Karen A. Homer

Analysis of Streptococcus mutans Proteins Modulated by Culture under Acidic Conditions

ABSTRACT Streptococcus mutans, a major etiological agent of dental caries, causes demineralization of the tooth tissue due to the formation of acids from dietary carbohydrates. Dominant among the virulence determinants of this organism are aciduricity and acidogenicity, the abilities to grow at low pH and to produce acid, respectively. The mechanisms underlying the ability of S. mutans to survive and proliferate at low pH are currently under investigation. In this study we cultured S. mutans at pH 5.2 or 7.0 and extracted soluble cellular proteins. These were analyzed using high-resolution two-dimensional gel electrophoresis, and replicate maps of proteins expressed under each of the two conditions were generated. Proteins with modulated expression at low pH, as judged by a change in the relative integrated optical density, were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption ionization mass spectrometry to generate peptide mass fingerprints, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Thirty individual proteins exhibited altered expression as a result of culture of S. mutans at low pH. Up-regulated proteins (n = 18) included neutral endopeptidase, phosphoglucomutase, 60-kDa chaperonin, cell division proteins, enolase, lactate dehydrogenase, fructose bisphosphate aldolase, acetoin reductase, superoxide dismutase, and lactoylglutathione lyase. Proteins down-regulated at pH 5.2 (n = 12) included protein translation elongation factors G, Tu, and Ts, DnaK, small-subunit ribosomal protein S1P, large-subunit ribosomal protein L12P, and components of both phosphoenolpyruvate:protein phosphotransferase and multiple sugar binding transport systems. The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.

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.

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 forStreptococcus 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.

Two Closely Related ABC Transporters in Streptococcus mutans Are Involved in Disaccharide and/or Oligosaccharide Uptake

Streptococcus mutans has a large number of transporters apparently involved in the uptake of carbohydrates. At least two of these, the multiple sugar metabolism transporter, MsmEFGK, and the previously uncharacterized MalXFGK, are members of the ATP-binding cassette (ABC) superfamily. Mutation analysis revealed that the MsmEFGK and MalXFGK transporters are principally involved in the uptake of distinct disaccharides and/or oligosaccharides. Furthermore, the data also indicated an unusual protein interaction between the components of these two related transporters. Strains lacking msmE (which encodes a solute binding protein) can no longer utilize raffinose or stachyose but grow normally on maltodextrins in the absence of MalT, a previously characterized EII(mal) phosphotransferase system component. In contrast, a mutant of malX (which encodes a solute binding protein) cannot utilize maltodextrins but grows normally on raffinose or stachyose. Radioactive uptake assays confirmed that MalX, but not MsmE, is required for uptake of [U-14C]maltotriose and that MalXFGK is principally involved in the uptake of maltodextrins with as many as 7 glucose units. Surprisingly, inactivation of the corresponding ATPase components did not result in an equivalent abolition of growth: the malK mutant can grow on maltotetraose as a sole carbon source, and the msmK mutant can utilize raffinose. We propose that the ATPase domains of these ABC transporters can interact with either their own or the alternative transporter complex. Such unexpected interaction of ATPase subunits with distinct membrane components to form complete multiple ABC transporters may be widespread in bacteria.

Production of specific glycosidase activities by Streptococcus intermedius strain UNS35 grown in the presence of mucin

An isolate of Streptococcus intermedius from a brain abscess showed neuraminidase (sialidase), beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase and N-acetyl-beta-D-galactosaminidase activities. The optimal pH values of these enzymes were 5.5-6.0, 5.5-6.0, 5.0-5.5 and 5.0-5.5, respectively. The km of the enzymes varied according to whether the type of substrate was chromogenic or fluorogenic; sialidase was most active at the lowest substrate concentrations, with a km of 0.01 mM. In semi-defined medium, with porcine gastric mucin--a model glycoprotein--as the sole source of fermentable carbohydrate, levels of the glycosidases were significantly increased. Addition of glucose to the mucin-containing medium, or growth of cells in media supplemented with glucose alone, repressed glycosidic activities and the majority of these were cell-associated. S. intermedius cells from cultures grown with mucin were able, simultaneously, to transport via sugar:phosphoenolpyruvate phosphotransferase (PTS) systems, monosaccharides which are constituents of carbohydrate side chains of glycoproteins. These cells also possessed significant levels of neuraminate-pyruvate lyase, involved in the intracellular catabolism of neuraminic acid; this was absent from cells grown with glucose. These mechanisms, collectively, may facilitate the persistence and growth of S. intermedius in vivo.

