Takahiko Oho

Invasion of human aortic endothelial cells by oral viridans group streptococci and induction of inflammatory cytokine production

Oral viridans group streptococci are the major commensal bacteria of the supragingival oral biofilm and have been detected in human atheromatous plaque. Atherosclerosis involves an ongoing inflammatory response, reportedly involving chronic infection caused by multiple pathogens. The aim of this study was to examine the invasion of human aortic endothelial cells (HAECs) by oral viridans group streptococci and the subsequent cytokine production by viable invaded HAECs. The invasion of HAECs by bacteria was examined using antibiotic protection assays and was visualized by confocal scanning laser microscopy. The inhibitory effects of catalase and cytochalasin D on the invasion of HAECs were also examined. The production of cytokines by invaded or infected HAECs was determined using enzyme-linked immunosorbent assays, and a real-time polymerase chain reaction method was used to evaluate the expression of cytokine messenger RNA. The oral streptococci tested were capable of invading HAECs. The number of invasive bacteria increased with the length of the co-culture period. After a certain co-culture period, some organisms were cytotoxic to the HAECs. Catalase and cytochalasin D inhibited the invasion of HAECs by the organism. HAECs invaded by Streptococcus mutans Xc, Streptococcus gordonii DL1 (Challis), Streptococcus gordonii ATCC 10558 and Streptococcus salivarius ATCC 13419 produced more cytokine(s) (interleukin-6, interleukin-8, monocyte chemoattractant protein-1) than non-invaded HAECs. The HAECs invaded by S.xa0mutans Xc produced the largest amounts of cytokines, and the messenger RNA expression of cytokines by invaded HAECs increased markedly compared with that by non-invaded HAECs. These results suggest that oral streptococci may participate in the pathogenesis of atherosclerosis.

Staphylococcus aureus SasA Is Responsible for Binding to the Salivary Agglutinin gp340, Derived from Human Saliva

ABSTRACT Staphylococcus aureus is a major human pathogen that can colonize the nasal cavity, skin, intestine, and oral cavity as a commensal bacterium. gp340, also known as DMBT1 (deleted in malignant brain tumors 1), is associated with epithelial differentiation and innate immunity. In the oral cavity, gp340 induces salivary aggregation with several oral bacteria and promotes bacterial adhesion to tissues such as the teeth and mucosa. S. aureus is often isolated from the oral cavity, but the mechanism underlying its persistence in the oral cavity remains unclear. In this study, we investigated the interaction between S. aureus and gp340 and found that S. aureus interacts with saliva- and gp340-coated resin. We then identified the S. aureus factor(s) responsible for binding to gp340. The cell surface protein SasA, which is rich in basic amino acids (BR domain) at the N terminus, was responsible for binding to gp340. Inactivation of the sasA gene resulted in a significant decrease in S. aureus binding to gp340-coated resin. Also, recombinant SasA protein (rSasA) showed binding affinity to gp340, which was inhibited by the addition of N-acetylneuraminic acid. Surface plasmon resonance analysis showed that rSasA significantly bound to the NeuAcα(2-3)Galβ(1-4)GlcNAc structure. These results indicate that SasA is responsible for binding to gp340 via the N-acetylneuraminic acid moiety.

A peptide domain of bovine milk lactoferrin inhibits the interaction between streptococcal surface protein antigen and a salivary agglutinin peptide domain

ABSTRACT The peptide domain of salivary agglutinin responsible for its interaction with cell surface protein antigen (PAc) of Streptococcus mutans or bovine lactoferrin was found in the same peptide, scavenger receptor cysteine-rich domain peptide 2 (SRCRP2). Inhibition studies suggest that PAc and lactoferrin, of which residues 480 to 492 seem important, competitively bind to the SRCRP2 domain of salivary agglutinin.

