Nucleotide sequence of the thermostable direct hemolysin gene of Vibrio parahaemolyticus.

The gene encoding the thermostable direct hemolysin of Vibrio parahaemolyticus was characterized. This gene (designated tdh) was subcloned into pBR322 in Escherichia coli, and the functional tdh gene was localized to a 1.3-kilobase HindIII fragment. This fragment was sequenced, and the structural gene was found to encode a mature protein of 165 amino acid residues. The mature protein sequence was preceded by a putative signal peptide sequence of 24 amino acids. A putative tdh promoter, determined by its similarity to concensus sequences, was not functional in E. coli. However, a promoter that was functional in E. coli was shown to exist further upstream by use of a promoter probe plasmid. A 5.7-kilobase SalI fragment containing the structural gene and both potential promoters was cloned into a broad-host-range plasmid and mobilized into a Kanagawa phenomenon-negative V. parahaemolyticus strain. In contrast to E. coli, where the hemolysin was detected only in cell lysates, introduction of the cloned gene into V. parahaemolyticus resulted in the production of extracellular hemolysin.     

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.     

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.     

Production of thermostable direct hemolysin by Vibrio parahaemolyticus enhanced by conjugated bile acids.

The effects of conjugated bile acids, glycocholic acid, and taurocholic acid (TC) on production of thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus were determined by a reversed passive latex agglutination assay against TDH. The amount of TDH excreted in growth medium containing either glycocholic acid or taurocholic acid (5 mM/liter) was, on a per-cell basis, 4- to 16-fold greater than that excreted in medium without the bile acids. The amounts of TDH released from lysed cells grown with the bile acids (5 mM/liter) were 4- to 32-fold greater than those from lysed cells grown without, suggesting that the bile acids enhanced synthesis of TDH within bacterial cells. These data imply that the conjugated bile acids play a key role in the pathogenicity of V. parahaemolyticus.     

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.     

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.     

Structural insights into thermostable direct hemolysin of Vibrio parahaemolyticus using single-particle cryo-EM

Thermostable direct hemolysin (TDH) is a ~19 kDa, hemolytic pore-forming toxin from the gram-negative marine bacterium Vibrio parahaemolyticus, one of the causative agents of seafood-borne acute gastroenteritis and septicemia. Previous studies have established that TDH exists as a tetrameric assembly in physiological state; however, there is limited knowledge regarding the molecular arrangement of its disordered N-terminal region (NTR)—the absence of which has been shown to compromise TDH's hemolytic and cytotoxic abilities. In our current study, we have employed single-particle cryo-electron microscopy to resolve the solution-state structures of wild-type TDH and a TDH construct with deletion of the NTR (NTD), in order to investigate structural aspects of NTR on the overall tetrameric architecture. We observed that both TDH and NTD electron density maps, resolved at global resolutions of 4.5 and 4.2 Å, respectively, showed good correlation in their respective oligomeric architecture. Additionally, we were able to locate extra densities near the pore opening of TDH which might correspond to the disordered NTR. Surprisingly, under cryogenic conditions, we were also able to observe novel supramolecular assemblies of TDH tetramers, which we were able to resolve to 4.3 Å. We further investigated the tetrameric and inter-tetrameric interaction interfaces to elaborate upon the key residues involved in both TDH tetramers and TDH super assemblies. Our current structural study will aid in understanding the mechanistic aspects of this pore-forming toxin and the role of its disordered NTR in membrane interaction.     

The thermostable direct hemolysin of Vibrio parahaemolyticus is a pore-forming toxin.

The hemolytic mechanism of thermostable direct hemolysin (TDH), a possible virulence factor of Vibrio parahaemolyticus, was studied. We demonstrated that TDH acts as a "pore-forming toxin" in temperature-dependent and -independent steps. The first temperature-dependent step requires only about 1-2 min incubation at 37 degrees C and makes a "pore" with a functional diameter of approximately 2 nm. The pore size was deduced from the molecular diameter of the colloidal inhibitory polysaccharides. The formation of the pores on TDH-treated erythrocyte membranes was also demonstrated by electron microscopic examination. The second step, which is a temperature-independent lytic step, causes the erythrocytes to swell owing to a colloidal osmotic influx of water via the "pores" into cells, resulting in erythrocyte lysis (or rupture) owing to increased intracellular pressure.     

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

A hemolysin (designated Vm-rTDH) from Vibrio mimicus (AQ0915-E13) was purified by ammonium sulfate fractionation and successive column chromatography with DEAE-Sephadex A-25, hydroxyapatite, Mono Q, Superose 12 and Phenyl-Superose. The Mr of the subunit was estimated to be about 22,000 by sodium dodecyl sulfate-slab gel electrophoresis. The isoelectric point of Vm-rTDH was approximately pH 4.9. The hemolytic activity of Vm-rTDH was stable upon heating at 100 degrees C for 10 min, similar to that of the thermostable direct hemolysin (Vp-TDH) of V. parahaemolyticus. Vm-rTDH also showed lytic activities similar to those of Vp-TDH. Immunological cross-reactivity between Vp-TDH and Vm-rTDH was demonstrated by the Ouchterlony double-diffusion test. Thus we conclude that V. mimicus produces a newly discovered type of hemolysin (Vm-rTDH) which is similar to Vp-TDH.     

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.     

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.     

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.     

Relationship between heat-induced fibrillogenicity and hemolytic activity of thermostable direct hemolysin and a related hemolysin of Vibrio parahaemolyticus

Microorganisms often use small chemicals or secondary metabolites as informational cues to regulate gene expression. It is hypothesized that microorganisms exploit these signals to gain a competitive advantage. Here, we present examples of pathogens that use this strategy to exclude other microorganisms from the site of infection. An emerging theme is that inhibiting these systems presents a novel approach to antimicrobial therapies.     

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.