Tyrosine tRNA synthetase as a novel extracellular immunomodulatory protein in Streptococcus anginosus.

Streptococcus anginosus is frequently detected in patients with infective endocarditis, abscesses or oral cancer. Although S. anginosus is considered the causative pathogen of these diseases, the pathogenic mechanisms of the bacterium have remained unclear. Previously, we suggested that an extracellular antigen from S. anginosus (SAA) serves as a pathogenic factor by inducing nitric oxide production in murine macrophages. In the present study, we identified SAA using LC-MS/MS and assessed the biological activities of His-tagged recombinant SAA in murine macrophages. SAA was identified as a tyrosine tRNA synthetase (SaTyrRS) that was isolated from the extracellular fraction of S. anginosus but not from other oral streptococci. In addition, inducible nitric oxide synthase and TNF-α mRNA expression was induced in recombinant SaTyrRS-stimulated murine macrophages. However, their mRNA expression was not induced in macrophages stimulated with truncated or heat-inactivated recombinant SaTyrRS, and the activation motif was identified as Arg264-Thr270. Consequently, these results indicated that SaTyrRS could be a novel and specific immunomodulatory protein in S. anginosus.

Aciduricity and acid tolerance mechanisms of Streptococcus anginosus.

Although Streptococcus anginosus constitutes a proportion of the normal flora of the gastrointestinal and genital tracts, and the oral cavity, it has been reported that S. anginosus infection could be closely associated with abscesses at various body sites, infective endocarditis, and upper gastrointestinal cancers. The colonization in an acidic environment due to the aciduricity of S. anginosus could be the etiology of the systemic infection of the bacteria. To elucidate the aciduricity and acid tolerance mechanisms of the microbe, we examined the viability and growth of S. anginosus under acidic conditions. The viabilities of S. anginosus NCTC 10713 and Streptococcus mutans ATCC 25175 at pH 4.0 showed as being markedly higher than those of Streptococcus sanguinis ATCC 10556, Streptococcus gordonii ATCC 10558, and Streptococcus mitis ATCC 49456; however, the viability was partially inhibited by dicyclohexylcarbodiimide, an H+-ATPase inhibitor, suggesting that H+-ATPase could play a role in the viability of S. anginosus under acidic conditions. In addition, S. anginosus NCTC 10713 could grow at pH 5.0 and showed a marked arginine deiminase (ADI) activity, unlike its ΔarcA mutant, deficient in the gene encoding ADI, and other streptococcal species, which indicated that ADI could also be associated with aciduricity. These results suggest that S. anginosus has significant aciduric properties, which can be attributed to these enzyme activities.

Establishment of a convenient sandwich-ELISA for direct quantification of glucosyltransferase-I: application for dual diagnosis of dental caries.

Previously, we established a convenient enzyme-linked immunosorbent assay (ELISA) system targeting glucosyltransferase (GTF)-B derived from Streptococcus mutans for diagnosing caries risk. However, it has been reported that S. sobrinus possesses high cariogenicity and is more frequently detected in highly caries-susceptible patients than S. mutans is. S. sobrinus can secrete GTF-I, an important cariogenic factor for dental plaque formation, as well as S. mutans GTF-B. Therefore, in this study, we developed another feasible ELISA system targeting S. sobrinus GTF-I that would ensure caries risk determination by combined GTF-I and GTF-B levels. A readily measurable sandwich-ELISA system was devised, which consisted of monoclonal and polyclonal antibodies against GTF-I. The developed sandwich-ELISA system quantified the purified GTF-I with sensitivity and specificity, and a positive correlation was observed between the amount of GTF-I extracted from clinical plaque samples and S. sobrinus levels. Furthermore, high levels of GTF-I and GTF-B were detected using the sandwich-ELISA system in caries-susceptible subjects. These results indicate that the sandwich-ELISA system against GTF-I developed in this study is useful, and that the dual detection of the caries risk factors GTF-I and GTF-B is helpful for predicting caries risk.

Structural insights into catalysis by ßC-S lyase from Streptococcus anginosus.

