Immunobiological characterization of genetic products, identified from whole genome lambda-display libraries of Pneumococcus

Objectives: Streptococcus pneumoniae is an important cause of morbidity and mortality worldwide, including meningitis. Here, we employed the isogenic Δspr0075-, Δspr1370- and Δspr1875-knock-out mutants of the DP1004 pneumococcal strain to investigate the role of these proteins in the interaction with microglia, the resident brain macrophages. Methods: By screening a whole genome phage display library with sera from patients, we identified clones carrying pneumococcal B-cell epitopes. Among these, we isolated six antigenic fragments of sequences matching three previously unidentified proteins, encoded by the ORFs spr0075, spr1370 and spr1875 of R6 strain genomic sequence. By using an in vitro infection model and a gentamicin protection assay we evaluated, respectively, the ability of microglial BV2 cells to phagocytose and kill the isogenic mutants and the survival of these bacteria inside the microglia. Results: All strains were efficiently internalized by BV2 cells; yet the levels of phagocytosis obtained with Δspr0075 strain were lower than those observed with the other groups. The survival of the deleted strains inside the microglia was significantly different. The residual CFU of Δspr1370 and Δspr1875 mutants were, indeed, respectively, higher and lower, than those observed with parental DP1004. Moreover, preliminary results indicate that a similar trend is also observed in a bactericidal assay using BV2 as effector cells. Conclusion: These findings indicate that the proteins encoded by spr1370 and spr1875 loci are involved in interaction between S. pneumoniae and microglia.