Production of monoclonal antibodies against thermostable direct hemolysin of Vibrio parahaemolyticus and application of the antibodies for enzyme-linked immunosorbent assay

A total nine hybridoma cell lines that produced monoclonal antibodies against thermostable direct hemolysin (Vp-TDH), a possible pathogenic toxin, of Kanagawa phenomenon-positive Vibrio parahaemolyticus was isolated and characterized. These monoclonal antibodies (mAbs) were divided into a minimum of five different specificity groups, including mAbs specific to Vp-TDH and common to Vp-TDH and Vp-TRH, a Vp-TDH-related hemolysin produced by Kanagawa phenomenon-negative V. parahaemolyticus. An enzyme-linked immunosorbent assay (ELISA) using mAb-1-D, a mAb specific for Vp-TDH, was developed for specific detection of Vp-TDH. On the other hand, the ELISA using mAb-9-D, and mAb common to both Vp-TDH and Vp-TRH, could be used for detection of both Vp-TDH and Vp-TRH. Thus, by combining these two ELISAs differential detection of Vp-TDH and Vp-TRH can be performed. Hence, the two ELISAs were applied for various strains of V. parahaemolyticus and it was found that most Kanagawa phenomenon-positive and -negative clinical isolates produced Vp-TDH and Vp-TRH, respectively, but all environmental strains, that were Kanagawa phenomenon-negative, produced neither toxin.Supported by a Grant-in-Aid for Scientific Research and a Grant-in-Aid for Developmental Scientific Research from the Ministry of Education, Science, and Culture of Japan     

Demonstration and characterization of simultaneous production of a thermostable direct hemolysin (TDH/I) and a TDH-related hemolysin (TRHx) by a clinically isolated Vibrio parahaemolyticus strain, TH3766.

Simultaneous production of a thermostable direct hemolysin (TDH)-like toxin (TDHx) and a TDH-related hemolysin (TRH)-like toxin (TRHx) by a clinical isolate (strain TH3766) of Kanagawa phenomenon-positive Vibrio parahaemolyticus was demonstrated and characterized. The two hemolysins were differentially purified by column chromatography on hydroxyapatite and immunoaffinity columns. The molecular weight of the two hemolysins were estimated to be 23,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). The purified TDHx was indistinguishable from the previously reported TDH/I (from strain TH012) but was different from the authentic TDH of a Kanagawa phenomenon-positive strain (T4750) physicochemically. The mobility of TRHx in nondenaturing PAGE differed from all the known TDHs and TRHs. The genes (tdhX and trhX) coding for TDHx and TRHx were cloned and sequenced. Homologies of nucleotide sequences of the coding regions between tdhX and tdhA (a gene for the authentic TDH) and between trhX and trh (a gene for the authentic TRH) were 98.1 and 99.1%, respectively, and homology between tdhX and trhX was 68.1%. At the amino acid level, TdhX was completely identical to TDH/I, although two base differences were found in the nucleotide sequences between tdhX and tdh/I. Two amino acid differences were observed between TrhX and Trh. Thus, these findings suggest that the TH3766 strain produces two types of hemolysins simultaneously. This is the first evidence that a strain of V. parahaemolyticus produces two types of toxins of the TDH-TRH family at the same time.     

Purification and partial characterization of a non-O1 Vibrio cholerae hemolysin that cross-reacts with thermostable direct hemolysin of Vibrio parahaemolyticus.

A newly identified hemolysin (NAG-rTDH), which is related to the thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus produced by non-O1 Vibrio cholerae, was studied. NAG-rTDH was purified by successive column chromatographies on DEAE-cellulose and an immunoaffinity column coupled with anti-Vp-TDH immunoglobulin. The molecular weight of NAG-rTDH was estimated as 18,500, similar to that of Vp-TDH, as judged by sodium dodecyl sulfate slab gel electrophoresis, but its charge or molecular shape was different, judging from its electrophoretic mobility. The lytic activities of NAG-rTDH on erythrocytes of most animals were essentially similar to those of Vp-TDH, but that on sheep erythrocytes was different. The hemolytic activity of NAG-rTDH was stable on heating at 100 degrees C for 10 min, as was that of Vp-TDH. Immunological cross-reactivity between NAG-rTDH and Vp-TDH was demonstrated by both the Ouchterlony test and the neutralization test. Thus, we conclude that non-O1 V. cholerae produce a new type of hemolysin that is similar but not identical to the thermostable direct hemolysin of V. parahaemolyticus.     

