Isolation from a coastal fish of Vibrio hollisae capable of producing a hemolysin similar to the thermostable direct hemolysin of Vibrio parahaemolyticus.

A vibrio isolated from the intestine of a coastal fish was identified as Vibrio hollisae by its biochemical characteristics. The isolate reacted with the gene probe for the thermostable direct hemolysin of Vibrio parahaemolyticus. The hemolysin produced by the isolate from the fish had traits identical to those of the thermostable direct hemolysin-like hemolysin produced by a clinical strain of V. hollisae.     

Analysis of the gene of Vibrio hollisae encoding the hemolysin similar to the thermostable direct hemolysin of Vibrio parahaemolyticus

The gene (designated as Vh-tdh) of Vibrio hollisae 9041 encoding a hemolysin similar to the thermostable direct hemolysin (TDH) of V. parahaemolyticus contained a 567-base-pair open reading frame (ORF), which was 93.3-93.5% homologous to those of the tdh genes of V. parahaemolyticus, V. cholerae non-01, and V. mimicus encoding TDH or similar hemolysins. Comparative analysis of the nucleotide sequence containing the Vh-tdh ORF with published nucleotide and amino acid sequences suggested that the Vh-tdh gene and other tdh genes diverged from a common ancestral gene, that the divergence was closely associated with the evolutionary divergence of V. hollisae from other species of genus Vibrio, and that strain-to-strain variation of the Vh-tdh gene exists in V. hollisae.     

Properties of a hemolysin related to the thermostable direct hemolysin produced by a Kanagawa phenomenon negative, clinical isolate of Vibrio parahaemolyticus

A hemolytic toxin (Vp-TRH) produced by a Kanagawa phenomenon negative, clinical isolate of Vibrio parahaemolyticus was further characterized. The purified Vp-TRH showed various biological activities, such as fluid accumulation in rabbit ileal loops, increase of rabbit skin vascular permeability, and cardiotoxicity on cultured myocardial cells, all of which are essentially similar to the activities found with thermostable direct hemolysin (Vp-TDH), a pathogenic toxin produced by Kanagawa phenomenon positive V. parahaemolyticus. Immunological similarities of Vp-TRH not only to Vp-TDH but also to hemolytic toxins produced by Vibrio hollisae and Vibrio cholerae non-O1, both of which are also enteropathogens closely related to V. parahaemolyticus, were demonstrated. The amino acid composition and sequence of N-terminal amino acids of Vp-TRH were determined. These results suggest that Vp-TRH has biological and immunological characters similar to Vp-TDH, although they are distinct molecules.     

Purification and partial characterization of a Vibrio hollisae hemolysin that relates to the thermostable direct hemolysin of Vibrio parahaemolyticus

Hemolysin produced by Vibrio hollisae (Vh-rTDH), which is related to the thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus, was studied. Vh-rTDH was purified by successive column chromatographies on diethylaminoethyl-cellulose and an immunoaffinity column coupled with anti Vp-TDH immunoglobulin. The purified toxin was homogeneous, as demonstrated by conventional and sodium dodecyl sulfate--polyacrylamide gel electrophoresis (PAGE). The molecular weight of Vh-rTDH was slightly smaller than that of Vp-TDH, as determined by sodium dodecyl sulfate--slab gel electrophoresis. Conventional PAGE also showed a difference between Vh-rTDH and Vp-TDH. Vp-TDH and Vh-rTDH showed different lytic activities on erythrocytes from various animals, in particular chicken, sheep, and calf. The hemolytic activity of Vh-rTDH was heat labile when heated at 70 degrees C for 10 min, unlike Vp-TDH. Immunological cross-reactivity between Vh-rTDH and Vp-TDH was demonstrated by both the Ouchterlony test and neutralization test.     

Comparative analysis of the hemolysin genes of Vibrio cholerae non-01, V. mimicus, and V. hollisae that are similar to the tdh gene of V. parahaemolyticus

The genes encoding the hemolysins similar to the thermostable direct hemolysin (tdh gene) of Vibrio parahaemolyticus were cloned from chromosomes of V. mimicus and V. hollisae. These cloned hemolysin genes and previously cloned tdh genes of V. parahaemolyticus and V. cholerae non-01 were compared by physical mapping and by hybridization with oligodeoxyribonucleotide probes. The nucleotide sequences in the coding regions of all the cloned hemolysin genes were very homologous and had only minor variations but the sequences flanking the homolysin genes were dissimilar, indicating that the hemolysin genes have a common ancestor and suggesting that they may have been transferred between Vibrio species as a descrete genetic unit.     

Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-O1 strains

Of 15 Vibrio cholerae non-O1 strains, 2 produced a hemolysin termed NAG-rTDH, which is very similar to the thermostable direct hemolysin of V. parahaemolyticus. These two strains contained DNA sequences which are homologous to a DNA probe for the V. parahaemolyticus thermostable direct hemolysin gene. A probe-positive 9-kilobase HindIII fragment was cloned from a plasmid of a V. cholerae non-O1 strain into plasmid pBR322, and the resulting Escherichia coli clones produced intracellular NAG-rTDH.     

