title: Streptococcus pneumoniae enolase is important for plasminogen binding despite low abundance of enolase protein on the bacterial cell surface.
abstract: Enolase represents one of the anchorless surface proteins of Streptococcus pneumoniae and has previously been identified as a plasminogen-binding protein, endowing this pathogen with host proteolytic activity. In this study the mAb 245,C-6 (IgG1) was produced in a BALB/c mouse after immunizing with a protein fraction from S. pneumoniae. The mAb reacted with recombinant pneumococcal enolase both under non-denaturing and denaturing conditions. The epitope for the mAb was mapped to residues (55)DKSRYGGLG(63) of pneumococcal enolase using a peptide array. By applying the previously reported structure of enolase, this epitope was localized in a surface-exposed loop in each of the monomers of the octameric enolase. Previous immunoelectron microscopic studies, using polyclonal rabbit antibodies against enolase, depicted enolase on the cell surface but did not quantify the amount of surface-exposed enolase on viable pneumococci. Here, flow cytometry revealed no binding of mAb 245,C-6 to viable pneumococci, including TIGR4 and its non-encapsulated isogenic mutant, and only a minor increase of fluorescence intensity was measured when the polyclonal anti-enolase antibodies were used. In contrast, control antibodies recognizing the choline-binding proteins (CBPs) PspA and PspC showed high reactivities. The non-encapsulated TIGR4 did not show increased levels of antibody binding for mAb 245,C-6 or polyclonal anti-enolase antibodies, but revealed increased binding of polyclonal antibodies reacting with PspA or PspC. These results suggest that, compared to other surface-exposed proteins such as CBPs, the amount of enolase under the selected conditions is low. Flow cytometry, however, with FITC-labelled plasminogen demonstrated that the amount of surface-exposed enolase is important for plasminogen binding and, therefore, is also important for pneumococcal pathogenesis.
authors.affiliation: Department of Bacteriology and Immunology, Norwegian Institute of Public Health, Box 4404 Nydalen, NO-0403 Oslo, Norway.
authors.forename: Jan
authors.initials: J
authors.lastname: Kolberg
authors.affiliation: Department of Bacteriology and Immunology, Norwegian Institute of Public Health, Box 4404 Nydalen, NO-0403 Oslo, Norway.
authors.forename: Audun
authors.initials: A
authors.lastname: Aase
authors.affiliation: Research Center for Infectious Diseases, University of Würzburg, Roentgenring 11, D-97070 Würzburg, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Bergmann
authors.affiliation: Department of Bacteriology and Immunology, Norwegian Institute of Public Health, Box 4404 Nydalen, NO-0403 Oslo, Norway.
authors.forename: Tove K
authors.initials: TK
authors.lastname: Herstad
authors.affiliation: Department of Bacteriology and Immunology, Norwegian Institute of Public Health, Box 4404 Nydalen, NO-0403 Oslo, Norway.
authors.forename: Gunnhild
authors.initials: G
authors.lastname: Rødal
authors.affiliation: Department of Chemical Biology, German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany.
authors.forename: Ronald
authors.initials: R
authors.lastname: Frank
authors.affiliation: Department of Microbial Pathogenesis, German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Rohde
authors.affiliation: Research Center for Infectious Diseases, University of Würzburg, Roentgenring 11, D-97070 Würzburg, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Hammerschmidt
mesh_terms: D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D018604:Epitope Mapping; D005434:Flow Cytometry; D008565:Membrane Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008958:Models, Molecular; D010751:Phosphopyruvate Hydratase / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000502:physiology
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D010958:Plasminogen; D010751:Phosphopyruvate Hydratase