Detection of the thermostable direct hemolysin gene and related DNA sequences in Vibrio parahaemolyticus and other vibrio species by the DNA colony hybridization test.

A specific gene probe for the Vibrio parahaemolyticus thermostable direct hemolysin gene was constructed and used to examine the presence or absence of the thermostable direct hemolysin gene or related DNA sequences in V. parahaemolyticus and other vibrios by the DNA colony hybridization method. The gene probe consisted of a 406-base-pair, completely internal fragment covering 71% of the structural gene with PstI linkers added to the ends. Six copies of this 415-base-pair PstI fragment were cloned into plasmid pBR322, which yielded large amounts of the probe DNA. One hundred forty-one V. parahaemolyticus strains were tested with the gene probe, and the results were compared with those of phenotypic assays for the thermostable direct hemolysin. All Kanagawa phenomenon-positive strains were gene positive. However, 86% of the strains that exhibited weak Kanagawa phenomenon and 16% of Kanagawa phenomenon-negative strains also reacted with the gene probe. Immunological methods for the detection of the thermostable direct hemolysin (modified Elek test, enzyme-linked immunosorbent assay) showed better correlation with gene probe results. All gene-positive strains produced hemolysin detectable in the enzyme-linked immunosorbent assay, although occasional strains showed weak reaction. The modified Elek test was slightly less sensitive than the enzyme-linked immunosorbent assay. All gene-negative strains were also negative in these immunological assays. One hundred twenty-one strains of Vibrio spp. other than V. parahaemolyticus were tested with the gene probe; only Vibrio hollisae strains reacted with the probe under stringent conditions.     

Molecular epidemiologic evidence for association of thermostable direct hemolysin (TDH) and TDH-related hemolysin of Vibrio parahaemolyticus with gastroenteritis.

The Kanagawa phenomenon induced by the thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus is almost exclusively associated with clinical strains, and TDH has been considered an important virulence factor. However, Kanagawa phenomenon-negative strains isolated from patients with diarrhea have recently been shown to produce TDH-related hemolysin (TRH). We studied the distribution of the tdh gene encoding TDH and the trh gene encoding TRH in vibrios by hybridization analyses. The presence or absence of the tdh gene and the trh gene in 285 strains of V. parahaemolyticus was examined by the DNA colony hybridization test with a tdh gene-specific probe and a newly constructed trh gene-specific probe. For assessment of the importance of TRH, many Kanagawa phenomenon-negative clinical strains (35.4% of all strains) were included. Of 214 clinical strains of V. parahaemolyticus, 112 strains (52.3%) had the tdh gene only, 52 strains (24.3%) had the trh gene only, and 24 strains (11.2%) carried both the tdh and the trh gene. The coexistence of the tdh and trh genes in these 24 strains was confirmed by Southern blot hybridization analysis. Of 71 environmental strains, 5 strains (7.0%) hybridized very weakly with the trh gene probe and none hybridized with the tdh gene probe. These results suggest that TRH as well as TDH is an important virulence factor of V. parahaemolyticus. Among 118 strains of other Vibrio species examined for the trh gene, only 1 strain of Vibrio furnissii gave a very weak hybridization signal. Among 48 representative trh gene-positive strains of V. parahaemolyticus, only 18 strains (37.5%) were found to produce TRH in culture medium when examined by a sensitive enzyme-linked immunosorbent assay method.     

Contribution of the tdh1 gene of Kanagawa phenomenon-positive Vibrio parahaemolyticus to production of extracellular thermostable direct hemolysin.

