OUTER-MEMBRANE PROTEIN-BINDING SITES OF COMPLEMENT COMPONENT 3 DURINGOPSONIZATION OF HAEMOPHILUS-INFLUENZAE

Complement component 3 (C3) binding to Haemophilus influenzae type b ( Hib) is an important step in host defense against invasive disease, bu t the details of this process remain poorly understood. We have shown that the P1 and P2 outer membrane proteins (OMPs) serve as binding sit es for C3 on serum-opsonized Hib. Whole-cell lysates of opsonized Hib were subjected to sodium dodecyl sulfate-polyacrylamide gel electropho resis, and the resolved proteins were transferred to nitrocellulose. I mmunoblot 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 H ib mutant still lacked C3 bound to the 98-kDa or P6 OMP, indicating th at the absence of C3 binding to these proteins was not the result of e pitope masking by the capsule. Studies with MAbs to C3 fragments confi rmed that the anti-P1- and anti-P2-reactive bands were C3 fragments bo und 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 pre sence of other anti-C3-reactive proteins indicated that several other proteins serve as C3 targets during the opsonization of Hib.