Involvement of N-acetyl-D-galactosamine-specific lectin in biofilm formation by the periodontopathogenic bacterium, Eikenella corrodens

Eikenella corrodens is known not only as one of the periodontopathogenic bacteria but also as a pathogen associated with many infectious diseases of humans. Dental plaque is a complex biofilm community comprised of many bacterial species. E. corrodens has a lectin on its cell surface that is thought to be involved in its pathogenicity. In this study, we found that E. corrodens forms a biofilm on a polystyrene surface. A biofilm was formed at the bottom of the wells in microtiter plates after 24 h. Microcolonies were observed as the amount of biofilm became larger. When anaerobic respiration was repressed due to nitrate limitation, the biofilm formed only at the air-water interface. Strain 1073 and HU, which have higher lectin activity, formed a biofilm more effectively than other strains. Biofilm formation was repressed by the addition of N-acetyl-D-galactosamine. These results suggest that the lectin on the surface of E. corrodens might be involved in biofilm formation.     

Purification and characterization of hemolysin from periodontopathogenic bacterium Eikenella corrodens strain 1073

Eikenella corrodens 1073 was found to show hemolytic activity when grown on sheep blood agar. A high and dose-dependent hemolytic activity was detected in the cell envelope fraction, which was further purified by ion-exchange and gel-filtration chromatography. Consequently, a 65-kDa protein with hemolytic activity was obtained, suggesting that this protein might be a hemolysin. Its N-terminal amino acid sequence was nearly identical to that of X-prolyl aminopeptidase from E. corrodens ATCC 23834. To confirm that X-prolyl aminopeptidase functions as a hemolytic factor, we expressed the hlyA gene, encoding X-prolyl aminopeptidase, in Escherichia coli. After induction with isopropyl β-D-1-thiogalactopyranoside, a protein of about 65 kDa was purified on a Ni column, and its hemolytic activity was confirmed. Meanwhile, a strain with a disrupted hlyA gene, which was constructed by homologous recombination, did not show any hemolytic activity. These results suggested that X-prolyl aminopeptidase might function as a hemolysin in E. corrodens.     

Genomic recombination through plasmid-encoded recombinase enhances hemolytic activity and adherence to epithelial cells in the periodontopathogenic bacterium Eikenella corrodens

The periodontopathogenic bacterium Eikenella corrodens has an N-acetyl-D-galactosamine (GalNAc)-specific lectin, that contributes significantly to the pathogenicity of the bacterium. Recently, we reported that plasmid-mediated genomic recombination enhances the activity of this lectin. In this study, we investigated the effects of genomic recombination on certain virulence factors. Introduction of the recombinase gene resulted in hemolysis and significantly increased bacterial adhesion to epithelial cells. It was suggested that the enhanced adhesion was attributable to increased lectin activity due to genomic recombination, because it was inhibited by the addition of GalNAc. In contrast, invasion of the epithelial cells was remarkably reduced by genomic recombination. Although we assumed that this decrease in invasion resulted from a loss of type-IV pili, the phase variant did not show any decrease in invasion activity. This suggests that type-IV pili do not contribute to the invasive ability of E. corrodens. Our results suggest that genomic recombination enhances the pathogenicity of E. corrodens.     

大蒜素对白色念珠菌生物被膜形成的作用

生物被膜的形成是白色念珠菌产生耐药性的重要原因之一。本研究首先构建白色念珠菌体外生物被膜模型,通过倒置显微镜和甲基四氮盐（XTT）法检测大蒜素对白色念珠菌生物被膜形成的影响,同时采用实时荧光定量PCR法（qRT-PCR）对白色念珠菌生物被膜相关基因ALS1、ALS3、HWP1、MP65、SUN41的表达水平进行检测。结果显示,当大蒜素浓度≥12.5μg/mL时,白色念珠菌生物被膜的生长被抑制,并且在生物被膜形成的早期,大蒜素干预能有效抑制其形成;大蒜素能下调白色念珠菌生物被膜相关基因ALS1、ALS3、HWP1、MP65、SUN41的表达水平。研究结果提示,大蒜素可有效抑制体外白色念珠菌生物被膜的形成,可能与其下调生物被膜相关基因的表达有关。     

The inhibitory effects of mushroom extracts on sucrose-dependent oral biofilm formation

Mushrooms contain large quantities of α-glucans. Shiitake (Lentinula edodes), Japan’s most popular edible mushroom, has been reported to contain about 6% (weight/dried weight) of α-(1,3)-glucan. This glucan is one of the major components of oral biofilm formed by the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. We found that extracts from shiitake and other edible mushrooms could reduce preformed biofilms of S. mutans and S. sobrinus in the presence of dextranase. We also investigated the α-glucanase activities of shiitake mushroom extracts and their effects on biofilm formation. The extracts possessed α-glucanase activity and degraded water-insoluble glucans from mutans streptococci. The extracts strongly inhibited the sucrose-dependent formation of biofilms by S. mutans and S. sobrinus in the presence of dextranase. Our results suggest that some components of mushrooms, including α-glucanases, might inhibit the sucrose-induced formation of oral biofilms.     

