The principal investigator and his colleagues have reported in 1989 that serum IgA1 initiates classical pathway-mediated lysis of N. meningitidis type C. Since IgA1 -initiated lysis is a unique event, the principal investigator now proposes to purify the outer membrane protein to which IgA1 binds as a possible mucosal vaccine candidate.
Four specific aims are proposed: (1) to purify the IgA1 binding protein by transposon mutagenesis of the IgA1-sensitive MC19 type C strain. Mutants will be screened for those colonies that are resistant to IgA1- induced lysis. Portions of the chromosome containing the transposon will be subcloned for expression as a fusion protein, which will be tested for binding of lytic IgA1. The gene encoding the target protein will be used to screen different meningococcal serotypes and other neisserial species, including N. lactamica, to determine the degree of conservation. Partial N- and C-terminal sequencing of the target protein will be performed to determine its similarity to conserved sequences in other OMP, particularly the leader sequence. If transposon mutagenesis does not prove to be feasible, three alternative methods for isolation are proposed: (1) traditional chromatography; (2) elution of IgA1binding proteins from OMP preparations on SDS-PAGE; and (3) use of OMP monoclonal antibodies to inhibit IgA1 bactericidal activity. (2) to determine the requirement for C1q binding in activation of the classical complement pathway by the binding of lytic IgA1. Lysis in C1q-depleted serum will be used to establish a requirement for C1q. C1q will be purified according to published procedures and its binding to lytic IgA1 will be quantified by ELISA. The Fc region of IgA1, relevant oligosaccharides, or the C1q triplet Gly-Xaa-Yaa will be used to inhibit C1q binding. (3) to determine a role for glycosylation of the Fc terminus of IgA1. Treatment with endoH will be used to remove N-linked sugars from two sites in the Fc portion of IgA1, and studies will be performed to determine whether deglycosylated IgA1 or its F(abc)'2 fragment retains lytic function. (4) to analyze the fragmentation patterns of C3 deposited in the presence of blocking versus lytic IgA1, in order to provide an explanation for the paradox between increased lysis in the absence of augmented C3 deposition.
