Multifunctional glyceraldehyde-3-phosphate dehydrogenase of Streptococcus pyogenes is essential for evasion from neutrophils

Streptococcus pyogenes is an important pathogen that causes pharyngitis, sepsis, and rheumatic fever. Cell-associated streptococcal C5a peptidase (ScpA) protects S. pyogenes from phagocytosis and has been suggested to interrupt host defenses by enzymatically cleaving complement C5a, a major factor in the accumulation of neutrophils at sites of infection. How S. pyogenes recognizes and binds to C5a, however, is unclear. We detected a C5a-binding protein in 8 M urea extracts of S. pyogenes by ligand blotting using biotinylated C5a. Searching of genome databases showed that the C5a-binding protein is identical to the streptococcal plasmin receptor (Plr), also known as streptococcal surface dehydrogenase (SDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the present study we identified a novel function of this multifunctional protein. Western blotting and immunofluorescence microscopy with anti-Plr/SDH/GAPDH showed that Plr/SDH/GAPDH is located on the bacterial surface and released into the culture supernatant. Next, we examined whether the streptococcal Plr/SDH/GAPDH inhibits the biological effects of C5a on human neutrophils. We found that soluble Plr/SDH/GAPDH inhibits C5a-activated chemotaxis and H2O2 production. Furthermore, our results suggested that soluble Plr/SDH/GAPDH captures C5a, inhibiting its chemotactic function. Also, cell-associated Plr/SDH/GAPDH and ScpA were both necessary for the cleavage of C5a on the bacterial surface. Together, these results indicate that the multifunctional protein Plr/SDH/GAPDH has additional functions that help S. pyogenes escape detection by the host immune system.     

Characterization of group B streptococcal glyceraldehyde-3-phosphate dehydrogenase: surface localization,enzymatic activity,and protein-protein interactions

During characterization of the surface antigens of serotype III group B streptococci (GBS), a protein with an apparent M(r) of approximately 173,500 migrating on a SDS--polyacrylamide gel was found to have an N-terminal amino acid sequence identical to that of the plasmin receptor (Plr) of group A streptococci, a surface-localized glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This work begins to characterize GBS GAPDH and to assess its functional activity on the cell surface. The 1.0-kb gapC gene of GBS was amplified by PCR. plr and gapC demonstrated 87% homology. An anti-Plr monoclonal antibody reacted with GBS whole cells, suggesting GBS GAPDH is surface localized. Multiple serotypes of GBS demonstrated functional GAPDH on their surfaces. The anti-Plr monoclonal antibody recognized GBS protein bands of approximately 41 and 173.5 kDa, by Western blot. Presumably, these represent monomeric and tetrameric forms of the GAPDH molecule. GBS GAPDH was demonstrated by Western blot analysis to interact with lys- and glu-plasminogens. Fluid-phase GBS GAPDH interacted, by means of ELISA, with immobilized lys-plasminogen, glu-plasminogen, actin, and fibrinogen. Enzymatically active GAPDH, capable of binding cytoskeletal and extracellular matrix proteins, is expressed on the surface of GBS.     

The potential role for nephritis-associated plasmin receptor in acute poststreptococcal glomerulonephritis

Immunoglobulin G from a patient convalescing from acute poststreptococcal glomerulonephritis (APSGN) bound specific antigenic sites in early APSGN glomeruli. A streptococcal cytoplasmic antigen (preabsorbing antigen, PA-Ag), could selectively preabsorb fluorescein isothiocyanate (FITC)-labeled IgG and prevented glomerular staining. The antigen was purified and identified as an M(r) approximately 43,000 protein with a pI of 4.7 that strongly activated complement C3 (N. Yoshizawa, S. Oshima, I. Sagel, J. Shimizu, and G. Treser, 1992, J. Immunol. 148, 3110-3116). In the present study, a nephritogenic antigen was purified by affinity chromatography using APSGN IgG-immobilized Sepharose followed by chromatography on an anion-exchange resin. Purification was monitored by ELISA and Western blotting using the binding characteristics of the specific antibodies present in APSGN serum. The molecular weight of the purified antigen, named nephritis-associated plasmin receptor (NAPlr), was an M(r) approximately 43,000 protein and the internal amino acid sequence was found to be homologous to those of the plasmin receptor (Plr) of group A streptococci strain 64/14 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus subtilis. The purified NAPlr exhibited GAPDH activity and plasmin(ogen) binding activity. Using FITC-labeled rabbit anti-NAPlr, the antigen was found to be present in the glomeruli of 22 of 22 patients in the early stage of APSGN. Bacterial Plr was also demonstrated in human APSGN glomeruli for the first time using monoclonal antibody to the recombinant Plr protein. Antibody to NAPlr was found in the sera of 46 of 50 (92%) patients within 3 months of onset. These results led us to speculate that NAPlr bound to the glomeruli may contribute to the pathogenesis of APSGN via plasmin and complement activation.     

