Effect of acidic pH on expression of surface-associated proteins of Streptococcus oralis

Abstract

Streptococcus oralis, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque. We undertook to identify the most abundant surface-associated proteins of S. oralis and to investigate changes in protein expression when the organism was grown under acidic culture conditions. Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Putative functions were assigned by homology to a translated genomic database of Streptococcus pneumoniae. A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase. The most abundant protein, phosphocarrier protein HPr, was present as three isoforms. Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis. Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from Lactococcus lactis, and the protein translation elongation factors G, Tu, and Ts. This study has extended the range of streptococcal proteins known to be expressed at the cell surface. Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions.

FIG. 1.

Surface-associated proteins of S. oralis separated by 2-D PAGE. Surface-associated proteins from pH 7.0- (A) and pH 5.2-grown (B) cultures were separated by isoelectric focusing in the pH range of 4 to 7 in the first dimension and by 12% SDS-PAGE in the second dimension. Resolved proteins were visualized following staining with CCBB, and the locations of molecular mass markers and pIs are shown. Spot numbering indicates those proteins identified by PMF and LC-MS/MS, and the numbers match the numbers in Table 1. *, proteins with altered expression at pH 5.2 compared with those extracted from cells cultured at pH 7.0; multiple arrows, proteins for which different isoforms were observed.

FIG. 2.

Cytoplasmic proteins were absent from the surface-associated protein preparation from pH 5.2-grown cultures separated by 2-D PAGE. Lactate dehydrogenase (LDH) and pyruvate oxidase (POX) were abundant in the whole-cell extract (A) but were absent on the 2-D gels of the surface-associated proteins (B). Fructose bisphosphate aldolase (FBA) was identified in both intracellular and surface-associated fractions.