The binding of C3b, the opsonic fragment of the third component of complement (C3) to bacterial surface structures prepares the microorganisms for ingestion by phagocytic cells. We propose to examine, at the molecular level, this interaction between C3 and pathogenic bacteria. We contend that a particular structure of C3--the reactive thiolester--mediates the attachment of the protein to the organism by means of a transacylation reaction. The structure of the organism, in turn, provides specific sites of acylation for the protein. The thiolester of C3 resides within a transiently generated binding site on the Alpha' chain of the C3 protein and is formed by the linking of a cysteinyl residue with a glutamyl residue. In our published studies of the covalent reaction of C3 with small carbohydrates and amines, we have shown that the glutamyl component of the thiolester is the site of transacylation reactions, donating its acyl group to form covalent ester or amide bonds with appropriate carbohydrates or amines. We propose that opsonization of virulent bacteria proceeds by the identical biochemical reaction. First, using radiolabeled, purified complement components, C4-deficient human serum, and polymorphonuclear leukocytes from normal adults, we shall analyze differences in the opsonization of virulent serotypes of Streptococcus pneumoniae (types 3, 4, 6, 8, 14, and 18) as a function of number of covalent binding sites for C3b per organism. Contributions of type-specific anticapsular antibody to the binding of C3b, as well as binding to polymorphonuclear leukocyte receptors for complement, will also be examined with these methods. Secondly, employing the methods which we used for the characterization of the transacylation reaction with small carbohydrates, we shall identify the constituents of the covalent bond formed in opsonic interactions. C3 will be covalently attached to pneumococcal polysaccharide, the peptide containing the binding site will be isolated, and the reactive species from both C3 and polysaccharide will be analyzed. Thirdly, we shall relate the reactivity of the thiolester to opsonic function in two human models of defective opsonization: neonates and patients homozygous for hemoglobin-SS.
