title: Outer membrane protein binding sites of complement component 3 during opsonization of Haemophilus influenzae.
abstract: Complement component 3 (C3) binding to Haemophilus influenzae type b (Hib) is an important step in host defense against invasive disease, but the details of this process remain poorly understood. We have shown that the P1 and P2 outer membrane proteins (OMPs) serve as binding sites for C3 on serum-opsonized Hib. Whole-cell lysates of opsonized Hib were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to nitrocellulose. Immunoblot analysis with monoclonal antibodies (MAbs) to the 49-kDa P1 and 39-kDa P2 OMPs demonstrated high-molecular-weight bands that were not present when the bacteria were opsonized with heat-inactivated or methylamine-treated serum. Immunoblot analysis with MAbs to the 98- or 16-kDa (P6) OMPs did not reveal additional bands. An unencapsulated Hib mutant still lacked C3 bound to the 98-kDa or P6 OMP, indicating that the absence of C3 binding to these proteins was not the result of epitope masking by the capsule. Studies with MAbs to C3 fragments confirmed that the anti-P1- and anti-P2-reactive bands were C3 fragments bound to these OMPs. The molecular weights of proteins reactive to anti-OMP and anti-C3 antibodies indicated that multiple C3 fragments may be bound to P1 or that C3 may be bound to P2 multimers. Finally, the presence of other anti-C3-reactive proteins indicated that several other proteins serve as C3 targets during the opsonization of Hib.
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38101-0318.
authors.forename: S V
authors.initials: SV
authors.lastname: Hetherington
authors.forename: C C
authors.initials: CC
authors.lastname: Patrick
authors.forename: E J
authors.initials: EJ
authors.lastname: Hansen
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D000954:Antigens, Surface / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003176:Complement C3 / Q000378:metabolism*; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D006193:Haemophilus influenzae / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009895:Opsonin Proteins / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D003176:Complement C3; D000939:Epitopes; D009895:Opsonin Proteins; C067559:outer membrane protein P1, Haemophilus influenzae type b; C074703:outer membrane protein P2, Haemophilus influenzae type b