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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

Effects of the cytoplasmic retention of SDH on the expression of virulence factors in GAS. (A) Transmission electron microscopy of the wild-type (M1-WT) and mutant (M1-SDHHBtail) GAS strains. (B) Relative expression levels of the M1 protein and streptokinase in the cell wall fractions and culture supernatants of the M1-WT and M1-SDHHBtail GAS strains as determined by type M1-reactive 10B6 monoclonal antibody (Anti-M 10B6 Ab) and antistreptokinase antibody (Anti-SKA Ab) in Western blot analysis. (C) Hyaluronic acid contents in M1-WT and M1-SDHHBtail GAS strains. (D) Relative percent hemolysin activity on sheep RBCs of the serially diluted (10-fold-diluted) culture supernatants of M1-WT and M1-SDHHBtail GAS strains. Lysis of RBCs in the undiluted culture supernatant of the M1-WT strain was set at 100%.
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f4: Effects of the cytoplasmic retention of SDH on the expression of virulence factors in GAS. (A) Transmission electron microscopy of the wild-type (M1-WT) and mutant (M1-SDHHBtail) GAS strains. (B) Relative expression levels of the M1 protein and streptokinase in the cell wall fractions and culture supernatants of the M1-WT and M1-SDHHBtail GAS strains as determined by type M1-reactive 10B6 monoclonal antibody (Anti-M 10B6 Ab) and antistreptokinase antibody (Anti-SKA Ab) in Western blot analysis. (C) Hyaluronic acid contents in M1-WT and M1-SDHHBtail GAS strains. (D) Relative percent hemolysin activity on sheep RBCs of the serially diluted (10-fold-diluted) culture supernatants of M1-WT and M1-SDHHBtail GAS strains. Lysis of RBCs in the undiluted culture supernatant of the M1-WT strain was set at 100%.

Mentions: Microarray analysis of the M1-SDHHBtail mutant revealed differential expression profiles for 33 of the most important and well-characterized virulence genes (Table 3). The latter include adenine dinucleotide glycohydrolase (Nga/SPy0165), streptolysin O (Slo/SPy0167), C3 family ADP-ribosyltransferase (SpyA/SPy0428), exotoxins (SpeJ/SPy0436, SpeB/SPy2039), streptolysin S (SagA operon/SPy0738-SPy00746), pullulanase (PulA/SPy1972), streptokinase A (Ska/SPy1979), collagen-like surface protein (Scl/SPy1983), inhibitor of complement-mediated lysis (Sic/SPy2016), M protein (Emm1/SPy2018), mitogenic factor (mf/SPy2043) and capsule (Has operon/SPy2200-SPy2202). These gene products have been shown to play an important role in S. pyogenes colonization, persistence, dissemination, and proliferation (1, 27). Many of these virulence factors which are displayed as surface proteins contribute to the outer, electron-dense, fuzzy layer on the surface of GAS. The mutant (0.94 µm) was significantly larger (34%) than the wild-type strain (~0.72 µm). In the mutant, this fuzzy layer was found to be absent, which suggests the loss of expression of several surface proteins, including M protein, which concurs with the microarray and qRT-PCR data (Fig. 4A; see also Table S4 in the supplemental material).


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

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

Effects of the cytoplasmic retention of SDH on the expression of virulence factors in GAS. (A) Transmission electron microscopy of the wild-type (M1-WT) and mutant (M1-SDHHBtail) GAS strains. (B) Relative expression levels of the M1 protein and streptokinase in the cell wall fractions and culture supernatants of the M1-WT and M1-SDHHBtail GAS strains as determined by type M1-reactive 10B6 monoclonal antibody (Anti-M 10B6 Ab) and antistreptokinase antibody (Anti-SKA Ab) in Western blot analysis. (C) Hyaluronic acid contents in M1-WT and M1-SDHHBtail GAS strains. (D) Relative percent hemolysin activity on sheep RBCs of the serially diluted (10-fold-diluted) culture supernatants of M1-WT and M1-SDHHBtail GAS strains. Lysis of RBCs in the undiluted culture supernatant of the M1-WT strain was set at 100%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Effects of the cytoplasmic retention of SDH on the expression of virulence factors in GAS. (A) Transmission electron microscopy of the wild-type (M1-WT) and mutant (M1-SDHHBtail) GAS strains. (B) Relative expression levels of the M1 protein and streptokinase in the cell wall fractions and culture supernatants of the M1-WT and M1-SDHHBtail GAS strains as determined by type M1-reactive 10B6 monoclonal antibody (Anti-M 10B6 Ab) and antistreptokinase antibody (Anti-SKA Ab) in Western blot analysis. (C) Hyaluronic acid contents in M1-WT and M1-SDHHBtail GAS strains. (D) Relative percent hemolysin activity on sheep RBCs of the serially diluted (10-fold-diluted) culture supernatants of M1-WT and M1-SDHHBtail GAS strains. Lysis of RBCs in the undiluted culture supernatant of the M1-WT strain was set at 100%.
Mentions: Microarray analysis of the M1-SDHHBtail mutant revealed differential expression profiles for 33 of the most important and well-characterized virulence genes (Table 3). The latter include adenine dinucleotide glycohydrolase (Nga/SPy0165), streptolysin O (Slo/SPy0167), C3 family ADP-ribosyltransferase (SpyA/SPy0428), exotoxins (SpeJ/SPy0436, SpeB/SPy2039), streptolysin S (SagA operon/SPy0738-SPy00746), pullulanase (PulA/SPy1972), streptokinase A (Ska/SPy1979), collagen-like surface protein (Scl/SPy1983), inhibitor of complement-mediated lysis (Sic/SPy2016), M protein (Emm1/SPy2018), mitogenic factor (mf/SPy2043) and capsule (Has operon/SPy2200-SPy2202). These gene products have been shown to play an important role in S. pyogenes colonization, persistence, dissemination, and proliferation (1, 27). Many of these virulence factors which are displayed as surface proteins contribute to the outer, electron-dense, fuzzy layer on the surface of GAS. The mutant (0.94 µm) was significantly larger (34%) than the wild-type strain (~0.72 µm). In the mutant, this fuzzy layer was found to be absent, which suggests the loss of expression of several surface proteins, including M protein, which concurs with the microarray and qRT-PCR data (Fig. 4A; see also Table S4 in the supplemental material).

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