Atsuo Sakurai

Comparative genomic analyses of Streptococcus mutans provide insights into chromosomal shuffling and species-specific content

BackgroundStreptococcus mutans is the major pathogen of dental caries, and it occasionally causes infective endocarditis. While the pathogenicity of this species is distinct from other human pathogenic streptococci, the species-specific evolution of the genus Streptococcus and its genomic diversity are poorly understood.ResultsWe have sequenced the complete genome of S. mutans serotype c strain NN2025, and compared it with the genome of UA159. The NN2025 genome is composed of 2,013,587 bp, and the two strains show highly conserved core-genome. However, comparison of the two S. mutans strains showed a large genomic inversion across the replication axis producing an X-shaped symmetrical DNA dot plot. This phenomenon was also observed between other streptococcal species, indicating that streptococcal genetic rearrangements across the replication axis play an important role in Streptococcus genetic shuffling. We further confirmed the genomic diversity among 95 clinical isolates using long-PCR analysis. Genomic diversity in S. mutans appears to occur frequently between insertion sequence (IS) elements and transposons, and these diversity regions consist of restriction/modification systems, antimicrobial peptide synthesis systems, and transporters. S. mutans may preferentially reject the phage infection by clustered regularly interspaced short palindromic repeats (CRISPRs). In particular, the CRISPR-2 region, which is highly divergent between strains, in NN2025 has long repeated spacer sequences corresponding to the streptococcal phage genome.ConclusionThese observations suggest that S. mutans strains evolve through chromosomal shuffling and that phage infection is not needed for gene acquisition. In contrast, S. pyogenes tolerates phage infection for acquisition of virulence determinants for niche adaptation.

Specific Behavior of Intracellular Streptococcus pyogenes That Has Undergone Autophagic Degradation Is Associated with Bacterial Streptolysin O and Host Small G Proteins Rab5 and Rab7

Streptococcus pyogenes (group A streptococcus (GAS)) is a pathogen that invades non-phagocytic host cells, and causes a variety of acute infections such as pharyngitis. Our group previously reported that intracellular GAS is effectively degraded by the host-cell autophagic machinery, and that a cholesterol-dependent cytolysin, streptolysin O (SLO), is associated with bacterial escape from endosomes in epithelial cells. However, the details of both the intracellular behavior of GAS and the process leading to its autophagic degradation remain unknown. In this study, we found that two host small G proteins, Rab5 and Rab7, were associated with the pathway of autophagosome formation and the fate of intracellular GAS. Rab5 was involved in bacterial invasion and endosome fusion. Rab7 was clearly multifunctional, with roles in bacterial invasion, endosome maturation, and autophagosome formation. In addition, this study showed that the bacterial cytolysin SLO supported the escape of GAS into the cytoplasm from endosomes, and surprisingly, a SLO-deficient mutant of GAS was viable longer than the wild-type strain although it failed to escape the endosomes. This intracellular behavior of GAS is unique and distinct from that of other types of bacterial invaders. Our results provide a new picture of GAS infection and host-cell responses in epithelial cells.

Streptococcus pyogenes Infection Induces Septic Arthritis with Increased Production of the Receptor Activator of the NF-κB Ligand

ABSTRACT Bacterial arthritis is a rapidly progressive and highly destructive joint disease in humans, with Staphylococcus aureus and Neisseria gonorrhoeae the major causative agents, although beta-hemolytic streptococci as well often induce the disease. We demonstrate here that intravenous inoculation of CD-1 mice with the group A streptococcus (GAS) species Streptococcus pyogenes resulted in a high incidence of septic arthritis. Signs of arthritis emerged within the first few days after injection, and bacterial examinations revealed that colonization of the inoculated GAS in the arthritic joints persisted for 21 days. Induction of persistent septic arthritis was dependent on the number of microorganisms inoculated. Immunohistochemical staining of GAS with anti-GAS antibodies revealed colonization in the joints of infected mice. Cytokine levels were quantified in the joints and sera of infected mice by using an enzyme-linked immunosorbent assay. High levels of interleukin-1β (IL-1β) and IL-6 were detected in the joints from 3 to 20 days after infection. We noted that an increase in the amount of receptor activator of NF-κB ligand (RANKL), which is a key cytokine in osteoclastogenesis, was also evident in the joints of the infected mice. RANKL was not detected in sera, indicating local production of RANKL in the infected joints. Blocking of RANKL by osteoprotegerin, a decoy receptor of RANKL, prevented bone destruction in the infected joints. These results suggest that GAS can colonize in the joints and induce bacterial arthritis. Local RANKL production in the infected joints may be involved in bone destruction.