Homogeneity of a hemolysin(Vh-rTDH) produced by clinical and environmental isolates of Vibrio hollisae

The characteristics of Vh-rTDH, a hemolysin similar to Vp-TDH of Vibrio parahaemolyticus produced by clinical and environmental isolates of Vibrio hollisae, were comparatively studied. All 7 strains of V. hollisae tested were found to produce indistinguishable Vh-rTDH when they were examined by heat-stability test, Western blotting analysis and conventional polyacrylamide slab gel electrophoresis.     

Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-O1 strains.

Of 15 Vibrio cholerae non-O1 strains, 2 produced a hemolysin termed NAG-rTDH, which is very similar to the thermostable direct hemolysin of V. parahaemolyticus. These two strains contained DNA sequences which are homologous to a DNA probe for the V. parahaemolyticus thermostable direct hemolysin gene. A probe-positive 9-kilobase HindIII fragment was cloned from a plasmid of a V. cholerae non-O1 strain into plasmid pBR322, and the resulting Escherichia coli clones produced intracellular NAG-rTDH.     

Comparison of hemolysins of Vibrio cholerae non-O1 and Vibrio hollisae with thermostable direct hemolysin of Vibrio parahaemolyticus

Hemolysin (Vh-rTDH) produced by Vibrio hollisae and hemolysin (NAG-rTDH) produced by Vibrio cholerae non-O1 were characterized and compared with hemolysin (Vp-TDH) produced by Vibrio parahaemolyticus. These three hemolysins are each composed of two subunits and have similar, but not identical, molecular weights. The amino acid compositions of Vp-TDH and NAG-rTDH are similar, but are different from that of Vh-rTDH. The three hemolysins showed similar lethal toxicities to mice. The effects of temperature on hemolysis and the time dependencies of hemolysis by the three hemolysins were similar. The three were concluded to be immunologically related, but not identical, and to have common and also unique antigenic determinants.     

Production by non-O1 Vibrio cholerae of hemolysin related to thermostable direct hemolysin of Vibrio parahaemolyticus

Abstract The production by non-O1 Vibrio cholerae of a hemolysin immunologically related to the thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus was demonstrated by enzyme-linked immunosorbent assay and a double gel diffusion test. Although results by the double gel diffusion test suggested the immunological identities of TDH and the TDH-related hemolysin of non-O1 V. cholerae , conventional polyacrylamide gel disc electrophoresis and immunoelectrophoresis suggested some differences between the two, at least with respect to charge. The TDH-related hemolysin of non-O1 V. cholerae was also shown to differ from the hemolysin of non-O1 V. cholerae reported previously.     

Comparative amino acid sequence analysis of hemolysins produced by Vibrio hollisae and Vibrio parahaemolyticus.

Vibrio hollisae produces a hemolysin (Vh-rTDH) that is related to the thermostable direct hemolysin of Vibrio parahaemolyticus (Vp-TDH). Although both hemolysins are essentially similar biologically and immunologically, they differ markedly in heat stability; Vp-TDH is heat stable, whereas Vh-rTDH is heat labile. To elucidate the relationships between their characteristics and molecular structures, we analyzed the amino acid sequence of Vh-rTDH and compared it with that of Vp-TDH. Vh-rTDH consisted of 165 residues, of which 23 residues, spread over the peptide chain, differed from those of Vp-TDH.     

Amino acid sequence of thermostable direct hemolysin produced by Vibrio parahaemolyticus

The complete amino acid sequence of the subunit of thermostable direct hemolysin, a dimeric protein composed of identical subunits isolated from Vibrio parahaemolyticus, was determined by sequencing BrCN-peptides, their tryptic peptides, and overlaps obtained by Achromobacter protease I digestion. The subunit consists of 165 amino acid residues with the sole disulfide bond between Cys 151 and Cys 161. It is deduced that the biologically active hemolysin is formed by noncovalent association of subunits which are not linked together by disulfide bonds. The primary structure of hemolysin elucidated in the present study is essentially the same as that deduced from the nucleotide sequence of a gene encoding the protein but differs in 9 amino acid residues, suggesting the possibility of the presence of multiple genes for the thermostable direct hemolysin in Vibrio parahaemolyticus.     

Purification of a TDH-related hemolysin produced by a Kanagawa phenomenon-negative clinical isolate of Vibrio parahaemolyticus 06: K46

A hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus 06: K46 was purified by 55% ammonium sulfate fractionation and successive column chromatographies on DEAE-cellulose, hydroxyapatite, Sepharose 4B and Mono Q. The purified hemolysin was physicochemically and immunologically identical with the Vp-TRH (V. parahaemolyticus thermostable direct hemolysin related hemolysin) recently described in V. parahaemolyticus 03: K6 (Honda et al. Infect. Immun. 56: 961-965, 1988). This indicates that V. parahaemolyticus of Kanagawa-negative clinical isolates possessing not only 03: K6 but also different serotypes such as 06: K46 produce Vp-TRH. Production of Vp-TRH by most clinical isolates of Kanagawa-negative V. parahaemolyticus was also demonstrated. These results suggest the importance of Vp-TRH among clinical isolates of Kanagawa-negative V. parahaemolyticus.     

