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We identified SA1684 as a Staphylococcus aureus virulence gene using a silkworm infection model. The SA1684 gene product carried the DUF402 domain, which is found in RNA-binding proteins, and had amino acid sequence similarity with a nucleoside diphosphatase, Streptomyces coelicolor SC4828 protein. The SA1684-deletion mutant exhibited drastically decreased virulence, in which the LD50 against silkworms was more than 10 times that of the parent strain. The SA1684-deletion mutant also exhibited decreased exotoxin production and colony-spreading ability. Purified SA1684 protein had Mn(2+)- or Co(2+)-dependent hydrolyzing activity against nucleoside diphosphates. Alanine substitutions of Tyr-88, Asp-106, and Asp-123/Glu-124, which are conserved between SA1684 and SC4828, diminished the nucleoside diphosphatase activity. Introduction of the wild-type SA1684 gene restored the hemolysin production of the SA1684-deletion mutant, whereas none of the alanine-substituted SA1684 mutant genes restored the hemolysin production. RNA sequence analysis revealed that SA1684 is required for the expression of the virulence regulatory genes agr, sarZ, and sarX, as well as metabolic genes involved in glycolysis and fermentation pathways. These findings suggest that the novel nucleoside diphosphatase SA1684 links metabolic pathways and virulence gene expression and plays an important role in S. aureus virulence., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

We previously reported that the rRNA methyltransferases RsmI and RsmH, which are responsible for cytidine dimethylation at position 1402 of 16S rRNA in the decoding center of the ribosome, contribute to Staphylococcus aureus virulence. Here we evaluated other 16S rRNA methyltransferases, including KsgA (RsmA), RsmB/F, RsmC, RsmD, RsmE, and RsmG. Knockout of KsgA, which methylates two adjacent adenosines at positions 1518 and 1519 of 16S rRNA in the intersubunit bridge of the ribosome, attenuated the S. aureus killing ability against silkworms. The ksgA knockout strain was sensitive to oxidative stress and had a lower survival rate in murine macrophages than the parent strain. The ksgA knockout strain exhibited decreased translational fidelity in oxidative stress conditions. Administration of N-acetyl-l-cysteine, a free-radical scavenger, restored the killing ability of the ksgA knockout strain against silkworms. These findings suggest that the methyl-modifications of 16S rRNA by KsgA contribute to maintain ribosome function under oxidative conditions and thus to S. aureus virulence., (Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Introduction: Streptococcus sobrinus exhibits marked dextran-dependent aggregation mediated by glucan-binding proteins (GBPs). In contrast to Streptococcus mutans, in which the gbpC gene responsible for dextran-dependent aggregation of this organism has been characterized, genes encoding the S. sobrinus GBPs have not yet been identified., Methods: Recently, we identified the gbpC gene homologue from Streptococcus macacae using polymerase chain reaction primers based on the conserved regions of the gbpC sequence exhibiting intraspecies variations. This method was applied to amplify a S. sobrinus homologue., Results: Unexpectedly, two gbpC gene homologues were identified in S. sobrinus strain 100-4. One homologue, named gbpC, was more similar to the S. mutans gbpC gene than the other and was approximately half the molecular size of its homologue with similar regions interrupted by several non-similar stretches. However, the dextran-binding activity of the protein expressed from gbpC in Escherichia coli was not detected in contrast to the other homologue, a protein designated as Dbl, expressing this activity. The gbpC gene was shown to be intact on the chromosome of strain OMZ176, which does not exhibit dextran-dependent aggregation, while the dbl gene of this strain contained a single adenine nucleotide insertion at approximately one-third the distance from the 5'-end. The insertion mutation in the dbl gene resulted in translation of a premature protein missing its LPXTG sequence signature sequence of the wall-anchored proteins., Conclusion: These results suggest that the dbl gene is very likely responsible for S. sobrinus dextran-dependent aggregation.