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains.

Infection by Streptococcus pyogenes Induces the Receptor Activator of NF-κB Ligand Expression in Mouse Osteoblastic Cells

ABSTRACT Group A Streptococcus pyogenes is known to induce nongonococcal septic arthritis in addition to pharyngitis, scarlet fever, and poststreptococcal sequelae. However, little is known about the interaction between S. pyogenes and bone cells. We report here that S. pyogenes strain JRS4 (M6) attached to and invaded mouse primary osteoblasts. Reverse transcription-PCR demonstrated that S. pyogenes infection of osteoblasts stimulated expression of mRNA for the receptor activator of NF-κB ligand (RANKL). Western blot analysis followed by ligand precipitation with the receptor activator of NF-κB receptor showed that there was an increase in RANKL protein in infected osteoblasts. Production of interleukin-6 was also stimulated, but no production of interleukin-1β or tumor necrosis factor alpha was observed. Stimulation of RANKL production was not observed in osteoblasts stimulated with heat-inactivated S. pyogenes, suggesting that an active interaction of S. pyogenes with osteoblasts is essential for this phenomenon. A Western blot analysis performed with antibodies specific for phosphorylated signal transduction proteins demonstrated that S. pyogenes infection induces phosphorylation of p38 mitogen-activated protein kinase. A specific inhibitor of this kinase, SB203580, inhibited RANKL production by infected osteoblasts. These results suggest that infection of osteoblasts by S. pyogenes stimulates RANKL production and may trigger bone destruction in infected bone tissue.

Hydrogen peroxide contributes to the epithelial cell death induced by the oral mitis group of streptococci.

Members of the mitis group of streptococci are normal inhabitants of the commensal flora of the oral cavity and upper respiratory tract of humans. Some mitis group species, such as Streptococcus oralis and Streptococcus sanguinis, are primary colonizers of the human oral cavity. Recently, we found that hydrogen peroxide (H2O2) produced by S. oralis is cytotoxic to human macrophages, suggesting that streptococcus-derived H2O2 may act as a cytotoxin. Since epithelial cells provide a physical barrier against pathogenic microbes, we investigated their susceptibility to infection by H2O2-producing streptococci in this study. Infection by S. oralis and S. sanguinis was found to stimulate cell death of Detroit 562, Calu-3 and HeLa epithelial cell lines at a multiplicity of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H2O2, inhibited S. oralis cytotoxicity, and H2O2 alone was capable of eliciting epithelial cell death. Moreover, S. oralis mutants lacking the spxB gene encoding pyruvate oxidase, which are deficient in H2O2 production, exhibited reduced cytotoxicity toward Detroit 562 epithelial cells. In addition, enzyme-linked immunosorbent assays revealed that both S. oralis and H2O2 induced interleukin-6 production in Detroit 562 epithelial cells. These results suggest that streptococcal H2O2 is cytotoxic to epithelial cells, and promotes bacterial evasion of the host defense systems in the oral cavity and upper respiratory tracts.