Purification of the thermostable direct hemolysin of Vibrio parahaemolyticus by immunoaffinity column chromatography

A simple method involving immunoaffinity column chromatography to purify the thermostable direct hemolysin of Vibrio parahaemolyticus was developed. The thermostable direct hemolysin purified from the culture supernatant of a strain isolated from the first reported case of V. parahaemolyticus infection in China in 1985 was indistinguishable from the hemolysins purified from strains isolated in Japan.     

Cloning and characterization of a gene encoding a new thermostable hemolysin from Vibrio parahaemolyticus

A new thermostable hemolysin (delta-VPH) gene was cloned from a Kanagawa-negative Vibrio parahaemolyticus strain into vector pBR322 in Escherichia coli K12. The nucleotide and amino acid sequences had no homology with those of the thermostable direct hemolysin (TDH) which causes the Kanagawa phenomenon, and of the thermolabile hemolysin (TLH) of V. parahaemolyticus. The gene was present in all V. parahaemolyticus strains tested and also in one strain of V. damsela.     

Disappearance of glyceraldehyde 3-phosphate dehydrogenase from erythrocyte membrane by hemolysis with thermostable direct hemolysin of Vibrio parahaemolyticus or Vibrio cholerae El Tor hemolysin.

It is believed that the thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus and El Tor hemolysin (ETH) of Vibrio cholerae damage erythrocytes and other cells by acting as pore-forming toxins. In this study, we found that a protein band with a molecular weight of 37,000 daltons specifically disappeared from erythrocyte membrane after hemolysis by TDH and ETH, but not after hypotonic hemolysis. The 37 kDa band was identified as glyceraldehyde 3-phosphate dehydrogenase (G3PD), a glycolytic enzyme, based on N-terminal 14 amino acid sequencing. The role of G3PD in hemolysis is discussed.     

Enzyme-labeled oligonucleotide probes for detection of the genes for thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus.

Alkaline phosphatase conjugated oligonucleotide probes were developed to detect the genes (tdh and trh) coding for the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus. Using dot blot hybridization, probes were tested with 94 clinical isolates of V. parahaemolyticus. Results agreed well with those obtained using radio-labeled recombinant DNA probes for the genes tdh and trh. Specificity and sensitivity of enzyme tdh probes for detection of the trh gene were 100 and 93%, respectively, and those of the trh probes for trh gene detection were 93 and 86%, respectively. The tdh probes also hybridized with tdh-like genes processed by all strains of V. hollisae, and some strains of V. mimicus and V. cholerae non-O1, but neither tdh nor trh probes reacted with other bacterial species isolated from diarrheal stools. However, some V. parahaemolyticus strains that were negative with the enzyme trh probe hybridized weakly with a radio-labeled trh DNA fragment probe at medium stringency, and a few strains that were negative in high stringency conditions with a radio-labeled trh DNA fragment probe hybridized with the enzyme trh probe. This suggests that some strains of V. parahaemolyticus may carry another gene resembling trh.     

Interaction of thermostable direct hemolysin of Vibrio parahaemolyticus with human erythrocytes.

The interaction between thermostable direct hemolysin produced by Vibrio parahaemolyticus WP-1 and human erythrocytes was studied. The lysis of human erythrocytes by the hemolysin was dependent of temperature and no hemolysis occurred at low temperature (0-4 C), but the hemolysin was adsorbed on human erythrocytes even at low temperature. No hemolysis was observed when antihemolysin antiserum was mixed with the hemolysin and human erythrocytes at zero time. On the other hand, lysis of the cells by hemolysin was not completely inhibited when the antiserum was added during the lag time and the inhibitory effect decreased with delay in the time of addition of antiserum. The inhibitory effect of the antiserum decreased with increase in the incubation temperature, increase in the concentration of divalent cations, and decrease in pH. These results suggest that lysis of human erythrocytes by the hemolysin is at least a two-step process consisting of adsorption of the hemolysin to human erythrocytes and the step(s) following adsorption.     

Ca2+independent cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin (TDH) on Intestine 407, a cell line derived from human embryonic intestine

Abstract The effects of Vibrio parahaemolyticus thermostable direct hemolysin on Intestine 407, a cell line derived from the intestine of human embryo, were investigated. The hemolysin was shown to be cytotoxic to Intestine 407. This cytotoxicity is accompanied by the damage of plasma membrane and lysosomes, as well as cellular degeneration in the form of large transparent blebs. Although an increase in cytosolic free Ca²⁺ due to the influx of extracellular Ca²⁺ was observed in cells treated with thermostable direct hemolysin, it was found to be irrelevant to any of the above effects. These results suggest that the effects of thermostable direct hemolysin observed in this study on Intestine 407 are not mediated by Ca²⁺-dependent pathways.