A role for the fibrinogen-binding regions of streptococcal M proteins in phagocytosis resistance

All virulent group A streptococcal isolates bind fibrinogen, a property that is closely linked to expression of type-specific antiphagocytic surface molecules designated M proteins. Here we show that although the M proteins from two different strains, M1 and M5, both bind fibrinogen with high affinity, they interact with different regions in the ligand. Moreover, mapping experiments demonstrated that the fibrinogen-binding regions in the M1 and M5 proteins are quite dissimilar at the amino acid sequence level and that they bind to different regions in the plasma protein. In spite of these differences, the fibrinogen-binding regions of M1 and M5 could both be shown to contribute to streptococcal survival in human blood, providing evidence for the distinct function of a plasma protein interaction in bacterial pathogenesis.     

M-related protein (Mrp) contributes to group A streptococcal resistance to phagocytosis by human granulocytes

The M protein has been postulated to be a major group A streptococcal (GAS) virulence factor because of its contribution to the bacterial resistance to opsono-phagocytosis. Direct evidence of this was only provided for GAS strains which expressed a single M protein. The majority of GAS express additional, structurally similar M-related proteins, Mrp and Enn, which have been described as IgG- and IgA-binding proteins. To determine the involvement of Mrp and M protein in phagocytosis resistance, the mrp and emm genes from serotypes M2, M4, and M49 as well as from M-untypeable strain 64/14 were insertionally inactivated. The mrp and emm mutants were subjected to direct bactericidal assays. As judged by numbers of surviving colony-forming units, all mutant strains with the exception of the mrp 4 mutant exhibited reduced multiplication factors as compared to the isogenic wild-type strains. Subsequent analysis of phagocytosis by flow cytometry, measuring association of BCECF/AM-labelled bacteria and granulocytes, paralleled the results from direct bactericidal assays regardless of whether isolated granulocytes or whole blood were utilized. Resistant wild-type GAS strains bound to less than 24% of granulocytes, whereas phagocytosis-sensitive controls attached to more than 90% of the white blood cells. 40 to 60% of the granulocytes associated with the mrp and emm mutants within 1 h of co-incubation. Kinetic data suggested that attachment to granulocytes proceeds faster for emm mutants than for corresponding mrp mutants. By adding a dihydro-rhodamine123 stain and measuring fluorescence induced by oxidative burst, the experimental data suggested that bacteria bound to granulocytes were also engulfed and integrated into phagolysosomes. Thus, these data indicated that, if present, both mrp and emm gene products contribute to phagocytosis resistance by decreasing bacterial binding to granulocytes.     

Mapping of the interaction site of CP12 with glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii. Functional consequences for glyceraldehyde-3-phosphate dehydrogenase

The 8.5 kDa chloroplast protein CP12 is essential for assembly of the phosphoribulokinase/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complex from Chlamydomonas reinhardtii. After reduction of this complex with thioredoxin, phosphoribulokinase is released but CP12 remains tightly associated with GAPDH and downregulates its NADPH-dependent activity. We show that only incubation with reduced thioredoxin and the GAPDH substrate 1,3-bisphosphoglycerate leads to dissociation of the GAPDH/CP12 complex. Consequently, a significant twofold increase in the NADPH-dependent activity of GAPDH was observed. 1,3-Bisphosphoglycerate or reduced thioredoxin alone weaken the association, causing a smaller increase in GAPDH activity. CP12 thus behaves as a negative regulator of GAPDH activity. A mutant lacking the C-terminal disulfide bridge is unable to interact with GAPDH, whereas absence of the N-terminal disulfide bridge does not prevent the association with GAPDH. Trypsin-protection experiments indicated that GAPDH may be also bound to the central alpha-helix of CP12 which includes residues at position 36 (D) and 39 (E). Mutants of CP12 (D36A, E39A and E39K) but not D36K, reconstituted the GAPDH/CP12 complex. Although the dissociation constants measured by surface plasmon resonance were 2.5-75-fold higher with these mutants than with wild-type CP12 and GAPDH, they remained low. For the D36K mutation, we calculated a 7 kcal.mol(-1) destabilizing effect, which may correspond to loss of the stabilizing effect of an ionic bond for the interaction between GAPDH and CP12. It thus suggests that electrostatic forces are responsible for the interaction between GAPDH and CP12.     