Background/aims: The past few decades have seen the isolation of certain glucosyltransferases and a number of proteins from mutans streptococci. Some of these proteins have been shown to possess glucan-binding capabilities which confer an important virulence property on mutans streptococci for the role of these bacteria play in dental caries. Among these proteins is glucan-binding protein C, which is encoded by the gbpC gene, and which we have identified as being involved in the dextran-dependent aggregation of Streptococcus mutans. However, gbpC homologues have yet to be identified in other mutans streptococci., Methods: We carried out polymerase chain reaction amplification of Streptococcus macacae using primers that were designed based on conserved sequences of S. mutans gbpC and identified a gbpC gene homologue. The gene of that homologue was then characterized., Results: Nucleotide sequencing of the S. macacae gbpC homologue revealed a 1854 bp open reading frame encoding a protein with an N-terminal signal peptide. The molecular mass of the processed protein was calculated to be 67 kDa. We also found an LPxTG motif, the consensus sequence for gram-positive cocci cell wall-anchored surface proteins, which was followed by a characteristic sequence at the carboxal terminal region of the putative protein. This suggests that the S. macacae GbpC homologue protein was tethered to the cell wall., Conclusion: Based on these results, together with the demonstrated glucan-binding ability of the S. macacae GbpC homologue protein, we suggest that S. macacae cells are capable of binding dextran via the GbpC homologue protein, which is similar to the S. mutans GbpC protein. In addition, Southern hybridization analysis using the S. macacae gbpC homologue as a probe showed a distribution of gbpC homologues throughout the mutans streptococci.

The Streptococcus mutans gbpC gene encoding cell wall-anchoring glucan-binding protein C is involved in the dextran(alpha-1,6 glucan)-dependent aggregation (ddag) of this organism. Unlike cells of other strains of S. mutans, strain GS-5 cells did not exhibit dextran(alpha-1,6 glucan)-dependent aggregation under any conditions. We therefore hypothesized that the gbpC gene may be mutated in strain GS-5. Sequencing analysis of the 1752-nucleotide GS-5 gbpC gene revealed a point mutation that switched codon 65 to a TAA termination codon. Strain GS-5 was previously reported also to have a mutation in the pac gene encoding the cell wall-anchored major protein antigen. The laboratory-maintained strain GS-5 is regarded as having lower cariogenicity than the original isolate. The decreased cariogenicity developed during the laboratory culture of strain GS-5 may have been caused by mutations in an environment lacking appropriate selective pressures.

Streptococcus mutans GbpC is a wall-anchored surface protein which is involved in dextran-dependent aggregation. The GbpC phenotype is observed only in cells grown under stress conditions. In order to detect the GbpC protein of S. mutans, we isolated the wall fraction following digestion of the cell wall of this organism by N-acetylmuramidase, and detected the GbpC protein from S. mutans cells by western analysis with anti-GbpC serum. Interestingly, S. mutans cells exhibiting the negative dextran(alpha-1,6 glucan)-dependent aggregation (ddag) phenotype expressed the protein and could bind to immobilized dextran.

Xylitol possesses a unique property distinct from the other caries-preventive sweeteners. This sugar alcohol cannot be metabolized to acids but is taken up by Streptococcus mutans and accumulated as a toxic sugar-phosphate in the cells, resulting in growth inhibition. Due to the accumulation, xylitol induces biological responses including the emergence of xylitol-insensitive populations. Therefore, we expected another response induced by xylitol and found a new phenomenon, that cells repeatedly cultured in the presence of xylitol evolved into those exhibiting an elevated dextran-dependent aggregation phenotype. This phenotype was found to result from expression of the gbpC gene, which was previously reported to be expressed only under certain stress conditions. Construction of a Strep. mutans isogenic mutant carrying the gbpC::lacZ fusion gene indicated that gbpC expression of cells repeatedly cultured in the presence of xylitol was elevated 20-fold. DNA transfer experiments indicated that this phenotypic change did not appear to be due to a mutation. These cells also exhibited decreased adhesion to glass surfaces when grown in the presence of sucrose. This may be one of the ways by which some populations of Strep. mutans are removed from dental plaques.

A previously unidentified 120-kDa protein was detected in Streptococcus mutans strain Z1 and was involved in the cold-agglutination of the strain. We have identified the gene, designated cnm, as being involved in the agglutination of strain Z1 following random mutagenesis. The amino acid sequence of the deduced Cnm protein exhibited high similarity to those of collagen-binding adhesins from staphylococci and other organisms. To confirm whether the protein is involved in collagen-binding, we cloned a cnm gene fragment, overexpressed it in E.coli, and prepared crude extracts. The extracts containing recombinant protein exhibited binding to immobilized collagen and laminin but not to fibronectin. Compared with the parental strain Z1, the cold-agglutination-negative mutant 05A02 exhibited reduced binding to collagen and laminin but retained that to fibronectin. This gene was detected in some strains of S. mutans. Therefore, the cnm gene encoded a new strain-specific member of the collagen-binding adhesin family. [ABSTRACT FROM AUTHOR]