Pyruvate Formate Lyase Is Required for Pneumococcal Fermentative Metabolism and Virulence

ABSTRACT Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and survival. Because the nutrient-limited, hypoxic host tissues favor mixed-acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed-acid fermentation, which is activated posttranslationally by PFL-activating enzyme (PFL-AE). Mutations were introduced to two putative pfl genes, SPD0235 and SPD0420, and two putative pflA genes, SPD0229 and SPD1774. End-product analysis showed that there was no formate, the main end product of the reaction catalyzed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 codes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in the upregulation of fba and pyk, encoding, respectively, fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wild type-infected group. Furthermore, the numbers of CFU of the SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer numbers of mutant CFU than of wild-type CFU were recovered from blood specimens after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.

Characterization of novel beta-galactosidase activity that contributes to glycoprotein degradation and virulence in Streptococcus pneumoniae.

ABSTRACT The pneumococcus obtains its energy from the metabolism of host glycosides. Therefore, efficient degradation of host glycoproteins is integral to pneumococcal virulence. In search of novel pneumococcal glycosidases, we characterized the Streptococcus pneumoniae strain D39 protein encoded by SPD_0065 and found that this gene encodes a β-galactosidase. The SPD_0065 recombinant protein released galactose from desialylated fetuin, which was used here as a model of glycoproteins found in vivo. A pneumococcal mutant with a mutation in SPD_0065 showed diminished β-galactosidase activity, exhibited an extended lag period in mucin-containing defined medium, and cleaved significantly less galactose than the parental strain during growth on mucin. As pneumococcal β-galactosidase activity had been previously attributed solely to SPD_0562 (bgaA), we evaluated the contribution of SPD_0065 and SPD_0562 to total β-galactosidase activity. Mutation of either gene significantly reduced enzymatic activity, but β-galactosidase activity in the double mutant, although significantly less than that in either of the single mutants, was not completely abolished. The expression of SPD_0065 in S. pneumoniae grown in mucin-containing medium or tissues harvested from infected animals was significantly upregulated compared to that in pneumococci from glucose-containing medium. The SPD_0065 mutant strain was found to be attenuated in virulence in a manner specific to the host tissue.

Isolation and characterisation of sialidase from a strain of Streptococcus oralis

Streptococcus oralis, the most virulent of the viridans streptococci, produces a sialidase and this exo-glycosidase has been implicated in the disease process of a number of pathogens. The sialidase of S. oralis strain AR3 was purified in order to understand the characteristics of this putative virulence determinant. The enzyme isolated as a high mol. wt aggregate (c. 325 kDa) was purified 4520-fold from late exponential phase cultures by a combination of ultrafiltration, ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. The sialidase component had a mol.wt of 144 kDa as determined by SDS-PAGE analysis. The purified sialidase released N-acetylneuraminic acid from a range of sialoglycoconjugates including human alpha1-acid glycoprotein, bovine submaxillary mucin, colominic acid and sialyl-alpha2,3- and sialyl-alpha2,6-lactose. Also, N-glycolylneuraminic acid was cleaved from bovine submaxillary mucin. The sialidase had a Km of 11.8 microM for alpha1-acid glycoprotein, was active over a broad pH range with a pH optimum of 6.0 and cleaved alpha2,3-, alpha2,6- and alpha2-8-sialyl glycosidic linkages with a marked preference for alpha2,3-linkages. The enzyme was competitively inhibited by the sialic acid derivative, 2,3-dehydro-N-acetylneuraminic acid, with a K(IC) of 1.2 microM. The characteristics of the purified sialidase would support a nutritional role for this enzyme that may be significant in the proliferation of this organism in the oral cavity and at extra-oral sites in association with life-threatening infections.

Fluorometric determination of bacterial protease activity using fluorescein isothiocyanate-labeled proteins as substrates☆

Intact fluorescein isothiocyanate-labeled proteins have relatively low background fluorescence at excitation and emission wavelengths of 495 and 525 nm, respectively. Degradation of these substrates leads to exposure of covalently linked fluorescein isothiocyanate molecules and to a concomitant increase in relative fluorescence at these wavelengths. The increase in relative fluorescence is proportional to the degree of protein degradation. This phenomenon provides the basis for a sensitive assay for bacterial protease activity. There is no requirement for the removal of undegraded substrate from the assay mixture prior to the measurement of fluorescence. Assays can be performed in 96-well microtiter trays, enabling a large number of samples and their respective controls to be processed simultaneously and repeated determinations of fluorescence values may be made on the same assay.

A Phosphoenolpyruvate-Dependent Phosphotransferase System Is the Principal Maltose Transporter in Streptococcus mutans

We report that a phosphoenolpyruvate-dependent phosphotransferase system, MalT, is the principal maltose transporter for Streptococcus mutans. MalT also contributes to maltotriose uptake. Since maltose and maltodextrins are products of starch degradation found in saliva, the ability to take up and ferment these carbohydrates may contribute to dental caries.