The C-terminal domain promotes the hemorrhagic damage caused by Vibrio vulnificus metalloprotease.

Vibrio vulnificus, an opportunistic human pathogen, produces a 45-kDa zinc metalloprotease (V. vulnificus protease; VVP) as an important virulence determinant. VVP injected intradermally into the dorsal skin causes the hemorrhagic damage through specific degradation of type IV collage in the vascular basement membrane. The N-terminal 35-kDa polypeptide (VVP-N), the catalytic domain, also evoked the hemorrhagic skin reaction within minutes. However, the hemorrhagic activity of VVP-N was one-third of that of VVP. Besides, the proteolytic activity of VVP-N toward the reconstituted basement membrane or type IV collagen was found to be about 50 % of VVP. VVP-N, like VVP, was quickly inactivated by an equimolar amount of alpha(2)-macroglobulin, a broad-spectrum plasma protease inhibitor. These findings indicate that the C-terminal 10-kDa polypeptide, the substrate-binding domain mediating the effective binding to protein substrates, functions to augment the hemorrhagic reaction of VVP.

Detection of virulence associated genes in clinical strains of vibrio mimicus.

A total of 42 clinical strains of Vibrio mimicus were examined for the presence of virulence associated genes toxR, toxS, toxT, tcpP, ctx and tcpA by PCR assay. Almost all strains were shown to have the toxR gene, while the toxS gene was found in 27 strains. On the other hand, five strains possessed both toxT and tcpP genes, but others had neither. Only two strains were positive for amplification of the ctx gene, whereas no PCR product with tcpA primers was detected. The results indicate the incomplete copies of virulence cascade in V mimicus strains. The pathogenesis and epidemic potential of this species is also discussed.

Analysis of seawaters for the recovery of culturable Vibrio parahaemolyticus and some other vibrios.

We investigated the recovery of dormant and injured cells along with the normally culturable cells of Vibrio species with special emphasis on V. parahaemolyticus using both selective and non-selective media at moderate (20 C) and standard (37 C) culture temperatures from a bay water environment. Culture temperatures (20 or 37 C) did not affect the recovery of V. parahaemolyticus but did for other vibrios. We observed similar seasonality of V parahaemolyticus as in most other environmental studies. V. parahaemolyticus and other Vibrio species were recovered in higher numbers by a replica plating method compared to most probable number (MPN) and direct TCBS (thiosulfate citrate bile-salt sucrose) agar counts. Even with the replica plating method, however, vibrios number goes down to a minimum level and V. parahaemolyticus was undetectable during the cool temperature period of the year, although total bacterial cells and CFU on nutrient agar (with 2% NaCl) did not vary so much during the study period.

Analysis of Vibrio mimicus clinical strains by arbitrarily primed polymerase chain reaction.

A total of 51 Vibrio mimicus clinical strains from different geographic locations were examined by arbitrarily primed polymerase chain reaction (AP-PCR). The primer VMH-3 divided them into 28 groups, although 18 groups consisted of a single strain at present. All groups had a common 1.0-kb amplification fragment. Most of the groups consisted of strains from same region, although two exceptional groups showed a few amplification fragments including strains from different regions. AP-PCR groups were not consistently associated with serogroups. AP-PCR is thought to be a valuable and easy method for the epidemiological study of V. mimicus.

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.

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.

The ability of Vibrio vulnificus to use a synthetic hydrophilic heme compound, Fe-TPPS, as a single iron source.

Vibrio vulnificus, an opportunistic human pathogen, can obtain iron from a variety of heme proteins. This process involves the digestion of heme proteins by an exoprotease to liberate protoheme (iron-protoporphyrin IX). In the present study, we tested whether this pathogen also uses a synthetic heme compound, Fe-alpha,beta,gamma,delta-tetraphenylporphine tetrasulfonic acid (Fe-TPPS), as an iron source. When inoculated into a medium containing Fe-TPPS, V. vulnificus L-180 multiplication was seen to be dependent on the concentration of the synthetic heme compound; a mutant lacking the ability to utilize protoheme did not multiply. Cells of the strain grown under the iron-restricted condition showed time-dependent uptake of Fe-TPPS. The ability to use either protoheme or Fe-TPPS was significantly reduced by the addition of an excess amount of free TPPS or Cu-TPPS. The data suggest that, V. vulnificus may assimilate Fe-TPPS, at least partially, through the same system as that for protoheme.

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.