The aerobic electron transport system of Eikenella corrodens

The respiratory system of the fastidious beta-proteobacterium Eikenella corrodens grown with limited oxygen was studied. Membranes showed the highest oxidase activity with ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) or succinate and the lowest activity with NADH and formate. The presence of a bc1-type complex was suggested by the inhibition exerted by 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), myxothiazol, and antimycin A on respiration with succinate and by the effect of the latter two inhibitors on the succinate-reduced difference spectra. Respiration with succinate or ascorbate-TMPD was abolished by low KCN concentrations, suggesting the presence of a KCN-sensitive terminal oxidase. Cytochromes b and c were spectroscopically detected after reduction with physiological or artificial electron donors, whereas type a and d cytochromes were not detected. The CO difference spectrum of membranes reduced by dithionite and its photodissociation spectrum (77 K) suggested the presence of a single CO compound that had the spectral features of a cytochrome o-like pigment. High-pressure liquid chromatography analysis of membrane haems confirmed the presence of haem B; in contrast, haems A and O were not detected. Peroxidase staining of membrane type c cytochromes using SDS-PAGE revealed the presence of five bands with apparent molecular masses of 44, 33, 30, 26, and 14 kDa. Based on our results, a tentative scheme of the respiratory chain in E. corrodens, comprising (i) dehydrogenases for succinate, NADH, and formate, (ii) a ubiquinone, (iii) a cytochrome bc1, and (iv) a type-cbb' cytochrome c oxidase, is proposed.     

Production of hydrolytic enzymes by oral isolates of Eikenella corrodens

Abstract Thermus thermophilus cells harboring an expression plasmid for the aqualysin I gene secrete the mature enzyme into the medium. In an Escherichia coli expression system, a precursor of the enzyme with the C-terminal pro-sequence is accumulated in the cells, and upon treatment at 65°C the active enzyme is produced. One- to 10-amino acid residue deletions, as well as complete 105-residue deletion of the C-terminal pro-sequence from the C-terminus, did not affect the production of the enzyme in T. coli cells. T. thermophilus cells harboring plasmids for mutant precursors with one- and three-residue deletions secreted the enzyme extracellularly. However, transformants harboring plasmids for mutant precursors with deletions of five or more amino acid residues could not be obtained. These results suggest that the C-terminal pro-sequence plays an important role in the extracellular secretion of the enzyme in T. thermophilus cells.     

Assessment of inhibitory effects of fluoride-coated tubes on biofilm formation by using the in vitro dental unit waterline biofilm model

This study aimed to establish an in vitro model to simulate biofilms formed in dental unit waterlines (DUWLs) and to investigate the ability of polyvinylidene fluoride (PVDF)-coated tubes to inhibit biofilm formation using this model. The water and biofilm samples were obtained from DUWLs which had been clinically used for 2.5 years, and the predominant bacteria were identified. A conventional polyurethane tube was incubated for 24 to 96 h in the mixed flora of isolated bacteria, and the optimal incubation conditions to simulate a clinically formed biofilm were determined by observation with a scanning electron microscope. Biofilm formation on a PVDF-coated tube was observed using this in vitro model, and the adherence of different bacterial species to conventional and PVDF-coated tubes was assessed. Sphingomonas paucimobilis, Acinetobacter haemolytics, and Methylobacterium mesophilicum were predominantly isolated from contaminated DUWLs. Incubation of the polyurethane tube with the mixed flora containing these three species for 96 h resulted in the formation of a mature biofilm similar to the one clinically observed. The PVDF-coated tube was significantly less adhesive to all three bacterial species than the polyurethane tube (P < 0.05 by the Mann-Whitney U test), and the attachment of small amounts of rods was observed even after incubation with the mixed flora for 96 h. In conclusion, an in vitro biofilm model was obtained by using a mixed flora of bacteria isolated from DUWLs, and the PVDF-coated tube was found to be effective in preventing biofilm formation using this model.     

Purification and partial characterization of a bacteriocin produced by Eikenella corrodens

Aims: The purpose of this study was to purify and characterize a bacteriocin produced by Eikenella corrodens A32E2. Methods and Results: Peptostreptococcus anaerobius ATCC27337 was used as indicator strain in antagonistic assays for bacteriocin‐producing E. corrodens A32E2. Protein extraction was influenced by pH and buffer composition. The protein was active in the pH range 6–8. Inhibitory activity was lost by both heating and treatment with proteolytic enzymes and decreased with organic solvents. The substance is rather unstable but maintains 100% of its activity after being exposed to acetone and when stored at ?70°C. The antagonistic substance was first precipitated by ammonium sulfate and further partially purified by Mono‐Q FPLC and C‐18 HPLC. Mass spectrometry analysis showed that the molecular mass was 23 625 Da, and the sequence obtained for the N‐terminus was: Met‐Asn‐Phe‐Asp‐Glu‐Lys‐Val‐Gly‐Lys‐Val‐X‐Phe‐Lys‐Val‐Gly‐Asp. Conclusions: The evidence presented in this study supports the idea that an antagonistic substance produced by E. corrodens A32E2 isolated from a periodontal diseased site is a novel bacteriocin, which we designate corrodecin. Significance and Impact of the Study:  We anticipated that corrodecin might play an important role at the periodontal site. This compound could also be attractive in biotechnological applications as an interesting tool for oral ecosystem control.     

