Comparative Proteomic Analysis of Hydrogen Peroxide-induced Protein Expression in Streptococcus pneumoniae D39

Streptococcus pneumoniae is a leading cause of human respiratory tract infection. Despite the lack of activities of antioxidative enzymes, including cytochromes, hemoproteins, and peroxidases/catalases, traits conferring the aerotolerant-anaerobic growth of this bacterium are conserved, with high efficacy of antioxidative actions, in an oxygen-rich environment. Through proteome analysis, this study’s intention was to evaluate differentially expressed proteins and/or gene products modeled in a highly virulent strain, S. pneumoniae D39, exogenously-treated with millimolar concentrations of H2O2. For two-dimensional gel electrophoresis (2-DE) analysis, following one dimensional isoelectric focusing with an immobilized pH gradient of pH 4-7, the most significantly mobilized proteins expressed were separated by SDS-PAGE in the second dimension. With a total of 431 protein spots detected, certain proteins were excised, in-gel trypsin digested, and analyzed by combination with MALDI-TOF and LC-ESI-MS/MS for mass spectrometric peptide mapping and protein identification. Utilizing mass spectrometry analysis of spots excised from 2-DE, the selected protein spots were identified with a variety of databases and MASCOT. With the aid of comparisons to proteome reference maps, the most differentially expressed 38 proteins, those with approximately 1.4-fold or more increase and/or decrease or with multiple isoforms exhibiting variable pI values, were induced by treatment of exogenous 2 mM H2O2. The identified proteins were seen to be involved in pneumococcal pathogenesis and primary metabolism, amongst others. This is the first study to convincingly document proteomic information associated with pathophysiological adaptation under the given oxidative conditions, and corresponding potential antioxidative mechanisms, in S. pneumoniae.