Glutathione transport systems of the budding yeast Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae was shown to have two kinetically distinguishable glutathione transport systems. While one with high affinity (GSH-P1; KT = 0.045 mM) was regulated, the other with low affinity (GSH-P2; KT > 2 mM) was not. GSH-P1 was highly specific to glutathione, and its activity was quickly lost by suspending the cells in buffer solutions. This activity loss was not observed if glucose-containing buffer was used. In addition, rho-isolates had only about one half of the glutathione transport activity of the original (rho+) strain. Therefore, it is concluded that GSH-P1 is an ATP-driven transport system. Strong and moderate inhibition of GSH-P1 by protonophores and ionophores, respectively, are attributed to competition for ATP between GSH-P1 and proton- and cation-pumps, respectively.

Characterization of the hemorrhagic reaction caused by Vibrio vulnificus metalloprotease, a member of the thermolysin family.

Vibrio vulnificus is an opportunistic human pathogen causing wound infections and septicemia, characterized by hemorrhagic and edematous damage to the skin. This human pathogen secretes a metalloprotease (V. vulnificus protease [VVP]) as an important virulence determinant. When several bacterial metalloproteases including VVP were injected intradermally into dorsal skin, VVP showed the greatest hemorrhagic activity. The level of the in vivo hemorrhagic activity of the bacterial metalloproteases was significantly correlated with that of the in vitro proteolytic activity for the reconstituted basement membrane gel. Of two major basement membrane components (laminin and type IV collagen), only type IV collagen was easily digested by VVP. Additionally, the immunoglobulin G antibody against type IV collagen, but not against laminin, showed sufficient protection against the hemorrhagic reaction caused by VVP. Capillary vessels are known to be stabilized by binding of the basal surface of vascular endothelial cells to the basement membrane. Therefore, specific degradation of type IV collagen may cause destruction of the basement membrane, breakdown of capillary vessels, and leakage of blood components including erythrocytes.

Preparation and characterization of everted membrane vesicles from cells of Staphylococcus aureus.

We developed a method for the preparation of everted membrane vesicles from cells of Staphylococcus aureus. The cells were first treated with ampicillin to weaken the peptidoglycan layer, then the cells were passed through a French press cell. The resulting vesicles were roughly 0.1 microm in diameter, judging from electron microscopic observations. We detected fairly high membrane-bound ATPase activity in the membrane vesicles. We observed respiratory-driven quenching of quinacrine fluorescence, which indicates that inward H+ transport took place. These results indicate that the vesicles are everted. We characterized the membrane-bound ATPase. We also detected Na+/H+ antiport, erythromycin/H+ antiport and chloramphenicol/H+ antiport activities in the membranes of S. aureus.

Detection of genes encoding cholera toxin (CT), zonula occludens toxin (ZOT), accessory cholera enterotoxin (ACE) and heat-stable enterotoxin (ST) in Vibrio mimicus clinical strains.

A total of 51 clinical strains of Vibrio mimicus were searched for the presence of virulence-associated genes, like ctx, zot or ace genes which locate in "cholera virulence cassette," and the st gene by polymerase chain reaction. Moreover, the pathological potential of each clinical strain was also examined by rabbit ileal loop (RIL). Three strains showed to have the ctx gene, of which only one strain was zot gene-positive. Meanwhile, one other strain was zot+ but ctx-. All of these four strains were found to have the ace gene and to belong to serogroup O115. Nine strains showed to carry the st gene. However, none of these ST-gene-positive strains was indicated to contain the genes located in the "cholera virulence cassette." It is of interest to note that all of the RIL-positive and/or virulence gene-positive strains were restricted to three serogroups, O20, O41 and O115. These results suggest a significant association between O antigens and enterotoxic activities in V. mimicus clinical strains, and clearly demonstrate multifactorial virulence potentials of this human pathogen.

Functional domains of a zinc metalloprotease from Vibrio vulnificus.