Role of the Eikenella corrodens pilA locus in pilus function and phase variation

The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolate E. corrodens VA1. Earlier work defined the pilA locus which includes pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin, whereas pilB predicts a putative pilus assembly protein. The role of hagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilA mutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated for pilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation of hagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function.     

In vivo inhibitory effect on the biofilm formation of Candida albicans by liverwort derived riccardin D

Riccardin D, a macrocyclic bisbibenzyl isolated from Chinese liverwort Dumortiera hirsute, has been proved to have inhibitory effect on biofilms formation of Candida albicans in in vitro study. Our present study aims to investigate the in vivo effect and mechanisms of riccardin D against C. albicans biofilms when used alone or in combination with clinical using antifungal agent fluconazole. XTT reduction assay revealed riccardin D had both prophylactic and therapeutic effect against C. albicans biofilms formation in a dose-dependent manner when using a central venous catheter related infective animal model. Scanning electron microscope and laser confocal scanning microscope showed that the morphology of biofilms was altered remarkably after riccardin D treatment, especially hypha growth inhibition. To uncover the underlying molecular mechanisms, quantitative real-time RT-PCR was performed to observe the variation of related genes. The downregulation of hypha-specific genes such as ALS1, ALS3, ECE1, EFG1, HWP1 and CDC35 following riccardin D treatment suggested riccardin D inhibited the Ras-cAMP-Efg pathway to retard the hypha formation, then leading to the defect of biofilms maturation. Moreover, riccardin D displayed an increased antifungal activity when administered in combination with fluconazole. Our study provides a potential clinical application to eliminate the biofilms of relevant pathogens.     

The inhibitory effect of Ulva fasciata on culturability,motility, and biofilm formation of Vibrio parahaemolyticus ATCC17802

International Microbiology - The outbreak of vibriosis from Vibrio parahaemolyticus (V. parahaemolyticus) is one of common pathogenic diseases found in the mariculture environment. In this study,...     

Identification of an antifungal peptide from Trapa natans fruits with inhibitory effects on Candida tropicalis biofilm formation

Due to recent emergence of fungal pathogens resistant to current antifungal therapies, several studies have been focused on screening of plant peptides to find novel compounds having antifungal activities. Here, a novel antifungal plant peptide, with molecular mass of 1230 Da was purified from fruits of Trapa natans by reverse phase high performance liquid chromatography using 300SB-C18 column and named as Tn-AFP1. Determination of complete amino acid sequences of this peptide by tandem mass spectrometry showed to contain following eleven amino acid residues: LMCTHPLDCSN. Purified Tn-AFP1 showed the inhibition of Candida tropicalis growth in vitro and disrupted the biofilm formation in a concentration dependent manner. It also showed downregulation of MDR1 and ERG11 gene expression in real time-PCR analysis. In silico molecular modeling predicted the structure of Tn-AFP1 as a single coil attached by a unique disulfide bond. Characterization of Tn-AFP1 could contribute in designing novel derivative(s) of this peptide for the development of more effective antimycotic compounds.     

Inhibitory effects of 7-epiclusianone on glucan synthesis, acidogenicity and biofilm formation by Streptococcus mutans

The aim of this study was to examine the effects of 7-epiclusianone, a new prenylated benzophenone isolated from the plant Rheedia gardneriana, on some of the virulence properties of Streptococcus mutans associated with biofilm development and acidogenicity. The synthesis of glucans by glucosyltransferases B (GTF B) and C (GTF C) was markedly reduced by 7-epiclusianone showing more than 80% inhibition of enzymatic activity at a concentration of 100 microg mL(-1). Double-reciprocal analysis (Lineweaver-Burk plots) revealed that the inhibition of GTF B activity was noncompetitive (mixed) while GTF C was inhibited uncompetitively. The glycolytic pH drop by S. mutans cells was also disrupted by 7-epiclusianone without affecting the bacterial viability, an effect that can be attributed, in part, to inhibition of F-ATPase activity (61.1+/-3.0% inhibition at 100 microg mL(-1)). Furthermore, topical applications (1-min exposure, twice daily) of 7-epiclusianone (at 250 microg mL(-1)) disrupted biofilm formation and physiology. The biomass (dry-weight), extracellular insoluble polysaccharide concentration and acidogenicity of the biofilms were significantly reduced by the test agent (P<0.05). The data show that 7-epiclusianone disrupts the extracellular and intracellular sugar metabolism of S. mutans, and holds promise as a novel, naturally occurring compound to prevent biofilm-related oral diseases.