 8 × 104 CFU/mg of tissue) of mice infected with M1-WT, no bacteria were recovered from the mice infected with the M1-SDHHBtail mutant, indicating its attenuation for virulence. Together, these results indicate that the mere retention of SDH in the cytoplasm by preventing its exportation onto the GAS surface adversely affect both GAS metabolism and virulence. Hence, the accumulation of SDH/GAPDH in the cytoplasm beyond its physiological concentration results in the attenuation of GAS virulence.

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