Altered protein expression of Streptococcus oralis cultured at low pH revealed by two-dimensional gel electrophoresis

Abstract

Streptococcus oralis is the predominant aciduric nonmutans streptococcus isolated from the human dentition, but the role of this organism in the initiation and progression of dental caries has yet to be established. To identify proteins that are differentially expressed by S. oralis growing under conditions of low pH, soluble cellular proteins extracted from bacteria grown in batch culture at pH 5.2 or 7.0 were analyzed by two-dimensional (2-D) gel electrophoresis. Thirty-nine proteins had altered expression at low pH; these were excised, digested with trypsin using an in-gel protocol, and further analyzed by peptide mass fingerprinting using matrix-assisted laser desorption ionization mass spectrometry. The resulting fingerprints were compared with the genomic database for Streptococcus pneumoniae, an organism that is phylogenetically closely related to S. oralis, and putative functions for the majority of these proteins were determined on the basis of functional homology. Twenty-eight proteins were up-regulated following growth at pH 5.2; these included enzymes of the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase), the polypeptide chains comprising ATP synthase, and proteins that are considered to play a role in the general stress response of bacteria, including the 60-kDa chaperone, Hsp33, and superoxide dismutase, and three distinct ABC transporters. These data identify, for the first time, gene products that may be important in the survival and proliferation of nonmutans aciduric S. oralis under conditions of low pH that are likely to be encountered by this organism in vivo.

FIG. 1

Growth of S. oralis strain 176N at pH 5.2 and 7.0. S. oralis was cultured in nutrient-rich media buffered to pH 5.2 (■) or 7.0 (⧫). Growth was monitored by determination of A₆₂₀, and error bars indicate standard deviation of the mean of experiments carried out in triplicate.

FIG. 2

2-D PAGE analysis of cellular proteins of S. oralis cultured at pH 5.2. Extracted proteins were separated by isoelectric focusing in the pI range of 4 to 7 in the first dimension and gradient (12 to 14%) SDS-PAGE in the second dimension. Resolved proteins were visualized following staining with colloidal Coomassie brilliant blue. Spot numbering indicates those proteins with altered expression at pH 5.2 compared with those extracted from cells cultured at pH 7.0. Proteins for which different isoforms were observed are indicated by multiple arrows.

FIG. 3

Representative S. oralis proteins up-regulated as a response to growth at low pH. S. oralis was cultured at pH 5.2 or 7.0, and cellular proteins were resolved by 2-D PAGE. Proteins were identified by peptide mass fingerprinting and comparison with the S. pneumoniae genomic database. (A) ABC transporter; (B) ATP synthase alpha chain; (C) ATP synthase beta chain.

FIG. 4

Isoforms of representative S. oralis proteins. Cellular proteins derived from cells cultured at pH 5.2 or pH 7.0 were resolved by 2-D PAGE. (A) Glyceraldehyde-3-phosphate dehydrogenase; (B) pyruvate oxidase; (C) 60-kDa chaperonin. Individual isoforms were identified by peptide mass fingerprinting and comparison with the S. pneumoniae genomic database.