Functional analysis of a murine monoclonal antibody against the repetitive region of the fibronectin-binding adhesins fibronectin-binding protein A and fibronectin-binding protein B from Staphylococcus aureus.
Fibronectin-binding proteins A and B are multifunctional LPXTG staphylococcal adhesins, comprising an N-terminal region that binds fibrinogen and elastin, and a C-terminal domain that interacts with fibronectin. The C-terminal domain of fibronectin-binding protein A is organized into 11 tandem repeats, six of which bind the ligand with high affinity; other sites bind more weakly. Fibronectin-binding protein B has been postulated to harbor 10 rather than 11 repeats, but it contains the same number of high-affinity fibronectin-binding sites as fibronectin-binding protein A. In this study, we confirm this prediction and show that six of 10 sites bind with dissociation constants in the nanomolar range. We also found that the full-length repetitive region of fibronectin-binding protein B stimulated the production of a mAb (15E11) that binds with high affinity to an epitope shared by repeats 9 and 10 from both adhesins. With the use of truncated fragments of repeat 9 of fibronectin-binding protein A, we mapped the antibody epitope to the N-terminal segment and the fibronectin-binding site to the C-terminal segment of the repeat. The distinct localization of the 15E11 epitope and the fibronectin-binding site suggests that the interfering effect of the antibody might result from steric hindrance or a conformational change in the structure that reduces the accessibility of fibronectin to its binding determinant. The epitope is well exposed on the surface of staphylococcal cells, as determined by genetic analyses, fluorescence microscopy, and flow cytometry. When incubated with cells of Staphylococcus aureus strains, 15E11 inhibits attachment of bacteria to surface-coated fibronectin by almost 70%.
Provenza
Giulio
G
Department of Biochemistry, University of Pavia, Pavia, Italy.
Provenzano
Maria
M
Visai
Livia
L
Burke
Fiona M
FM
Geoghegan
Joan A
JA
Stravalaci
Matteo
M
Gobbi
Marco
M
Mazzini
Giuliano
G
Arciola
Carla Renata
CR
Foster
Timothy J
TJ
Speziale
Pietro
P
D018829:Adhesins, Bacterial / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity / Q000276:immunology; D001422:Bacterial Adhesion / Q000276:immunology; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D000939:Epitopes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D005434:Flow Cytometry; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011961:Receptors, Fc / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics / Q000276:immunology; D013205:Staphylococcal Protein A / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism
D018829:Adhesins, Bacterial; D000911:Antibodies, Monoclonal; D000939:Epitopes; D005353:Fibronectins; C045281:IgA receptor; D011961:Receptors, Fc; D013205:Staphylococcal Protein A