Vibrio vulnificus, an opportunistic human pathogen causing wound infection and septicemia, secretes a 45-kDa metalloprotease (V. vulnificus protease; VVP). A plasmid which carries the entire vvp gene subcloned into pBluescriptIIKS+ was transformed into Escherichia coli DH5alpha for overproduction of the protease. The 45-kDa recombinant protease (rVVP) was isolated from the periplasmic fraction of the transformant by ammonium sulfate precipitation followed by column chromatography on phenyl Sepharose. Biochemical characterization of the isolated rVVP showed that the recombinant protease was identical to that produced by V. vulnificus. When rVVP was incubated at 37 degrees C, a 35-kDa fragment was generated through autoproteolytic removal of the C-terminal peptide. This 35-kDa fragment (rVVP-N) was found to have sufficient proteolytic activity toward oligopeptides and soluble proteins but had markedly reduced activity toward insoluble proteins. Lineweaver-Burk plot analysis indicated increased Km values of rVVP-N for all of the protein substrates. rVVP, but not rVVP-N, was shown to agglutinate rabbit erythrocytes, bind to the erythrocyte ghosts, and digest the ghost membrane proteins. These results strongly suggest that rVVP (and VVP) consists of at least two functional domains: an N-terminal 35-kDa polypeptide mediating proteolysis and a C-terminal 10-kDa polypeptide which may be essential for efficient attachment to protein substrates and erythrocyte membranes.

Some properties of nicked Vibrio vulnificus hemolysin.

Vibrio vulnificus, an opportunistic human pathogen, secretes the 50 kDa single-chain hemolysin. When incubated with an exocellular protease from this vibrio, the 50 kDa hemolysin was cleaved in some peptides joined with the disulfide bond(s); the 40 kDa fragment and the small fragment(s) undetectable in SDS-PAGE. The nicked hemolysin induced comparable hemolysis through the same process as that of the intact toxin. However, the nicked hemolysin was found to be more stable against inactivation due to autoaggregation, so that it formed a larger precipitation zone in the single radial immunodiffusion test using the antiserum against the intact hemolysin. These results suggest that V. vulnificus hemolysin is modified to be a more hydrophilic protein by nicking, while it is not accompanied by loss of the hemolytic activity.

Hemagglutination is a novel biological function of lipopolysaccharide (LPS), as seen with the Vibrio cholerae O139 LPS.

It has been generally thought that the polysaccharide moiety of lipopolysaccharide (LPS) maintains only serological specificity, while the lipid A portion determines various biological functions. However, we found that hemagglutination was a common function of the polysaccharide moiety of LPSs from important human enteropathogenic bacteria. Of the LPSs examined, Vibrio cholerae O139 LPS showed the highest hemagglutinating activity. Glycoproteins, such as mucin and fetuin, showed efficient inhibition of the hemagglutinating ability. Since cell-mediated hemagglutination is known to be correlated with bacterial adherence, hemagglutination induced by the polysaccharide moiety is interpreted to indicate that cell-surface LPS is a potential adhesin.

Vibrio mimicus attaches to the intestinal mucosa by outer membrane hemagglutinins specific to polypeptide moieties of glycoproteins.

Vibrio mimicus is the closest organism to Vibrio cholerae. V. mimicus E-33, which is a highly adhesive and enteropathogenic strain, is known to produce three types of hemagglutinins (HAs), i.e., a 31-kDa exocellular metalloprotease (Vm-HA/protease), lipopolysaccharide (Vm-LPSHA), and a 39-kDa major outer membrane protein (Vm-OMPHA). Hemagglutination induced by Vm-LPSHA and Vm-OMPHA was inhibited by glycoproteins, including mucin, fetuin, and asialofetuin, but not by monosaccharides, disaccharides, or N-acetylated saccharides. The inhibitory potential of each glycoprotein for Vm-OMPHA was greatly augmented by treatment with a glycolytic enzyme such as beta-D-galactosidase or beta-D-glucosidase, while pronase treatment achieved complete abolition of the inhibitory potential. The inhibitory ability of the glycoproteins for Vm-LPSHA was also abolished by pronase treatment; however, glycolytic enzyme treatment showed no effect. Hence, the polypeptide portion of glycoproteins may directly associate with Vm-OMPHA and Vm-LPSHA, but the sugar moiety may act as a barrier to interaction with Vm-OMPHA. The glycoproteins as well as Fab antibodies against Vm-OMPHA and Vm-LPSHA eliminated the ability of E-33 cells to agglutinate rabbit erythrocytes and to attach to rabbit intestinal mucosa. Additionally, expression of the hemagglutinating ability by the bacterial cells was accompanied by efficient bacterial adherence to the intestinal mucosa. Finally, the hemagglutinating activity of Vm-OMPHA was markedly increased by incubation with Vm-HA/protease. These results indicate that all three HAs may have significant roles in the glycoprotein-mediated intestinal adherence of V. mimicus E-33.

Purification and characterization of a hemolysin produced by Vibrio mimicus.

