Interactions of C3 with Bacterial Pathogens

In the pathogenesis of pneumococcal infection, early events including attachment of the organism to resting epithelial cells have received considerably less attention than later aspects such as opsonization and phagocytosis. Our laboratory has recently demonstrated that several types of epithelial cells synthesize C3 as part of the pericellular environment. We have further shown that virulent serotypes of S. pneumoniae, including serotypes 2,3,4,14 and 19, synthesize as cell-bound C3-degrading proteinase during exponential growth. In vitro, degradation of 50% of soluble C3 molecules occurs rapidly within 5 minutes by preferential attack of the proteinase at the V3 beta-chain. Of those C3 moleculs not completely degraded, more than 40% are functionally inactivated at the thiolester site. Standard cleavage fragments such as C3b, iC3b, or C3d are not observed. The biochemical action of the pneumococcal proteinase therefore differs unequivocally from traditional C3 cleavage due to serine proteinases, complement convertases, or organisms such as Pseudomonas aeruginosa or Entamoeba histolytica. Our recent experiments have shown that C3 degrading activity correlates directly with pneumococcal adhesion to resting pulmonary epithelial cells (A549). We therefore hypothesize that the C3-degrading proteinase of S. pneumoniae is a novel enzyme that not only inactivates C3 in plasma, but also facilitates epithelial adhesion. In Specific Aim One, we shall clone the gene encoding the C3-degrading proteinase by insertional inactivation, by screening our library of 8,000 pneumococcal transformants for loss of proteinase production in a C3 antibody-binding ELISA assay. Three insertionally inactivated mutants that are deficient in C3 degradation have already been identified. Excision of the recombinant plasmid at low frequency allows its use as a probe (a) to obtain the entire gene for sequencing and overexpression and (b) to examine a variety of clinical isolates for the presence of the gene and its level of expression. In Specific Aim Two, we shall purify the C3-degrading proteinase from mutanolysin digests of pneumococcal surface proteins by ion exchange and affinity chromatography, in quantities sufficient to permit sequencing of the amino-terminus or internal peptides and generation of polyclonal antibodies. In Specific Aim Three, we shall further characterize the interactions of the C3-degrading proteinase with C3 at the epithelial surface and in plasma. Functions of the proteinase in facilitating pneumococcal adhesion to epithelial cells, in inhibiting C3- mediated opsonization, and in releasing cytokines from leukocytes and epithelium will be assessed with purified proteinase and proteinase- deficient mutants. These studies should yield new information regarding a novel C3-degrading enzyme from S. pneumoniae and its role in the pathogenesis of pneumococcal infection.