Increase in detectable opportunistic bacteria in the oral cavity of orthodontic patients

OBJECTIVESnThis study was performed to detect the opportunistic bacteria and fungi from the oral cavities of orthodontic patients and examine the ability of the organisms to adhere to saliva-coated metallic brackets.nnnMETHODSnOpportunistic bacteria and fungi were isolated from 58 patients (orthodontic group: 42; non-orthodontic group: 16) using culture methods and were identified based on their biochemical and enzymatic profiles. Seven opportunistic and four streptococcal strains were tested for their ability to adhere to saliva-coated metallic brackets.nnnRESULTSnMore opportunistic bacteria and fungi were detected in the orthodontic group than in the non-orthodontic group (P < 0.05). Opportunistic bacteria adhered to saliva-coated metallic brackets to the same degree as oral streptococci.nnnCONCLUSIONSnThe isolation frequencies of opportunistic bacteria and fungi increase during orthodontic treatment, suggesting the importance of paying special attention to oral hygiene in orthodontic patients to prevent periodontal disease and the aggravation of systemic disease in immunocompromised conditions.

Experimental Abscess Formation Caused by Human Dental Plaque

Human dental plaque consists of a wide variety of microorganisms, some of which are believed to cause systemic infections, including abscesses, at various sites in the body. To confirm this hypothesis experimentally, we examined the abscess‐forming ability of native dental plaque in mice, the microbial features of the infectious locus produced by the plaque, and the anti‐phagocytic property of microbial isolates. Aliquots of a suspension of supragingival dental plaque containing 6 × 106 colony‐forming unit of bacteria were injected subcutaneously into the dorsa of mice. Abscess formation was induced in 76 of 85 mice using ten different plaque samples. Thirteen microorganisms were isolated from pus samples aspirated from abscess lesions. The microbial composition of pus, examined in 17 of 76 abscesses, was very simple compared to that of the plaque sample that had induced the abscess. The majority of the isolates belonged to the Streptococcus anginosus group, normally a minor component of plaque samples. S. anginosus was the most frequently detected organism and the most prevalent in seven abscesses, and Streptococcus intermedius and Streptococcus constellatus were predominant in one and three abscess samples, respectively. Each isolate of S. anginosus group produced abscesses in mice, and heat‐treated supragingival dental plaque influenced the abscess‐forming ability of S. anginosus isolate. These isolates possessed a high anti‐phagocytic capacity against human polymorphonuclear leukocytes. Our results suggest that human supragingival dental plaque itself is a source of the infectious pathogens that cause abscess formation.

GlmS and NagB regulate amino sugar metabolism in opposing directions and affect Streptococcus mutans virulence.

Streptococcus mutans is a cariogenic pathogen that produces an extracellular polysaccharide (glucan) from dietary sugars, which allows it to establish a reproductive niche and secrete acids that degrade tooth enamel. While two enzymes (GlmS and NagB) are known to be key factors affecting the entrance of amino sugars into glycolysis and cell wall synthesis in several other bacteria, their roles in S. mutans remain unclear. Therefore, we investigated the roles of GlmS and NagB in S. mutans sugar metabolism and determined whether they have an effect on virulence. NagB expression increased in the presence of GlcNAc while GlmS expression decreased, suggesting that the regulation of these enzymes, which functionally oppose one another, is dependent on the concentration of environmental GlcNAc. A glmS-inactivated mutant could not grow in the absence of GlcNAc, while nagB-inactivated mutant growth was decreased in the presence of GlcNAc. Also, nagB inactivation was found to decrease the expression of virulence factors, including cell-surface protein antigen and glucosyltransferase, and to decrease biofilm formation and saliva-induced S. mutans aggregation, while glmS inactivation had the opposite effects on virulence factor expression and bacterial aggregation. Our results suggest that GlmS and NagB function in sugar metabolism in opposing directions, increasing and decreasing S. mutans virulence, respectively.

Streptococcus anginosusl-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 (βC-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 βC-S lyase, in the pus of abscesses that formed in BALB/c mice following subcutaneous injection of S.xa0anginosus 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.xa0anginosus FW73 plus l-cysteine was greater than that of abscesses caused by S.xa0anginosus FW73 in the absence of l-cysteine. These findings demonstrate that the lcd gene of S.xa0anginosus is upregulated in mouse abscesses and that hydrogen sulfide, the product of a reaction catalyzed by βC-S lyase, plays an etiological role in odontogenic abscess formation.