The modification of the cell wall proteins in group A streptococci type M 29 under the influence of spermidine contained in the culture medium

The addition of spermidine into growth medium used for the cultivation of group A streptococci, type M 29, leads to changes in the amino acid composition of cell walls and surface proteins isolated by the method of E. H. Beachey et al. The separation of surface proteins into fibrinogen-binding proteins and fibrinogen receptors by affinity chromatography techniques on cellulose with covalently bound fibrinogen indicates that the proportion of these proteins in pepsin extracts obtained from different strains varies. Both spermidine and avirulent strains have similar content of fibrinogen-binding proteins, although these proteins are absent in virulent strains. Different amounts of fibrinogen receptors are extracted from all strains. As shown in the enzyme immunoassay, fibrinogen receptors contain no group-specific polysaccharide A, Fc-receptors and interact with total antiserum to group A streptococci, type M 29 [correction of 28]. Fibrinogen receptors isolated from the strains under study have been found to have similar amino acid composition. On the basis of these results we believe that neither receptor capacity to fibrinogen nor amino acid composition is indicative of the protective properties of protein M.     

Anti-phagocytic mechanisms of Streptococcus pyogenes: binding of fibrinogen to M-related protein

A key attribute of invasive Streptococcus pyogenes is their ability to resist phagocytosis and multiply in human blood. M-related protein (Mrp) is a major anti-phagocytic factor but the mechanism whereby it helps streptococci to evade phagocytosis has not been demonstrated. We investigated phagocytosis resistance in a strain of serotype M4 by inactivating the mrp gene and also the emm, enn, sof and sfbX genes and by analysing the effect on streptococcal growth in blood and on complement deposition on the bacterial surface. Inactivation of enn4 and sfbX4 had little impact on growth in blood, but ablation of mrp4, emm4 or sof4 reduced streptococcal growth in human blood, confirming that Mrp and Emm are required for optimal resistance to phagocytosis and providing the first indication that Sof may be an anti-phagocytic factor. Moreover, antisera against Mrp4, Emm4 and Sof4 promoted the killing of S. pyogenes, but anti-SfbX serum had no effect. Growth of S. pyogenes in blood was dependent on the presence of fibrinogen and in the absence of fibrinogen there was a twofold increase in complement deposition. Inactivation of mrp4 resulted in a loss of fibrinogen-binding and caused a twofold increase in the binding of C3b that was inhibited by Mg-EGTA. Mrp contained two fibrinogen-binding sites, one of which is within a highly conserved region. These findings indicate that Mrp-fibrinogen interactions prevent surface deposition of complement via the classical pathway, thereby contributing to the ability of these streptococci to resist phagocytosis. This may be a common mechanism for evasion of phagocytosis because Mrp is expressed by approximately half of the clinical isolates of S. pyogenes.     

Promotion of phagocytosis of Streptococcus pyogenes in human blood by a fibrinogen-binding peptide

The binding of fibrinogen to M-related protein (Mrp) is known to contribute to the ability of Streptococcus pyogenes to evade phagocytosis by preventing the deposition of complement on the streptococcal surface. The objectives of this investigation were to map the common fibrinogen-binding domain of Mrp and to determine if this domain has a therapeutic potential to enhance phagocytosis of S. pyogenes in human blood. Using a series of recombinant, truncated proteins of Mrp, two fibrinogen-binding domains (FBD) were mapped. FBD1 was contained within amino acid residues 1-55 of Mrp and FBD2 within residues 81-138. FBD2 is found in all Mrp sequenced to date whereas FBD1 is not. Both FBD1 and FBD2 peptides but not a control peptide blocked the binding of fibrinogen to S. pyogenes and promoted phagocytosis of the streptococci in human blood. The data support the hypothesis that the binding of fibrinogen by S. pyogenes is centrally involved in their resistance to phagocytosis in human blood and suggest that treatments that interfere with the binding of fibrinogen to S. pyogenes may help in fighting infections by these organisms.     

