Modification of heterotrimeric G-proteins in Swiss 3T3 cells stimulated with Pasteurella multocida toxin.

Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G(q), G(12) and G(i). PMT has been shown by others to lead to the deamidation of recombinant Gα(i) at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the G(q) and G(i) families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα(11), a member of the G(q) family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα(13) was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. G(s) was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gα(i), but not Gα(q). We also show that PMT inhibits the GTPase activity of G(q).

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

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 beta-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 beta-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 beta-galactosidase activity had been previously attributed solely to SPD_0562 (bgaA), we evaluated the contribution of SPD_0065 and SPD_0562 to total beta-galactosidase activity. Mutation of either gene significantly reduced enzymatic activity, but beta-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.

Pyruvate formate lyase is required for pneumococcal fermentative metabolism and virulence.

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.

An orthologue of Bacteroides fragilis NanH is the principal sialidase in Tannerella forsythia.

Sialidase activity is a putative virulence factor of the anaerobic periodontal pathogen Tannerella forsythia, but it is uncertain which genes encode this activity. Characterization of a putative sialidase, SiaHI, by others, indicated that this protein alone may not be responsible for all of the sialidase activity. We describe a second sialidase in T. forsythia (TF0035), an orthologue of Bacteroides fragilis NanH, and its expression in Escherichia coli. Sialidase activity of the expressed NanH was confirmed by using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid as a substrate. Biochemical characterization of the recombinant T. forsythia NanH indicated that it was active over a broad pH range, with optimum activity at pH 5.5. This enzyme has high affinity for 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (K(m) of 32.9 +/- 10.3 microM) and rapidly releases 4-methylumbelliferone (V(max) of 170.8 +/- 11.8 nmol of 4-methylumbelliferone min(-1) mg of protein(-1)). E. coli lysates containing recombinant T. forsythia NanH cleave sialic acid from a range of substrates, with a preference for alpha2-3 glycosidic linkages. The genes adjacent to nanH encode proteins apparently involved in the metabolism of sialic acid, indicating that the NanH sialidase is likely to be involved in nutrient acquisition.

Maturation of Streptococcus pneumoniae lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence.

Cell surface lipoproteins are important for the full virulence of several bacterial pathogens, including Streptococcus pneumoniae. Processing of prolipoproteins seems to be conserved among different bacterial species, and requires type II signal peptidase (Lsp) mediated cleavage of the N-terminal signal peptide to form the mature lipoprotein. Lsp has been suggested as a target for new antibiotic therapies, but at present there are only limited data on the function of Lsp for Gram-positive bacterial pathogens. We have investigated the function and role during disease pathogenesis of the S. pneumoniae Lsp, which, blast searches suggest, is encoded by the gene Sp0928. Expression of Sp0928 protected Escherichia coli against the Lsp antagonist globomycin, and proteomics and immunoblot analysis demonstrated that deletion of Sp0928 prevented processing of S. pneumoniae prolipoproteins to mature lipoproteins. These data strongly suggest that Sp0928 encodes the S. pneumoniae Lsp. However, immunoblots of membrane-associated proteins, immunoelectron microscopy and flow cytometry assays all confirmed that in the absence of Lsp, immature lipoproteins were still attached to the cell surface. Despite preservation of lipoprotein attachment to the cell membrane, loss of S. pneumoniae Lsp resulted in several phenotypes associated with impaired lipoprotein function and reduced S. pneumoniae replication in animal models of infection.

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.

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.

Effect of biofilm growth on expression of surface proteins of Actinomyces naeslundii genospecies 2.

The predominant surface proteins of biofilm and planktonic Actinomyces naeslundii, a primary colonizer of the tooth surface, were examined. Seventy-nine proteins (the products of 52 genes) were identified in biofilm cells, and 30 of these, including adhesins, chaperones, and stress-response proteins, were significantly up-regulated relative to planktonic cells.

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

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.

Analysis of Streptococcus mutans proteins modulated by culture under acidic conditions.

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.