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

Abstract

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.

FIG 1

Growth curves of the wild-type M1-SF370 (M1-WT) and its isogenic mutant (M1-SDH_HBtail) GAS strains in chemically defined medium supplemented with glucose, sucrose, fructose, maltose, maltodextrin, and mannose. Error bars represent standard error of the mean OD_600nm obtained from three independent experiments.

FIG 2

(A) Intracellular ATP concentrations in the wild-type M1-SF370 (M1-WT) and its isogenic mutant (M1-SDH_HBtail) 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-SDH_HBtail) 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 OD_600nm obtained from three independent experiments.

FIG 3

Western blot analysis of the whole-cell lysate (CL) of the overnight-grown wild-type M1-SF370 (M1-WT) and mutant (M1-SDH_HBtail) 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-SDH_HBtail 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-SDH_HBtail GAS strains.

FIG 4

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-SDH_HBtail) 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-SDH_HBtail 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-SDH_HBtail GAS strains. (D) Relative percent hemolysin activity on sheep RBCs of the serially diluted (10-fold-diluted) culture supernatants of M1-WT and M1-SDH_HBtail GAS strains. Lysis of RBCs in the undiluted culture supernatant of the M1-WT strain was set at 100%.

FIG 5

Relationship of the surface export of SDH and SpeB secretion. (A) Determination of the relative cysteine protease activities of SpeB present in the culture supernatants of M1-WT and M1-SDH_HBtail GAS using FITC-labeled casein. The released fluorescence activity by the culture supernatant of the wild-type GAS strain was set at 100% activity, and its specificity was determined in the presence of cysteine protease inhibitor E-64. Values are means plus standard errors of the means (error bars) of three to six independent experiments. (B) Determination of SpeB-specific cysteine protease activity (hydrolysis of milk casein) present in the culture supernatants of M1-WT, M1-SDH_HBtail, M1-SDH_HBtail::sdh, and M1-WT::sdh_HBtail GAS strains using milk agar. (C) Western blot analysis showing the effect on the secretion of SpeB in the culture supernatants of M1-WT, M1-SDH_HBtail, M1-SDH_HBtail::sdh, and M1-WT::sdh_HBtail strains and the ability of SDH to bind secreted SpeB as determined by the blot overlay method.

FIG 6

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-SDH_HBtail (20 mice per group) and their corresponding complemented strains (10 mice per group), M1-SDH_HBtail::sdh and M1-WT::sdh_HBTail (complemented strains created with pDC123 plasmid containing genes encoding SDH_HBtail 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-SDH_HBtail::sdh versus M1-SDH_HBtail and M1-WT::sdh_HBTail versus M1-WT) after normalization with the housekeeping gene.