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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

Western blot analysis of the whole-cell lysate (CL) of the overnight-grown wild-type M1-SF370 (M1-WT) and mutant (M1-SDHHBtail) GAS strains for the presence of SDH, CcpA, HPr, and CovR, using protein-specific antibodies. Equal amounts (50 µg total protein [25 ul] as the starting concentration) of cell lysates of M1-WT and M1-SDHHBtail were used for the assay. (A) Western blot analysis of equal volume (25ul) of the four serially 2-fold diluted samples of whole-cell lysates to determine the concentrations of different proteins as indicated in the wild-type and mutant GAS strains. (B) Reactivity of mono- and doubly phosphorylated forms of HPr (HPr-P1 and HPr-P2) in the whole-cell lysates of M1-WT and M1-SDHHBtail GAS strains.
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f3: Western blot analysis of the whole-cell lysate (CL) of the overnight-grown wild-type M1-SF370 (M1-WT) and mutant (M1-SDHHBtail) GAS strains for the presence of SDH, CcpA, HPr, and CovR, using protein-specific antibodies. Equal amounts (50 µg total protein [25 ul] as the starting concentration) of cell lysates of M1-WT and M1-SDHHBtail were used for the assay. (A) Western blot analysis of equal volume (25ul) of the four serially 2-fold diluted samples of whole-cell lysates to determine the concentrations of different proteins as indicated in the wild-type and mutant GAS strains. (B) Reactivity of mono- and doubly phosphorylated forms of HPr (HPr-P1 and HPr-P2) in the whole-cell lysates of M1-WT and M1-SDHHBtail GAS strains.

Mentions: To examine the relative changes in the phosphorylation status of Hpr in the M1-SDHHBtail mutant strain in comparison to that in the wild-type GAS strain, we determined expression levels of differentially resolved phosphorylated forms of HPr by native PAGE analysis. This analysis allowed us to differentiate between fast-migrating Ser-phosphorylated HPr (HPr-Ser-P or HPr-P1) and/or His/Ser-phosphorylated HPr (HPr-His/Ser-P or HPr-P2). Parallel Western blot analysis of the whole-cell lysates of M1-SDHHBtail mutant and M1-WT strains was carried out to determine relative endogenous protein levels of HPr and CcpA. Although the protein expression profiles of CcpA and Hpr were comparable in both GAS strains (Fig. 3A), a significant increase in the amount of doubly phosphorylated Hpr (Hpr-Ser-P, -His-P [HPr-P2]) in the M1-SDHHBtail mutant was observed (Fig. 3B). This indicated that the retention of SDH in the cytoplasm results in increased phosphorylation of Hpr possibly due to increased ATP production (Fig. 2A) as also reported previously (31).


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

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

Western blot analysis of the whole-cell lysate (CL) of the overnight-grown wild-type M1-SF370 (M1-WT) and mutant (M1-SDHHBtail) GAS strains for the presence of SDH, CcpA, HPr, and CovR, using protein-specific antibodies. Equal amounts (50 µg total protein [25 ul] as the starting concentration) of cell lysates of M1-WT and M1-SDHHBtail were used for the assay. (A) Western blot analysis of equal volume (25ul) of the four serially 2-fold diluted samples of whole-cell lysates to determine the concentrations of different proteins as indicated in the wild-type and mutant GAS strains. (B) Reactivity of mono- and doubly phosphorylated forms of HPr (HPr-P1 and HPr-P2) in the whole-cell lysates of M1-WT and M1-SDHHBtail GAS strains.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Western blot analysis of the whole-cell lysate (CL) of the overnight-grown wild-type M1-SF370 (M1-WT) and mutant (M1-SDHHBtail) GAS strains for the presence of SDH, CcpA, HPr, and CovR, using protein-specific antibodies. Equal amounts (50 µg total protein [25 ul] as the starting concentration) of cell lysates of M1-WT and M1-SDHHBtail were used for the assay. (A) Western blot analysis of equal volume (25ul) of the four serially 2-fold diluted samples of whole-cell lysates to determine the concentrations of different proteins as indicated in the wild-type and mutant GAS strains. (B) Reactivity of mono- and doubly phosphorylated forms of HPr (HPr-P1 and HPr-P2) in the whole-cell lysates of M1-WT and M1-SDHHBtail GAS strains.
Mentions: To examine the relative changes in the phosphorylation status of Hpr in the M1-SDHHBtail mutant strain in comparison to that in the wild-type GAS strain, we determined expression levels of differentially resolved phosphorylated forms of HPr by native PAGE analysis. This analysis allowed us to differentiate between fast-migrating Ser-phosphorylated HPr (HPr-Ser-P or HPr-P1) and/or His/Ser-phosphorylated HPr (HPr-His/Ser-P or HPr-P2). Parallel Western blot analysis of the whole-cell lysates of M1-SDHHBtail mutant and M1-WT strains was carried out to determine relative endogenous protein levels of HPr and CcpA. Although the protein expression profiles of CcpA and Hpr were comparable in both GAS strains (Fig. 3A), a significant increase in the amount of doubly phosphorylated Hpr (Hpr-Ser-P, -His-P [HPr-P2]) in the M1-SDHHBtail mutant was observed (Fig. 3B). This indicated that the retention of SDH in the cytoplasm results in increased phosphorylation of Hpr possibly due to increased ATP production (Fig. 2A) as also reported previously (31).

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