Vibrio mimicus is a causative agent of human gastroenteritis. This pathogen secretes a pore-forming toxin, V. mimicus hemolysin (VMH), which causes hemolysis by three sequential steps: binding to an erythrocyte membrane, formation of a transmembrane pore, and disruption of the cell membrane. VMH with a molecular mass of 63 kDa was purified by ammonium sulfate precipitation and column chromatography with phenyl Sepharose HP and Superose 6 HR. The hemolytic reaction induced by VMH continued up to disruption of all erythrocytes in the assay system. Moreover, VMH that bound preliminarily to erythrocyte ghosts showed a sufficient ability to attack intact erythrocytes. These results suggest reversible binding of the toxin molecule to the membrane. The final cell-disrupting stage was effectively inhibited by various divalent cations. Additionally, some cations, such as Zn2+ and Cu2+, blocked the pore-forming stage at high concentrations. Although VMH could disrupt all kinds of mammalian erythrocytes tested, those from horses were most sensitive to the hemolysin. Horse erythrocytes were found to have the most toxin-binding sites and to be hemolyzed by the least amount of membrane-bound toxin molecules, suggesting that toxin binding to and pore formation on erythrocytes are more effective in horses than in other mammals. Purified VMH induced fluid accumulation in a ligated rabbit ileal loop in a dose-dependent manner. In addition, the antibody against the hemolysin obviously reduced enteropathogenicity of living V. mimicus cells. These findings clearly demonstrate that VMH is probably involved in the virulence of this human pathogen.

Characterization of a mutant of Vibrio vulnificus for heme utilization.

Vibrio vulnificus, an opportunistic human pathogen, can obtain iron from a variety of heme proteins. This process involves the digestion of heme proteins by an exoprotease to liberate protoheme (iron-protoporphyrin IX). In the present study, we isolated and characterized a mutant for protoheme utilization. One mutant isolated by treatment with a chemical mutagen was shown to be unable to use either protoheme or heme proteins, but multiplied in a medium supplemented with an iron siderophore, such as iron-vulnibactin. Like a wild-type strain, the mutant sensed iron depletion, so that the 74- and 79-kDa outer membrane proteins were expressed under iron-regulated conditions. Both the parent and mutant strains secreted hemolysin independent of the iron concentration of the medium. Whole cells of either of the strains were equally capable of binding of hematin. Taken together, the data suggest that the mutant may have a mutation in a gene encoding an inner membrane or a periplasmic protein which transports protoheme or iron dissociated from protoporphyrin IX into the cell.

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

The urine in the mouse bladder was found to be acidic, ranging from pH 5.3 to 5.5 in the daytime and pH 6.0 to 6.3 at night. Administration of bafilomycin A1 or concanamycin A, specific inhibitors of vacuolar-type H(+)-ATPase, into bladder lumen caused neutralization of urinary pH at least for 36 h, whereas inhibitors of mitochondrial ATP synthase (F-type H(+)-ATPase) or P-type H(+)-ATPases did not. Bafilomycin A1-sensitive proton secretion from isolated inside-out bladder was also observed. Immuno-electron microscopy with antibodies against vacuolar H(+)-ATPase revealed that vacuolar-type H(+)-ATPase is rich in luminal plasma membrane and endosomes of superficial cells of the bladder epithelium. These results indicate that vacuolar-type H(+)-ATPases present in luminal plasma membrane of the superficial epithelial cells secrete protons so as to acidify the urine in mouse bladder.

Analysis of the structural gene encoding a hemolysin in Vibrio mimicus.

An environmental isolate of V. mimicus, strain E-33, has been reported to produce and secrete a hemolysin of 63 kDa. The hemolysin is enterotoxic in test animals. The nucleotide sequence of the structural gene of the hemolysin was determined. We found a 2,232 bp open reading frame, which codes a peptide of 744 amino acids, with a calculated molecular weight of 83,903 Da. The sequence for the structural gene was closely related to the V. cholerae el tor hlyA gene, coding an exocellular hemolysin. The amino terminal amino-acid sequence of the 63 kDa hemolysin, purified from V. mimicus, was determined by the Edman degradation method and found to be NH2-S-V-S-A-N-N-V-T-N-N-N-E-T. This sequence is identified from S-152 to T-164 predicted from the nucleotide sequence. So, it seems that the mature hemolysin in V. mimicus is processed upon deleting the first 151 amino acids, and the molecular mass is 65,972 Da. Analyzing the deduced amino-acid sequence, we also found a potential signal sequence of 24 amino acids at the amino terminal. Our results suggest that, like V. cholerae hemolysin, two-step processing also exists in V. Mimicus hemolysin.