Effect of saliva viscosity on the co-aggregation between oral streptococci and Actinomyces naeslundii

BACKGROUNDnThe co-aggregation of oral bacteria leads to their clearance from the oral cavity. Poor oral hygiene and high saliva viscosity are common amongst the elderly; thus, they frequently suffer from pneumonia caused by the aspiration of oral microorganisms.nnnOBJECTIVESnTo examine the direct effect of saliva viscosity on the co-aggregation of oral streptococci with actinomyces.nnnMATERIALS AND METHODSnFifteen oral streptococcal and a single actinomyces strain were used. Co-aggregation was assessed by a visual assay in phosphate buffer and a spectrophotometric assay in the same buffer containing 0-60% glycerol or whole saliva.nnnRESULTSnNine oral streptococci co-aggregated with Actinomyces naeslundii ATCC12104 in the visual assay and were subsequently used for the spectrophotometric analysis. All tested strains displayed a decrease in co-aggregation with increasing amounts of glycerol in the buffer. The co-aggregation of Streptococcus oralis with A. naeslundii recovered to baseline level following the removal of glycerol. The per cent co-aggregation of S. oralis with A. naeslundii was significantly correlated with the viscosity in unstimulated and stimulated whole saliva samples (correlation coefficients: -0.52 and -0.48, respectively).nnnCONCLUSIONnThis study suggests that saliva viscosity affects the co-aggregation of oral streptococci with actinomyces and that bacterial co-aggregation decreases with increasing saliva viscosity.

Streptococcus oralis coaggregation receptor polysaccharides induce inflammatory responses in human aortic endothelial cells.

Streptococcus oralis, belonging to the oral viridans group streptococci, has been detected in human cardiovascular lesions including infective endocarditis and atheromatous plaques. The organism has coaggregation receptor polysaccharides (RPS) on the cell wall, which function as receptors for surface adhesins on other members of the oral biofilm community. The present study examined the capacity of S. oralis RPS to induce inflammatory responses in human aortic endothelial cells (HAECs). Purified RPS was used to stimulate HAECs, and the induction of cytokines, adhesion molecules and Toll-like receptors (TLRs) was examined. Involvement of RPS in HAEC invasion by S. oralis was also examined. RPS-stimulated HAECs produced more cytokines (interleukin-6, interleukin-8 and monocyte chemoattractant protein-1) and intercellular adhesion molecule-1 than non-stimulated HAECs. The messenger RNA (mRNA) expression of cytokines and adhesion molecules in RPS-stimulated HAECs increased markedly compared with that in non-stimulated HAECs. Upregulation of TLR-2 mRNA expression was demonstrated in RPS-stimulated HAECs. Moreover, TLR-2 mRNA expression and cytokine production were reduced by the incubation of HAECs with inhibitors against p38 mitogen-activated protein kinase and nuclear factor-κB. An RPS-defective mutant of S. oralis showed greater invasion into HAECs than an RPS-possessing strain. However, HAECs invaded by the RPS-defective mutant produced less cytokines than HAECs invaded by the RPS-possessing strain, indicating that RPS can stimulate HAECs intracellularly. These results suggest that S. oralis RPS may be an important contributor to the pathogenesis of cardiovascular diseases such as infective endocarditis and atherosclerosis.

Identification and characterization of a fibronectin-binding protein from Granulicatella adiacens.

The interaction of microorganisms with fibronectin plays an important role in infective endocarditis. Granulicatella adiacens is a member of the oral microbiota, formerly known as nutritionally variant streptococci, and is often isolated from endocarditis patients. In the present study we identified a surface protein, designated Cha, which binds to fibronectin, by a plaque hybridization procedure using the cshA sequence as probe, which encodes a fibronectin-binding molecule of Streptococcus gordonii DL1. The cha sequence was highly homologous to cshA and encoded a product of 2351 amino acid residues. The protein comprised a unique sequence in the N-terminal half region. The C-terminal region contained nine complete, and one incomplete, 115-amino acid residue repeat blocks. Among eight strains of nutritionally variant streptococci, three G.xa0adiacens strains and one Abiotrophia defectiva strain carried the cha gene. Heterologous expression studies suggested that Cha adhered to immobilized fibronectin, and that this function was located in the unique region. Recombinant Cha protein also adhered to immobilize fibronectin and partially inhibited adherence of G.xa0adiacens to fibronectin in a dose-dependent manner. These results suggest that Cha is a cell surface protein that mediates adherence of G.xa0adiacens to fibronectin.