Hydrogen sulfide (H(2)S) is a causative agent of oral malodor and may play an important role in the pathogenicity of oral bacteria such as Streptococcus anginosus. In this microorganism, H(2)S production is associated with ßC-S lyase (Lcd) encoded by lcd gene, which is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the α,ß-elimination of sulfur-containing amino acids. When Lcd acts on L-cysteine, H(2)S is produced along with pyruvate and ammonia. To understand the H(2)S-producing mechanism of Lcd in detail, we determined the crystal structures of substrate-free Lcd (internal aldimine form) and two reaction intermediate complexes (external aldimine and α-aminoacrylate forms). The formation of intermediates induced little changes in the overall structure of the enzyme and in the active site residues, with the exception of Lys234, a PLP-binding residue. Structural and mutational analyses highlighted the importance of the active site residues Tyr60, Tyr119, and Arg365. In particular, Tyr119 forms a hydrogen bond with the side chain oxygen atom of L-serine, a substrate analog, in the external aldimine form suggesting its role in the recognition of the sulfur atom of the true substrate (L-cysteine). Tyr119 also plays a role in fixing the PLP cofactor at the proper position during catalysis through binding with its side chain. Finally, we partly modified the catalytic mechanism known for cystalysin, a ßC-S lyase from Treponema denticola, and proposed an improved mechanism, which seems to be common to the ßC-S lyases from oral bacteria.

Streptococcus anginosus l-cysteine desulfhydrase gene expression is associated with abscess formation in BALB/c mice.

Streptococcus anginosus, an anginosus group bacterium, is frequently isolated from odontogenic abscesses, and is the oral bacterium that is primarily responsible for producing hydrogen sulfide from l-cysteine through the action of its l-cysteine desulfhydrase (ßC-S lyase) enzyme. However, the relationship between its production of hydrogen sulfide and abscess formation has not been investigated. To elucidate the etiological role of hydrogen sulfide in abscess formation, we initially measured, using specific primers, expression of the lcd gene, which encodes ßC-S lyase, in the pus of abscesses that formed in BALB/c mice following subcutaneous injection of S. anginosus into the dorsa. Expression of lcd was >15-fold higher when l-cysteine was present than when it was absent. A mouse virulence assay revealed that the mean diameter of abscesses caused by S. anginosus FW73 plus l-cysteine was greater than that of abscesses caused by S. anginosus FW73 in the absence of l-cysteine. These findings demonstrate that the lcd gene of S. anginosus is upregulated in mouse abscesses and that hydrogen sulfide, the product of a reaction catalyzed by ßC-S lyase, plays an etiological role in odontogenic abscess formation.

Crystallization and preliminary X-ray analysis of betaC-S lyases from two oral Streptococci.

Hydrogen sulfide, which causes oral malodour, is generally produced from L-cysteine by the action of betaC-S lyase from oral bacteria. The betaC-S lyases from two oral bacteria, Streptococcus anginosus and S. gordonii, have been cloned, overproduced, purified and crystallized. X-ray diffraction data were collected from the two types of crystals using synchrotron radiation. The crystal of S. anginosus betaC-S lyase belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.0, b = 111.1, c = 216.4 A, and the crystal of S. gordonii betaC-S lyase belonged to the same space group, with unit-cell parameters a = 58.0, b = 73.9. c = 187.6 A. The structures of the betaC-S lyases were solved by molecular-replacement techniques.

Molecular and enzymatic characterization of betaC-S lyase in Streptococcus constellatus.

