Arihide Kamaguchi

Studies on the Toxin of Aspergillus fumigatus

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 gel 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 approximately 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, 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+.

Effect of Porphyromonas gingivalis vesicles on coaggregation of Staphylococcus aureus to oral microorganisms.

Vesicles from the outer membrane of Porphyromonas gingivalis have the ability to aggregate a wide range of Streptococcus spp., Fusobacterium nucleatum, Actinomyces naeslundii, and Actinomyces viscosus. We found that in the presence of P. gingivalis vesicles, Staphylococcus aureus coaggregated with Streptococcus spp., and the mycelium-type Candida albicans, but not the yeast type. Autoaggregation of S. aureus in the presence of P. gingivalis vesicles is inhibited by L-arginine, L-lysine, and L-cysteine. Both the methicillin-sensitive (MSSA) and -resistant (MRSA) strains of S. aureus were able to coaggregate with Streptococcus spp., A. naeslundii, and A. viscosus when they were treated with P. gingivalis vesicles. P. gingivalis vesicle-treated mycelium-type C. albicans coaggregated with S. aureus, but the yeast-type did not. These results indicate that strains of S. aureus, including MRSA, could adhere to oral biofilms in dental plaque on the tooth surface or in the gingival crevice when P. gingivalis is present.

Coaggregation of Porphyromonas gingivalis and Prevotella intermedia.

Porphyromonas gingivalis cells coaggregated with Prevotella intermedia cells. The coaggregation was inhibited with L‐arginine, L‐lysine, Nα‐p‐tosyl‐L‐lysine chloromethyl ketone, trypsin inhibitor, and leupeptin. Heat‐ and proteinase K‐treated P. gingivalis cells showed no coaggregation with P. intermedia cells, whereas heat and proteinase K treatments of P. intermedia cells did not affect the coaggregation. The vesicles from P. gingivalis culture supernatant aggregated with P. intermedia cells, and this aggregation was also inhibited by addition of L‐arginine or L‐lysine and by heat treatment of the vesicles. The rgpA rgpB, rgpA kgp, rgpA rgpB kgp, and rgpA kgp hagA mutants of P. gingivalis did not coaggregate with P. intermedia. On the other hand, the fimA mutant lacking the FimA fimbriae showed coaggregation with P. intermedia as well as the wild type parent. These results strongly imply that a heat‐labile and proteinous factor on the cell surface of P. gingivalis, most likely the gingipain‐adhesin complex, is involved in coaggregation of P. gingivalis and P. intermedia.

Roles of Arg- and Lys-gingipains in coaggregation of Porphyromonas gingivalis: Identification of its responsible molecules in translation products of rgpA, kgp, and hagA genes

Abstract Arg- (Rgp) and Lys-gingipains (Kgp) are two individual cysteine proteinases produced by Porphyromonas gingivalis, an oral anaerobic bacterium, and are implicated as major virulence factors in a wide range of pathologies of adult periodontitis. Coaggregation of this bacterium with other oral bacteria is an initial and critical step in infectious processes, yet the factors and mechanisms responsible for this process remain elusive. Here we show that the initial translation products of the rgpA, kgp and hemagglutinin hagA genes are responsible for coaggregation of P. gingivalis and that the proteolytic activity of Rgp and Kgp is indispensable in this process. The rgpA rgpB kgp- and rgpA kgp hagA-deficient triple mutants exhibited no coaggregation activity with Actinomyces viscosus, whereas the kgp-null and rgpA rgpB-deficient double mutants significantly retained this activity. Consistently, the combined action of Rgp- and Kgp-specific inhibitors strongly inhibited the coaggregation activity of the bacterium, although single use of Rgp- or Kgp-specific inhibitor significantly retained this activity. We also demonstrate that the 47- and 43-kDa proteins produced from the translation products of the rgpA, kgp, and hagA genes by proteolytic activity of both Rgp and Kgp are responsible for the coaggregation of P. gingivalis.

Construction of recombinant hemagglutinin derived from the gingipain-encoding gene of Porphyromonas gingivalis, identification of its target protein on erythrocytes, and inhibition of hemagglutination by an interdomain regional peptide

Porphyromonas gingivalis, an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however, P. gingivalis-mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of P. gingivalis is intragenically encoded by rgpA, kgp, and hagA, direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44(720-1081), a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44(720-1138), did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44(720-1138) but not in HGP44(720-1081), could bind HGP44(720-1081) in a dose-dependent manner and effectively inhibited HGP44(720-1081)-mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44(720-1081)-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44(720-1081)-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44(720-1081) could bind to asialoglycophorin with a dissociation constant of 3.0 x 10(-7) M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.

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.

Veillonella tobetsuensis sp. nov., an anaerobic, Gram-negative coccus isolated from human tongue biofilms

Four previously unknown, gram-negative, anaerobic coccal strains were isolated from the tongue biofilm of healthy human adults (ages 22-29 years). The isolates displayed all phenotypic characteristics of the genus Veillonella. Comparative analysis of the 16S rRNA, dnaK and rpoB gene sequences indicated that the four strains were phylogenetically homogeneous and comprised a distinct novel lineage within the genus Veillonella. The production of major cellular fatty acids (C13 : 0 and C17 : 1ω8) was consistent with that of other members of the genus Veillonella. Based on these observations, strains B16(T), A16, B4 and Y6 represent a novel species, for which the name Veillonella tobetsuensis sp. nov. is proposed, with the type strain B16(T) ( = JCM 17976(T) = ATCC BAA-2400(T)).

Coaggregation between Porphyromonas gingivalis and mutans streptococci

Coaggregation occurred between Porphyromonas gingivalis and mutans streptococci. The coaggregation was completely inhibited by l‐arginine, Nα‐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.

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. 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.