Ken Ichi Tomochika

Distribution of Virulence-Associated Genes in Vibrio mimicus Isolates from Clinical and Environmental Origins

Distribution of virulence‐associated genes in Vibrio mimicus was studied including the toxin genes ctxA, tdh, st and vmh and the genes necessary for regulation of toxin production, toxR, toxS, toxT, tcpA and tcpP. Approximately half of clinical V. mimicus isolates possessed one or more genes encoding V. cholerae enterotoxic factors such as ctxA, tdh and st. All of the clinical and environmental isolates possessed vmh encoding V. mimicus hemolysin (VMH). The ctxA encoding cholera toxin was detected in only 2 strains, 5% of the clinical isolates. Furthermore, there were very few strains possessing tcpP and toxT needed for the expression of ctxA. These results may suggest that VMH is a more important pathogenic factor than well recognized toxins such as cholera toxin (CT) in V. mimicus infection.

Purification of a serine protease of Vibrio parahaemolyticus and its characterization

A 50 kDa protease designated as VPP1 was purified from the culture supernatant of a clinical strain of Vibrio parahaemolyticus by ammonium sulfate fractionation, Sephacryl S‐200 HR gel filtration and Fractogel EMD TMAE 650 ion‐exchange chromatography. VPP1 was inhibited by EDTA, EGTA and serine protease inhibitors, suggesting that it is a calcium‐dependent serine protease. N‐terminal amino acid sequence of VPP1 was quite similar to that of V. metschnikovii protease and antibody against VPP1 inhibited the activity of V. metschnikovii protease, suggesting the similarity of the two proteases. It was demonstrated that VPP1 or its related protease widely distribute in not only V. parahaemolyticus but also V. alginolyticus.

Bacterial Proteases as Pathogenic Factors, with Special Emphasis on Vibrio Proteases

AbstractBacterial proteases play various pathogenic roles in infection. Pathogenic species of the genus Vibrio, such as V. cholerae, V. parahaemolyticus or V. vulnificus also produce exocellular proteases, and almost of them are metalloproteases having a zinc atom. V. vulnificus is an opportunistic human pathogen causing septicemia or wound infections characterized by the formation of edema, necrotic ulcer and cellulitis on the skin. The protease produced by the vibrio (VVP) enhanced vascular permeability through activation of the Hageman factor-plasma kallikrein-kinin cascade and/or exocytotic histamine release from mast cells, and formed hemorrhagic lesions which subsequently provoke severe dermonecrosis. Thus, VVP is the most probable candidate for edema formation in the infection. Other pathogenic roles of VVP were also demonstrated.The VVP gene was cloned, and the amino acid sequence was deduced from the nucleotide sequence. The mature protein was demonstrated to be composed of 413 amino acid residue...

Histamine-releasing reaction induced by the N-terminal domain of Vibrio vulnificus metalloprotease.

A zinc metalloprotease secreted by Vibrio vulnificus, an opportunistic human pathogen causing septicemia and wound infection, stimulates exocytotic histamine release from rat mast cells. This protease consists of two functional domains: the N-terminal domain that catalyzes proteolytic reaction and the C-terminal domain that promotes the association with a protein substrate or cell membrane. Like the intact protease, the N-terminal domain alone also induced histamine release from rat peritoneal mast cells in a dose- and time-dependent manner. However, the reaction induced was apparently weak and went on more slowly. The nickel-substituted protease or its N-terminal domain, each of which has the reduced proteolytic activity due to decreased affinity to a substrate, showed much less histamine-releasing activity. When injected into the rat dorsal skin, the N-terminal domain also evoked enhancement of the hypodermic vascular permeability, while the activity was comparable to that of the protease. Taken together, the protease may stimulate histamine release through the action of the catalytic center of the N-terminal domain on the target substance(s) on the mast cell membrane. The C-terminal domain may support the in vitro action of the N-terminal domain by coordination of the association of the protease with the membrane, but it may not modulate the in vivo action.

Vacuolar-type H+-ATPase in mouse bladder epithelium is responsible for urinary acidification

© 1997 Federation of European Biochemical Societies.