Expression of different group A streptococcal M proteins in an isogenic background demonstrates diversity in adherence to and invasion of eukaryotic cells

The M protein of group A streptococcus (GAS) is considered to be a major virulence factor because it renders GAS resistant to phagocytosis and allows bacterial growth in human blood. There are more than 80 known serotypes of M proteins, and protective opsonic antibodies produced during disease in humans are serotype specific. M proteins also mediate bacterial adherence to epithelial cells of skin and pharynx. GAS strains vary in the genomic organization of the mga regulon, which contains the genes encoding M and M-like proteins and other virulence factors. This diversity of organization makes it difficult to assess virulence of M proteins of different serotypes, unless they can be expressed in an isogenic background. Here, we express M proteins of different serotypes in the M protein- and protein F1-deficient GAS strain, SAM2, which also lacks M-like proteins. Genes encoding M proteins of different serotypes (emmXs) have been integrated into the SAM2 chromosome in frame with the emm6.1 promoter and its mga regulon, resulting in similar levels of emmX expression. Although SAM2 exhibits a very low level of adherence to and invasion of HEp-2 and HaCaT cells, a SAM2-derived strain expressing M6 protein adheres to and invades both cell types. In contrast, the isogenic strain expressing M18 protein adheres to both cell types, but invades with a very low efficiency. A strain expressing M3 protein adheres to both types of cells, but its invasion of HEp-2 cells is serum dependent. A GAS strain expressing M6 protein does not compete with the isogenic strain expressing M18 protein for adherence to or invasion of HaCaT cells. We conclude that M proteins of different serotypes recognize different repertoires of receptors on the surfaces of eukaryotic cells.     

Relationship between Expression of the Family of M Proteins and Lipoteichoic Acid to Hydrophobicity and Biofilm Formation in Streptococcus pyogenes

Background

Hydrophobicity is an important attribute of bacteria that contributes to adhesion and biofilm formation. Hydrophobicity of Streptococcus pyogenes is primarily due to lipoteichoic acid (LTA) on the streptococcal surface but the mechanism(s) whereby LTA is retained on the surface is poorly understood. In this study, we sought to determine whether members of the M protein family consisting of Emm (M protein), Mrp (M-related protein), Enn (an M-like protein), and the streptococcal protective antigen (Spa) are involved in anchoring LTA in a manner that contributes to hydrophobicity of the streptococci and its ability to form biofilms.

Methodology/Principal Findings

Isogenic mutants defective in expression of emm, mrp, enn, and/or spa genes of eight different serotypes and their parental strains were tested for differences in LTA bound to surface proteins, LTA released into the culture media, and membrane-bound LTA. The effect of these mutations on the ability of streptococci to form a hydrophobic surface and to generate biofilms was also investigated. A recombinant strain overexpressing Emm1 was also engineered and similarly tested. The serotypes tested ranged from those that express only a single M protein gene to those that express two or three members of the M protein family. Overexpression of Emm1 led to enhanced hydrophobicity and biofilm formation. Inactivation of emm in those serotypes expressing only a single emm gene reduced biofilm formation, and protein-bound LTA on the surface, but did not alter the levels of membrane-bound LTA. The results were more varied in those serotypes that express two to three members of the M protein family.

Conclusions/Significance

Our findings suggest that the formation of complexes with members of the M protein family is a common mechanism for anchoring LTA on the surface in a manner that contributes to hydrophobicity and to biofilm formation in S. pyogenes, but these activities in some serotypes are dependent on a trypsin-sensitive protein(s) that remains to be identified. The need for interactions between LTA and M proteins may impose functional constraints that limit variations in the sequence of the M proteins, major virulence factors of S. pyogenes.     

Molecular mechanism of NADPH-glyceraldehyde-3-phosphate dehydrogenase regulation through the C-terminus of CP12 in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) consists of four GapA subunits. This A4 GAPDH is not autonomously regulated, as the regulatory cysteine residues present on GapB subunits are missing in GapA subunits. The regulation of A4 GAPDH is provided by another protein, CP12. To determine the molecular mechanisms of regulation of A4 GAPDH, we mutated three residues (R82, R190, and S195) of GAPDH of C. reinhardtii. Kinetic studies of GAPDH mutants showed the importance of residue R82 in the specificity of GAPDH for NADPH, as previously shown for the spinach enzyme. The cofactor NADPH was not stabilized through the 2'-phosphate by the serine 195 residue of the algal GAPDH, unlike the case in spinach. The mutation of R190 also led to a structural change that was not observed in the spinach enzyme. This mutation led to a loss of activity for NADPH and NADH, indicating the crucial role of this residue in maintaining the algal GAPDH structure. Finally, the interaction between GAPDH mutants and wild-type and mutated CP12 was analyzed by immunoblotting experiments, surface plasmon resonance, and kinetic studies. The results obtained with these approaches highlight the involvement of the last residue of CP12, Asp80, in modulating the activity of GAPDH by preventing access of the cofactor NADPH to the active site. These results help us to bridge the gap between our knowledge of structure and our understanding of functional biology in GAPDH regulation.     

