ABSTRACT
The normally cytosolic glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, (GAPDH) has been reported to be expressed on the surface of Streptococcus pyogenes, group A, where it can act as a plasmin binding protein (Plr), and potentially a signaling molecule. In studies of wild-type and isogenic mutants, an association between surface expression of antigenic GAPDH/Plr and M and M-related fibrinogen-binding proteins was identified. Inactivation of the mga gene, whose product controls expression of M and M-related proteins also influenced expression of surface GAPDH/Plr. Revertants or pseudorevertants of mga mutants led to concomitant re-expression of surface GAPDH/Plr and M and M-related proteins. Using surface enhanced laser desorption ionization (SELDI) mass spectroscopy, a physical association between GAPDH/Plr and streptococcal fibrinogen-binding proteins was demonstrated. These studies support the hypothesis that surface M and M-related proteins are involved in anchoring GAPDH/Plr on the surface of group A streptococci.
Subject(s)
Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Receptors, Peptide/metabolism , Streptococcus pyogenes/enzymology , Bacterial Proteins/metabolism , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cytosol/enzymology , Fibrinogen/metabolism , Fibrinolysin/metabolism , Mass Spectrometry , Recombinant Proteins/metabolism , Streptococcus pyogenes/genetics , Streptococcus pyogenes/ultrastructureABSTRACT
A series of methods for analyzing the interaction of group A streptococci with the human plasminogen system are described. Examples of group A streptococcal isolates capable of assembling surface plasminogen activator activity when grown in human plasma are presented and the key requirements for this process are evaluated. The stabilities of cell-associated plasmin and plasminogen activator complexes are compared and a model for the interaction of group A streptococci with the plasminogen system in an infected host is presented.
Subject(s)
Fibrinolysin/metabolism , Plasminogen/metabolism , Streptococcal Infections/blood , Streptococcus pyogenes/physiology , Streptokinase/metabolism , Adult , Blotting, Western , Fibrinogen/metabolism , Fibrinogen/physiology , Humans , Peptide Fragments/metabolismABSTRACT
Group A streptococcal isolate 187061 incubated in human plasma or serum reconstituted with fibrinogen but not plasminogen-depleted plasma or serum alone acquired a surface plasminogen activator activity. Assembly of the surface plasminogen activator was inhibited by the presence of neutralizing antibodies to streptokinase. Once assembled, the bacterial-associated plasminogen activator could generate plasmin when incubated in human plasminogen, plasmin or serum which could bind to bacterial surface plasmin-binding structures despite the presence of host physiological inhibitors. These studies provide evidence that the pathways by which group A isolates interact with human plasmin(ogen) are potentially linked and may provide a mechanism for bacteria to acquire host enzymatic activity efficiently in the infected host.
Subject(s)
Bacterial Proteins/metabolism , Fibrinolysin/metabolism , Plasminogen Activators/metabolism , Streptococcus pyogenes/metabolism , Aminocaproic Acid/analysis , Aminocaproic Acid/metabolism , Antibodies, Bacterial/administration & dosage , Fibrinogen/chemistry , Humans , Plasma , Streptococcus pyogenes/enzymology , Streptococcus pyogenes/pathogenicity , Streptokinase/metabolism , alpha-Macroglobulins/metabolismABSTRACT
A recombinant plasmin receptor (Plr) gene product originally cloned from group A streptococcal isolate 64/14 was analysed for its ability to bind plasmin(ogen) and to account for all the surface plasmin-binding properties of streptococcal isolate 64/14. Functional analysis of recombinant Plr demonstrated that the protein exhibited equal reactivity with human Lys-plasmin and Lys-plasminogen, but significantly lower reactivity with Glu-plasminogen. Plasmin-binding was both inhibitable and elutable by lysine or lysine analogs, and active plasmin bound to recombinant Plr was not neutralized by alpha 2-antiplasmin. Thus, the plasmin-binding properties of recombinant Plr correlated with the plasmin-binding phenotype of the intact streptococcal isolate 64/14. In addition, fluid-phase recombinant Plr could completely inhibit binding of plasmin to either immobilized recombinant Plr or group A streptococcal isolate 64/14 with equal efficiency, indicating that surface-expressed Plr could account for all the plasmin-binding properties of the intact organism. An IgM monoclonal antibody to recombinant Plr that specifically recognized a surface structure on streptococcal isolate 64/14 significantly inhibited the binding of plasmin to the recombinant protein; however, the antibody was not successful at inhibiting plasmin-binding to the intact bacteria, indicating the presence of other plasmin-binding structures on the bacterial surface in addition to Plr.