Streptococcus sanguinis induces foam cell formation and cell death of macrophages in association with production of reactive oxygen species

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, is a common streptococcal species implicated in infective endocarditis. Herein, we investigated the effects of infection with S. sanguinis on foam cell formation and cell death of macrophages. Infection with S. sanguinis stimulated foam cell formation of THP-1, a human macrophage cell line. At a multiplicity of infection >100, S. sanguinis-induced cell death of the macrophages. Viable bacterial infection was required to trigger cell death because heat-inactivated S. sanguinis did not induce cell death. The production of cytokines interleukin-1β and tumor necrosis factor-α from macrophages was also stimulated during bacterial infection. Inhibition of the production of reactive oxygen species (ROS) resulted in reduced cell death, suggesting an association of ROS with cell death. Furthermore, S. sanguinis-induced cell death appeared to be independent of activation of inflammasomes, because cleavage of procaspase-1 was not evident in infected macrophages.

Streptococcus pyogenes degrades extracellular matrix in chondrocytes via MMP-13.

Group A streptococcus (GAS) causes a wide range of human diseases, including bacterial arthritis. The pathogenesis of arthritis is characterized by synovial proliferation and the destruction of cartilage and subchondral bone in joints. We report here that GAS strain JRS4 invaded a chondrogenic cell line ATDC5 and induced the degradation of the extracellular matrix (ECM), whereas an isogenic mutant of JRS4 lacking a fibronectin-binding protein, SAM1, failed to invade the chondrocytes or degrade the ECM. Reverse transcription-PCR and Western blot analysis revealed that the expression of matrix metalloproteinase (MMP)-13 was strongly elevated during the infection with GAS. A reporter assay revealed that the activation of the AP-1 transcription factor and the phosphorylation of c-Jun terminal kinase participated in MMP-13 expression. These results suggest that MMP-13 plays an important role in the destruction of infected joints during the development of septic arthritis.

Identification, characterization, and expression of dentin matrix protein 1 gene in Xenopus laevis

Dentin matrix protein 1 (DMP1) is an acidic extracellular matrix protein expressed mainly in bone and dentin, and is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family. The DMP1 gene, however, appears to evolve rapidly in comparison with other SIBLING genes, even though such functionally important molecules usually evolve more slowly than less important ones. The purpose of this study was to identify and characterize an ortholog of the DMP1 gene in an amphibian (Xenopus laevis; X. laevis) to clarify molecular evolutionary alterations in DMP1 associated with calcified tissues in tetrapods. Furthermore, we analyzed the mRNA expression of this gene to elucidate its functional change in bone and developing tooth germ in comparison with amniote DMP1s. The similarities of the deduced amino acid sequence of X. laevis DMP1 to that of the corresponding amniote proteins were low, although they did share several unique features specific to DMP1 and have similar properties. Expression of X. laevis DMP1 mRNA was predominant in osteocytes and odontoblasts, but only transiently observed in ameloblasts, as in amniotes. These results suggest that DMP1 has conserved several functions during tetrapod evolution. This indicates that continuity of biochemical properties has been more important in maintaining DMP1 functionality than that of the sequence of amino acid residues, which has undergone change over the course of molecular evolution.

Influence of Lifestyle Factors on Risk of Dental Caries among Children Living in Urban China

The prevalence of dental caries has been decreasing among kindergarten children in Shanghai, China, over recent years, although it still remains at an unacceptably high level. The purpose of this study was to identify which factors were important in providing oral health guidance and achieving further improvement in the oral health status of kindergarten children in urban China. A survey was conducted on dental caries in 128 Japanese and 368 Chinese kindergarten children and a questionnaire given to their parents/guardians on each childs lifestyle and dietary habits from birth to the present. Correlations between responses to each questionnaire item and the status of dental caries were statistically analyzed. The dft index score (p=0.0016), prevalence of dental caries (p=0.0002), and percentages of children with decayed (untreated caries-affected) teeth (p<0.0001) were significantly higher in the Chinese than in the Japanese children. Many differences were observed in lifestyle factors between the two groups. The percentage of parents failing to control the childs snacking habits between meals was higher in China, and weaning was significantly delayed in China compared with in Japan. These lifestyle factors were considered to be associated closely with the high risk of dental caries in Chinese kindergarten children. These findings indicate that oral health guidance for kindergarten children in Shanghai, China, should focus on control of dietary habits, including control of inter-meal snacking, and breastfeeding practices. The results of this study may help improve the status of dental caries among Chinese children.