Identification of genes encoding glycosyltransferases involved in lipopolysaccharide synthesis in Porphyromonas gingivalis.

Porphyromonas gingivalis can synthesize both A-LPS and O-LPS lipopolysaccharides, which contain anionic O-polysaccharides and conventional O-polysaccharides, respectively. A-LPS can anchor virulence proteins to the cell surface, so elucidating the mechanism of A-LPS synthesis is important for understanding the pathogenicity of this bacterium. To identify the genes involved in LPS synthesis, we focused on uncharacterized genes encoding the glycosyltransferases, PGN_0361, PGN_1239, PGN_1240 and PGN_1668, which were tentatively named gtfC, gtfD, gtfE and gtfF, respectively, and characterized their mutants. We found that disruption of gtfC and gtfF resulted in A-LPS deficiency. In addition, a gtfD mutant had abnormal A-LPS synthesis, and a gtfE mutant exhibited a rough-type LPS that possesses a short oligosaccharide with lipid A-core. We then constructed a gtfC and gtfD double mutant, because their amino acid sequences were very similar, and this mutant similarly possessed a rough-type LPS. Cross-complementation analysis revealed that the GtfD protein is a functional homologue of the Escherichia coli WbbL protein, which is a rhamnosyltransferase. These results suggested that the GtfE protein is essential for the synthesis of both O-LPS and A-LPS, and that GtfC and GtfD proteins may work together to synthesize the two kinds of LPS. In addition, the GtfF protein was essential for A-LPS synthesis, although this may be achieved in a strain-specific manner.

Identification of oral species of the genus Veillonella by polymerase chain reaction.

INTRODUCTION: Members of the genus Veillonella cannot be reliably distinguished by their biochemical characteristics and phenotypic features. Moreover, DNA-DNA hybridization and sequence analyses of the 16S ribosomal RNA gene including random fragment length polymorphism analysis, are complex and time-consuming procedures that are not well-suited to identifying oral species of Veillonella: Veillonella atypica, Veillonella denticariosi, Veillonella dispar, Veillonella parvula, and Veillonella rogosae. METHODS: In this study, five forward primers and a reverse primer were designed for polymerase chain reaction (PCR) according to the partial sequences of the rpoB genes of these oral Veillonella species. RESULTS: The forward primers were species-specific for these five Veillonella species, and could produce specific amplicons when used together with reverse primer and individual DNA templates of these species in PCR. These primer pairs were also found to discriminate between the respective species, and the Veillonella strains isolated from human oral cavities were successfully assigned to one of the five oral species of the genus Veillonella based on their specific products by PCR. CONCLUSION: A simple two-step PCR procedure using the five sets of primer pairs developed in the present study is a rapid and reliable method for the identification of the recognized oral Veillonella species.

Inhibitory effect of green tea catechins on cysteine proteinases in Porphyromonas gingivalis.

The purpose of this study was to examine the effects of catechins and their derivatives on the activities of Arg-gingipain (Rgp) and Lys-gingipain (Kgp) in Porphyromonas gingivalis. Catechin derivatives, which included (-)-epigallocatechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, and (-)-catechin gallate, significantly inhibited the Rgp activity. The 50% inhibitory concentrations (IC50s) of these catechin derivatives for Rgp ranged from 3 to 5 microm. While (-)-epigallocatechin and (-)-gallocatechin moderately inhibited Rgp activity (IC50s, 20 microm), (-) -epicatechin, (+)-catechin, and gallic acid were not effective, with IC50s greater than 300 microm. Further, some of the catechin derivatives tested also inhibited the Kgp activity, though to a lesser extent than inhibition of the Rgp activity. These findings suggest that green tea catechins may have the potential to reduce periodontal breakdown resulting from the potent proteinase activity of P. gingivalis.

Effect of Porphyromonas gingivalis ATCC 33277 vesicle on adherence of Streptococcus mutans OMZ 70 to the experimental pellicle.

The vesicles of Porphyromonas gingivalis ATCC 33277 strongly aggregated Streptococcus cricetus, S. rattus, and S. mutans, but poorly aggregated S. sobrinus. The adherence of S. mutans OMZ 70 to hydroxyapatite (HA) coated with whole saliva was increased in parallel with the quantity of the vesicles. The significant increase of adherence of S. mutans OMZ 70 by the vesicles was also observed on the HA coated with parotid saliva, submandibular saliva, serum, and type I collagen. These findings suggest that the vesicles may act as a bridge between mutans streptococcus and the tooth surface.

