Showing papers by "Sumio Shinoda published in 1997"

Functional domains of a zinc metalloprotease from Vibrio vulnificus.

Abstract: 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.

Purification and characterization of a hemolysin produced by Vibrio mimicus.

Abstract: 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.

Bacterial Metalloprotease as the Toxic Factor in Infection

Abstract: Metalloproteases, in which zinc metal ion is essential for catalytic activity, are produced by various human pathogenic bacteria. These metalloproteases have the consensus sequence HEXXH, in which two histidine residues function as the first and second zinc ligands. However, on the basis of the location of the third zinc ligand, they are classified into three families: the thermolysin, serralysin and neurotoxin families. Members of the thermolysin family are synthesized as an inactive precursor with a N-terminal propeptide, and maturation of the precursor is achieved by several processing stages. The metalloprotease in the serralysin family is generated without a conventional propeptide, and its transport is supported by additional secreted proteins.A wide variety of pathological actions of bacterial metalloproteases have been documented. In local bacterial infections, such as keratitis, dermatitis and pneumonia, the metalloprotease functions as a decisive virulence determinant. The protease gener...

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

Abstract: 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.

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

Abstract: 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 identical 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.

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

Abstract: 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.

Cloning and sequencing of the Vibrio parahaemolyticus fur gene

Abstract: A ferric uptake regulatory gene (fur) was cloned from Vibrio parahaemolyticus WP1 by a polymerase chain reaction-based technique followed by functional complementation of a fur mutation in Escherichia coli. A sequence analysis showed that, at the amino acid level, the V. parahaemolyticus Fur protein is 81% identical with the Fur protein from E. coli and over 90% identical with those of the Vibrio species.

Characterization of a mutant of Vibrio vulnificus for heme utilization

Abstract: 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.

Enterohemorrhagic Escherichia coli O157: H7 infection

Abstract: The first recognized outbreaks of hemorrhagic colitis occurred in 1982 in the United State and its etiologic agent was identified to be Escherichia coli O157 : H7, a serotype not previously associated with diseases in humans. In Japan, isolates of the serotype O157 : H7 from contaminated drinking water were first implicated in an outbreak occurred in 1990 in a kindergarten of the Saitama Prefecture, and at least other 12 such outbreaks have been recorded in 1993-1995. In the year 1996, unprecedentedly large outbreaks and many sporadic cases of disease caused by E. coli O157 : H7 occurred in various parts of Japan, affecting more than 9000 people in total (11 deaths). In most cases, however, the ultimate source of the infection could not be traced. Although many different serotypes of E. coli, which are collectively referred to as enterohemorrhagic E. coli (EHEC), were found to cause bloody diarrhea, the serotype O157 : H7 has been recognized worldwide as the pathogen associated most frequently with serious complications known as hemolytic colitis and hemolytic uremic syndrome. E. coli O157 : H7 is characteristic of a low infectious dose, on the order of a few hundred organisms, which contributes to the spread of the infection in outbreak situations. Cattle are considered to be the major reservoir of EHEC including O157 : H7. EHEC strains produce at least two immunologically distinct cytotoxins that closely resemble the Shiga toxin produced by the Shigella dysenteriae type 1 strains. These toxins (called Shiga-like toxins or Vero toxins) appear to be responsible for causing many pathological effects associated with EHEC infections. However, how the toxins move from the intestinal tract lumen to the sites where they damage the kidney remains to be evaluated. Moreover, there are some doubts about the value of antibiotic therapy in such infections because of the observation that some antibiotics can increase the toxin expression in vitro and because of the concern that EHEC which are lysing due to their actions in the gastrointestinal tract lumen may actually release more toxin than do intact bacterial cells. Unique biochemical characteristics of E. coli O157 : H7-it ferments sorbitol very slowly and usually does not make β-glucuronidase- are used to differentiate this strain from other enteric E. coli strains. Alternative methods based on the detection of the toxins themselves by enzyme immunoassay are employed in parallel. This review describes the current understanding of the infectious disease caused by E. coli O157 : H7, with an emphasis on the main diagnostic tests and epidemiology for this serotype and the role of the toxins in pathogenesis.