An Exocellular Cytolysin Produced by Vibrio vulnificus CDC B3547, a Clinical Isolate in Biotype 2 (Serovar E)

Vibrio vulnificus biotype 2, a primary eel pathogen, is also an opportunistic pathogen for humans. The strains in this biotype secrete a cytolysin into the culture medium. The cytolysin from the strain CDC B3547 (ATCC 33817), which was originally isolated from a human leg wound, can disrupt various kinds of eukaryotic cells including erythrocytes and mast cells, and artificial vesicles, liposomes. The cytolysin is a 50 kDa single‐chain protein and is categorized into the pore‐forming toxins. After binding tightly to the cell‐membrane cholesterol in a temperature‐independent manner, the toxin molecules assemble each other in a temperature‐dependent manner, forming a small transmembrane pore. When incubated with a metalloprotease from the same species, the cytolysin is converted to the nicked toxin composed of some peptide chains, joined with disulfide bond(s). This nicked toxin is more hydrophilic while maintaining comparable cytolytic activity.

Role of the sulphate assimilation pathway in utilization of glutathione as a sulphur source by Saccharomyces cerevisiae

Mutants unable to grow on medium containing glutathione as a sole source of sulphur (GSH medium) were isolated from Saccharomyces cerevisiae strains carrying met17(deficiency of O‐acetylserine and O‐acetylhomoserine sulphydrylase). They were defective in the high‐affinity glutathione transport system, GSH‐P1. Newly acquired mutations belonged to the same complementation group, gsh11. However, it became apparent that gsh11 conferred the mutant phenotype not by itself but in collaboration with met17. Moreover, mutations conferring the defect in sulphate assimilation made the cell unable to grow on GSH medium in collaboration with gsh11. From this finding, we propose that the sulphate assimilation pathway acts as a sulphur‐recycling system and that this function is especially vital to the cell when the supply of glutathione is limited. Copyright

The Hemagglutinating Action of Vibrio vulnificus Metalloprotease

Vibrio vulnificus protease (VVP), a 45‐kDa zinc metalloprotease, consists of two functional domains: an N‐terminal 35‐kDa polypeptide having endoproteinase activity, and a C‐terminal 10‐kDa polypeptide that mediates the binding of VVP to the erythrocyte membrane. Therefore, VVP, but not its N‐terminal endoproteinase domain alone, has agglutinating activity to rabbit erythrocytes. When a single zinc atom in the catalytic center was substituted by treatment with CuCl2 or NiCl2, proteolytic and hemagglutinating activities were reduced by Ni substitution but not by Cu substitution. Cu‐treated 35‐kDa polypeptide showed sufficient affinity of the catalytic center and weak binding ability to the erythrocyte membrane, but the Ni‐treated polypeptide did not. These results suggest that the binding of endoproteinase domain to membrane is also necessary for hemagglutination.

Purification and characterization of 2-ethoxyphenol-induced cytochrome P450 from Corynebacterium sp. strain EP1

A soluble cytochrome P450 (P450EP1A) induced by 2-ethoxyphenol was purified to apparent homogeneity from Corynebacterium sp. strain EP1. The P450EP1A showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of about 45 kDa. The CO-reduced difference spectra of P450EP1A had a Soret maximum at 447.6 nm. The substrate difference spectra with 2-ethoxyphenol showed an absorption maximum at 394.0 nm. The purified P450EP1A degraded 2-ethoxyphenol in an assay system composed of spinach ferredoxin-NADP+ oxidoreductase and NADPH. The reaction activity decreased to 1.4% of its original activity by addition of CO. The existence of catechol in the reaction mixture was confirmed after the metabolic reaction, indicating that P450EP1A catalyzes O-dealkylation of 2-ethoxyphenol. In addition to 2-ethoxyphenol, the P450EP1A metabolized 2-methoxyphenol, 1,1,1-trichloroethane, carbon tetrachloride, benzene, and toluene.

Detection and isolation of nanobacteria-like particles from urinary stones: long-withheld data.

Objectives:u2003 To report our experimental results on detection and isolation of nanobacteria‐like particles (NLP) from urinary stone samples.