Two DNA-binding domains of Mga are required for virulence gene activation in the group A streptococcus

Mga is a DNA-binding protein that activates expression of several important virulence genes in the group A streptococcus (GAS), including those encoding M protein (emm), C5a peptidase (scpA) and Mga (mga). To determine the functionality of four potential helix-turn-helix DNA-binding motifs (HTH1-HTH4) identified within the amino-terminus of Mga, alanine substitutions were introduced within each domain in a MBP-Mga fusion allele and purified proteins were assayed for binding to Mga-specific promoter fragments (Pmga, PscpA and Pemm) in vitro. Although HTH-1 and HTH-2 mutations showed wild type DNA-binding activity, an altered HTH-3 domain resulted in reduced binding to the three promoters and an HTH-4 mutant was devoid of detectable binding activity. Plasmid-encoded expression of the HTH-3 and HTH-4 alleles from a constitutive promoter (Pspac) in the mga-deleted GAS strain JRS519 demonstrated that Mga-regulated emm expression correlated directly to the DNA-binding activity observed for each mutant protein in vitro. Single-copy expression of HTH-3 and HTH-4 from their native Pmga resulted in a dramatic reduction in autoregulated mga expression in both mutant strains. Thus, Mga appears to contain two DNA-binding domains (HTH-3 and HTH-4) that are required for direct activation of the Mga virulence regulon in vivo.     

Cell surface proteins of a group A streptococcus type M4: The IgA receptor and a receptor related to M proteins are coded for by closely linked genes

Summary Two genes coding for cell surface proteins were cloned from a group A streptococcus type M4: the gene for an IgA binding protein and the gene for a fibrinogen binding protein. Both proteins were purified and partially characterized after expression in Escherichia coli. There was no immunological cross-reaction between the two proteins. The IgA binding protein, called protein Arp4, is similar to an IgA receptor previously purified from another strain of group A streptococci, but the proteins are not identical. Characterization of many independent clones showed that the two proteins described here are coded for by closely linked genes. Bacterial mutants have been found which have simultaneously lost the ability to express both genes, and a simple method to isolate such mutants is described. The existence of these variants indicates that expression of the two cell surface proteins may be coordinately regulated. Binding of fibrinogen is a characteristic property of streptococcal M proteins, and the available evidence suggests that the fibrinogen binding protein is indeed an M protein.     

Cloning of a cDNA fragment encoding part of the protein moiety of the 58-kDa fibrinogen-binding mannoprotein of Candida albicans

Immunoscreening of a Candida albicans expression library with antibodies against the 58 kDa fibrinogen-binding mannoprotein (mp58) of the fungus resulted in the isolation of clones encoding the protein moiety of this molecule. Sequence of the 0.9 kb cDNA of one of the clones selected for further analysis, revealed an open reading frame coding for 292 amino acids, which displays sequence similarity to proteins belonging to a family of immunodominant antigens of Aspergillus spp. The gene corresponding to this cDNA was named FBP1 (fibrinogen-binding protein). These results represent the first report on the identification of C. albicans genes encoding surface receptors for host proteins.     

Involvement of two positively charged residues of Chlamydomonas reinhardtii glyceraldehyde-3-phosphate dehydrogenase in the assembly process of a bi-enzyme complex involved in CO2 assimilation.

The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the chloroplast of Chlamydomonas reinhardtii is part of a complex that also includes phosphoribulokinase (PRK) and CP12. We identified two residues of GAPDH involved in protein-protein interactions in this complex, by changing residues K128 and R197 into A or E. K128A/E mutants had a Km for NADH that was twice that of the wild type and a lower catalytic constant, whatever the cofactor. The kinetics of the mutant R197A were similar to those of the wild type, while the R197E mutant had a lower catalytic constant with NADPH. Only small structural changes near the mutation may have caused these differences, since circular dichroism and fluorescence spectra were similar to those of wild-type GAPDH. Molecular modelling of the mutants led to the same conclusion. All mutants, except R197E, reconstituted the GAPDH-CP12 subcomplex. Although the dissociation constants measured by surface plasmon resonance were 10-70-fold higher with the mutants than with wild-type GAPDH and CP12, they remained low. For the R197E mutation, we calculated a 4 kcal/mol destabilizing effect, which may correspond to the loss of the stabilizing effect of a salt bridge for the interaction between GAPDH and CP12. All the mutant GAPDH-CP12 subcomplexes failed to interact with PRK and to form the native complex. The absence of kinetic changes of all the mutant GAPDH-CP12 subcomplexes, compared to wild-type GAPDH-CP12, suggests that mutants do not undergo the conformation change essential for PRK binding.