Streptococcus sanguinis Noncoding cia-Dependent Small RNAs Negatively Regulate Expression of Type IV Pilus Retraction ATPase PilT and Biofilm Formation.

Small noncoding RNAs (sRNAs) have been identified as important regulators of gene expression in various cellular processes. cia-dependent small RNAs (csRNAs), a group of sRNAs that are controlled by the two-component regulatory system CiaRH, are widely conserved in streptococci, but their targets have been identified only in Streptococcus pneumoniaeStreptococcus sanguinis, a pioneer colonizer of teeth and one of the most predominant bacteria in the early oral biofilm, has been shown to have six csRNAs. Using computational target prediction and the luciferase reporter assay, we identified pilT, a constituent of the type IV pilus operon, as a negative regulatory target for one of the csRNAs, namely, csRNA1-1, in S. sanguinis RNA-RNA electrophoretic mobility shift assay using a nucleotide exchange mutant of csRNA1-1 revealed that csRNA1-1 binds directly to pilT mRNA. In addition, csRNA1-1 and csRNA1-2, a putative gene duplication product of csRNA1-1 that is tandemly located in the S. sanguinis genome, negatively regulated S. sanguinis biofilm formation. These results suggest the involvement of csRNAs in the colonization step of S. sanguinis.

Influence of excess branched-chain amino acid uptake by Streptococcus mutans in human host cells.

Oral streptococci, including cariogenic bacterium Streptococcus mutans, comprise a large percentage of human supragingival plaque, which contacts both tooth surfaces and gingiva. Eukaryotic cells are able to take up macromolecules and particles, including bacteria, by endocytosis. Increasing evidence indicates endocytosis may be used as an entry process by bacteria. We hypothesized that some endocytosed bacteria might survive and obtain nutrients, such as amino acids, until they are killed. To verify this hypothesis, we focused on bacterial utilization of branched-chain amino acids (BCAAs; isoleucine, leucine and valine) in host cells. A branched-chain aminotransferase, IlvE (EC 2.6.1.42), has been suggested to play an important role in internal synthesis of BCAAs in S. mutans UA159. Therefore, we constructed an ilvE-deficient S. mutans 109c strain and confirmed that it had similar growth behavior as reported previously. 14C radioactive leucine uptake assays showed that ilvE-deficient S. mutans took up more leucine both inside and outside of host cells. We further clarified that a relative decrease of BCAAs in host cells caused enhanced endocytic and autophagic activity. In conclusion, S. mutans is endocytosed by host cells and may survive and obtain nutrients, such as BCAAs, inside the cells, which might affect cellular functions of host cells.

Effects of Hangeshashinto on Growth of Oral Microorganisms.

Oral mucositis (OM) in cancer patients induced by chemotherapy or radiotherapy has a significant impact on quality of life, and causes considerable morbidity. Oral microorganisms are likely to intensify the inflammatory process and aggravate the formation of ulcers. Hangeshashinto (HST), a Japanese kampo medicine, has been reported to be effective when used as a gargle for the treatment of OM. To clarify the effects of HST on oral microorganisms, we assessed its antimicrobial activity against 27 microbial species, including 19 oral bacteria and one fungus. HST extract inhibited the growth of Gram-negative bacteria, including Fusobacterium nucleatum, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, Prevotella melaninogenica, Tannerella forsythia, Treponema denticola, and Porphyromonas asaccharolytica, though inhibitory effects were less pronounced for Gram-positive bacteria and the fungal strain. We then investigated the effects of antibacterial activities on 15 purified ingredients of HST and determined that baicalein, berberine, coptisine, [6]-shogaol, and homogentisic acid actively inhibited the growth of these bacteria. These findings showed that HST inhibits the growth of specific Gram-negative periodontopathogenic bacteria, which are significant pathogens in OM, without disturbing the normal oral flora. Our data suggest that HST may be a useful treatment for OM in patients undergoing anticancer treatment.

Lipoproteins of Actinomyces viscosus induce inflammatory responses through TLR2 in human gingival epithelial cells and macrophages.

Actinomyces viscosus has been suggested to be associated with periodontal disease. However, the pathogenicity of this bacterium is not known. In this study, we examined inflammation-inducing activity by A. viscosus. Whole cells and a lipophilic fraction of A. viscosus ATCC19246 induced production of interleukin-8 and tumor necrosis factor alpha from both human oral epithelial cells and human monocytoid cells. This cytokine production was blocked by lipoprotein lipase treatment of the lipophilic fraction. In addition, anti-Toll-like receptor 2 antibody blocked the cytokine production. These results suggest that lipoprotein of A. viscosus triggers inflammatory responses in periodontitis by activation of Toll-like receptor 2.

Biological activities of Bacteroides forsythus lipoproteins and their possible pathological roles in periodontal disease.

Bacteroides forsythus is a gram-negative, anaerobic, fusiform bacterium and is considered to be an etiological agent in periodontal disease. A lipoprotein fraction prepared from B. forsythus cells by Triton X-114 phase separation (BfLP) activated human gingival fibroblasts and a human monocytic cell line, THP-1, to induce interleukin-6 production and tumor necrosis factor alpha production. BfLP was found to be capable of inducing nuclear factor-kappaB translocation in human gingival fibroblasts and THP-1 cells. By using Chinese hamster ovary K1 cells transfected with Toll-like receptor genes together with a nuclear factor-kappaB-dependent CD25 reporter plasmid, it was found that signaling by BfLP was mediated by Toll-like receptor 2 but not by CD14 or Toll-like receptor 4. BfLP induced apoptotic cell death in human gingival fibroblasts, KB cells (an oral epithelial cell line), HL-60 cells (a human myeloid leukemia cell line), and THP-1 cells but not in MOLT4 cells (a T-cell leukemia cell line). Caspase-8, an initiator caspase in apoptosis, was found to be activated in these cells in response to BfLP stimulation. Thus, this study suggested that BfLP plays some etiological roles in oral infections, especially periodontal disease, by induction of cell activation or apoptosis.