Coaggregation between Porphyromonas gingivalis and mutans streptococci.

Coaggregation occurred between Porphyromonas gingivalis and mutans streptococci. The coaggregation was completely inhibited by L-arginine, N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), and a trypsin inhibitor, and weakly inhibited by L-lysine, N-ethylmaleimide, lysozyme, and human whole saliva. The results of heat and proteinase K treatment suggested that a heat-labile proteinaceous substance of P. gingivalis and a heat-stable substance of mutans streptococci may play a role in the coaggregation. Mutans streptococci also aggregated in the presence of the heat-labile factor in the supernatant of P. gingivalis. The aggregation was also inhibited by L-arginine, TLCK, and a trypsin inhibitor.

Studies on the toxin of Aspergillus fumigatus. XVIII. Photooxidation of asp-hemolysin in the presence of various dyes and its relation to the site of hemolytic activity.

The site of hemolytic activity of a toxin isolated from Aspergillus fumigatus designated Asp-hemolysin was determined by photooxidation techniques. The hemolytic activity of this toxin was strongly inhibited by photooxidation with methylene blue, rose bengal, riboflavin, or eosin G as a sensitizer, whereas crystal violet, hematoxylin, naphthol yellow S, bromothymol blue, methyl orange, and cresol red had no effect. pH dependence of the inactivation with methylene blue was observed in the narrow range of pH values from 7.0 to 8.0, like that of the inactivation with rose bengal or riboflavin. The histidine, cysteine, methionine, tryptophan, and tyrosine content of methylene blue-photooxidized Asp-hemolysin was significantly decreased, while other amino acids were not affected. The hemolytic activity of the toxin was lost more slowly than the histidine residue, being maintained at about 50% even at the time when the histidine residue was completely lost after 30 min. Photooxidation of Asp-hemolysin in the presence of rose bengal also caused a decrease in histidine, methionine, and threonine content. These findings suggest that residues of cysteine, methionine, threonine, tryptophan, and/or tyrosine but not histidine may play an important role through stereostructure in the manifestation of the hemolytic activity of Asp-hemolysin.

Production of contiguously arranged chlamydospores in Candida albicans.

Contiguous arrangements of two to three chlamydospores occurred in a clinical isolate of Candida albicans. Time-lapse photography showed that the terminal cell of a cell chain was first transformed into a chlamydospore and such transformation proceeded centripetally to the next cell in the chain. Ultrathin sections revealed that the outermost layer of the three-layered chlamydospore wall was continuous throughout the interconnected spores, with the other layers surrounding each spore separately. Chlamydospore chains were common in this organism.

Studies on the toxin of Aspergillus fumigatus. VII. Purification and some properities of hemolytic toxin (asp-hemolysin) from culture filtrates and mycelia.

A hemolytic toxin has been obtained from mycelia and culture filtrates of Aspergillus fumigatus by the procedures that included precipitation with ammonium sulfate, chromatography of DEAE-Sephadex, affinity chromatography on Concanavalin A-Sepharose and gell filtration on Sephadex G-50, G-100 AND G-150. The purified homolytic toxin was homogeneous on immunological and disk electrophoretic analysis, and the toxin from culture filtrates was identical with that from mycelia by the immunodiffusion technique. The hemolytic toxin was obtained for the first time from fungi and designated as Asp-hemolysin. The molecular weight of Asp-hemolysin was estimated to be appoximately 30,000 by the gel-filtration technique and its isoelectric point was found to be around pH 4.0. This Asp-hemolysin contained large amounts of protein and very small amounts of carbohydrate. The UV absorption spectrum of Asp-hemolysin showed a maximum absorption at 280 nm and minimum absorption at 251 nm. The extinction coefficient at 280 nm and minimum absorption at 251 nm. The extinction coefficient at 280 nm, E 1% 1CM, was 12.4 and the ratio of absorbance at 280 nm to that at 260 nm was 2.3. The optimum pH for the hemolytic activity of the toxin toward chicken erythrocytes was 5.0 at room temperature and it was active in the pH range of 3.5 to 10.5. The optimum temperature was 21 C and about 50% of the activity was lost by incubation at 50 C for 5 min or 45 C for 23 min. The hemolytic activity was remarkably inhibited by Hg2+, Cu2+, Fe2+, Ag1+, iodine and p-CMB, but enhanced slightly by Zn2+ and Co2+.