BACKGROUND/AIMS: Streptococcus anginosus and Streptococcus constellatus are frequently isolated from dental abscesses and other suppurative lesions. We previously reported that betaC-S lyase from a strain of S. anginosus produced significantly more hydrogen sulfide than betaC-S lyases from other streptococci. The purpose of this study was to establish the molecular and enzymatic features of the betaC-S lyase in S. constellatus and to elucidate whether this unique capacity is common to many strains of S. constellatus and S. anginosus. METHODS: The capacity of crude extract to produce hydrogen sulfide was evaluated among 16 strains of S. constellatus, S. anginosus, and Streptococcus gordonii. The lcd gene encoding betaC-S lyase was cloned from the genomic DNA of each strain to compare the deduced amino acid sequences. The recombinant betaC-S lyases of three representative strains were purified and characterized. RESULTS: Incubation of crude extracts from all strains of S. constellatus and S. anginosus with l-cysteine resulted in the production of a large amount of hydrogen sulfide. The primary sequence of betaC-S lyase was very similar among strains of S. constellatus and S. anginosus. The kinetic properties of the betaC-S lyases purified from S. constellatus resembled those for betaC-S lyases purified from S. anginosus. In contrast, the betaC-S lyases of S. constellatus and S. gordonii differed in terms of their hydrogen sulfide production, with the former producing much more. CONCLUSION: A high level of hydrogen sulfide production, which appears to be a common feature in both S. constellatus and S. anginosus, may be associated with their abscess formation.

Homogeneous enzymatic assay for L-cysteine with betaC-S lyase.

We have developed a new enzymatic assay for determining L-cysteine concentration. The method involves the use of betaC-S lyase from Streptococcus anginosus, which catalyzes the alpha,beta-elimination of L-cysteine to hydrogen sulfide, pyruvate, and ammonia. The production of pyruvate is measured by D-lactate dehydrogenase and NADH. The decrease in NADH was proportional to the L-cysteine concentration up to 1.0 mM. When serum samples were used, within-day and day-to-day coefficient variations were below 4%. This method is simple, and can easily and reliably be used for accurate determination of L-cysteine concentration in serum or other samples.

Dipeptidyl peptidase IV of Streptococcus anginosus: purification and characterization.

We found that N-unblocked nine p-nitroanilde derivatives of amino acids or peptides were hydrolyzed by the crude cell extracts of Streptococcus anginosus NCTC 10713. Then dipeptidyl peptidase IV was purified 323-fold by the procedures including ammonium sulfate concentration, anion exchange chromatography (twice), gel filtration (twice), hydrophobic interaction chromatography, and isoelectric focusing. The molecular weight was calculated as 84 kDa, and the isoelectric point was 4.9. The enzyme hydrolyzed mainly dipeptides containing proline residues at P1 position. It was strongly inhibited by serine enzyme inhibitors. General protease inhibitors, metal chelators, thiol alkylating agent, reducing agent, and several metal ions had no effect on the enzyme activity. Optimum pH for the activity was found at 7.0. The enzyme was mostly inactivated by heating at 50 degrees C for 15 min.

Homocysteine biosynthesis pathways of Streptococcus anginosus.

A gene (cgs) encoding cystathionine gamma-synthase was cloned from Streptococcus anginosus, and its protein was purified and characterized. The cgs gene and the immediately downstream lcd gene were shown to be cotranscribed as an operon. High-performance liquid chromatography analyses showed that the S. anginosus Cgs not only has cystathionine gamma-synthase activity, but also expresses O-acetylhomoserine sulfhydrylase activity. These results suggest that S. anginosus has the capacity to utilize both the transsulfuration and direct sulfhydrylation pathways for homocysteine biosynthesis.

Differences in the betaC-S lyase activities of viridans group streptococci.

betaC-S Lyase catalyzes the alpha,beta-elimination of L-cysteine to hydrogen sulfide, which is one of the main causes of oral malodor and is highly toxic to mammalian cells. We evaluated the capacity of six species of oral streptococci to produce hydrogen sulfide. The crude enzyme extract from Streptococcus anginosus had the greatest capacity. However, comparative analysis of amino acid sequences did not detect any meaningful differences in the S. anginosus betaC-S lyase. The capacity of S. anginosus purified betaC-S lyase to degrade L-cysteine was also extremely high, while its capacity to degrade L-cystathionine was unremarkable. These findings suggest that the extremely high capacity of S. anginosus to produce hydrogen sulfide is due to the unique characteristic of betaC-S lyase from that organism.