Analysis of Streptococcus pneumoniae secreted antigens by immuno-proteomic approach

Streptococcus pneumoniae is an important human pathogen that causes a variety of diseases in both adults and children, such as pneumonia, bacteremia, meningitis, otitis media, and sinusitis. Despite their clinical importance, to date, there have been few proteomic studies of these strains for screening of virulence factors or diagnostic markers. In the present study, secreted proteins (secretome) of Streptococcus pneumoniae strains were enriched using ammonium sulfate precipitation and identified by the shotgun proteomic method using 1-dimensional electrophoresis liquid chromatography-mass spectrometry/mass spectrometry analysis. Characterization of the identified proteins revealed that 17.8% (42) of the secreted proteins possessed signal peptides. Twenty-one secreted proteins belonged to the extracellular group, and 4 secreted proteins belonged to the cell wall group. Well-known virulence factors (PrtA, PspC, PsaA, PbpA, PhtD, AmiA, ZmpB, Eno, and Ply) were included in the secreted protein fraction. Western blotting using antiserum against secreted protein mixtures showed that Gsp-781, Sphtra, NagA, PhtD, ZmpB, and Eno were strongly immunogenic. Our data suggest that the immuno-proteomic approach is a useful method for high-throughput identification of secreted proteins and screening of candidate vaccine antigens or diagnostic markers. Gsp-781 is introduced as a novel secreted antigen of Streptococcus pneumoniae.