Kanagawa phenomenon-positive strains of Vibrio parahaemolyticus contain two copies of the tdh gene (tdh 1 and tdh 2) encoding thermostable direct hemolysin (TDH). Previous studies suggested that the tdh 2 gene, but not the tdh 1 gene, was responsible for production of extracellular TDH. In this study, a tdh 2-deficient isogenic mutant of Kanagawa phenomenon-positive strain AQ3815 was constructed by a suicide vector-mediated in vivo recombination method. The intact tdh 1 gene in the mutant contributed little to Kanagawa phenomenon on Wagatsuma agar but produced TDH in broth media, accounting for 0.5-9.4% of total extracellular TDH of AQ3815.     

Cloning of the thermostable direct or Kanagawa phenomenon-associated hemolysin of Vibrio parahaemolyticus

Genes encoding the thermostable direct or Kanagawa phenomenon hemolysin of Vibrio parahaemolyticus were cloned in Escherichia coli. DNA hybridization experiments with the cloned genes showed that none of the five Kanagawa phenomenon-negative environmental isolates tested possessed DNA sequences homologous to the probe.     

Immunological methods for detection of Kanagawa phenomenon of Vibrio parahaemolyticus.

A modified Elek test and an immuno-halo test were developed for detection of the thermostable direct hemolysin produced by Kanagawa phenomenon-positive strains of Vibrio parahaemolyticus. The sensitivity of the modified Elek test was almost the same as that of the Kanagawa phenomenon on Wagatsuma medium, and the results of both reactions correlated very well. The immuno-halo test was a little less sensitive than the modified Elek test. Since the Kanagawa phenomenon on Wagatsuma medium sometimes give false-positive results due to instability of the blood used in the medium, we recommend the immunological techniques described here as reproducible methods for identification of the thermostable direct hemolysin by V. parahaemolyticus.     

Identification of lethal toxin with the thermostable direct hemolysin produced by Vibrio parahaemolyticus, and some physicochemical properties of the purified toxin.

Lethal toxin was purified extensively from the culture filtrate of a Kanagawa phenomenon-positive strain of Vibrio parahaemolyticus. The purified toxin was a protein, and its homogeneity was demonstrated by sodium dodecyl sulfate-polyacrylamide gel disc electrophoresis and analytical ultracentrifugation. It was demonstrated that the thermostable direct hemolysin was identical to the lethal toxin and that it was the main, if not only, lethal toxin in the culture filtrate. The purified toxin had a lethal effect when injected into mice either intravenously or intraperitoneally. Its lethal effect was very rapid, a dose of 5 mug of toxin per mouse killing the animals within 1 min. The lethal activity was inhibited by a ganglioside mixture. Some physicochemical properties of the purified toxin are reported.     

Existence of Two Distinct Hemolysins in Vibrio parahaemolyticus

Two distinct hemolysins were demonstrated in Vibrio parahaemolyticus. A thermostable direct hemolysin purified from V. parahemolyticus WP-1, a Kanagawa phenomenon (KP)-positive strain, is antigenically different from a thermolabile hemolysin produced by V. parahaemolyticus T-3454, a KP-negative strain. The thermostable direct hemolysin was found in KP-positive strains but not in KP-negative strains. On the other hand, the thermolabile hemolysins were found in both KP-positive and -negative strains, although some KP-positive strains did not produce this hemolysin.     

Contribution of the tdh 1 gene of Kanagawa phenomenon-positive Vibrio parahaemolyticus to production of extracellular thermostable direct hemolysin

Kanagawa phenomenon-positive strains of Vibrio parahaemolyticus contain two copies of the tdh gene (tdh1 and tdh2) encoding thermostable direct hemolysin (TDH). Previous studies suggested that the tdh2 gene, but not the tdh1 gene, was responsible for production of extracellular TDH. In this study, a tdh2-deficient isogenic mutant of Kanagawa phenomenon-positive strain AQ3815 was constructed by a suicide vector-mediated in vivo recombination method. The intact tdh1 gene in the mutant contributed little to Kanagawa phenomenon on Wagatsuma agar but produced TDH in broth media, accounting for 0.5–9.4% of total extracellular TDH of AQ3815.     

Identification of proteins secreted via Vibrio parahaemolyticus type III secretion system 1

Vibrio parahaemolyticus, a gram-negative marine bacterium, is an important pathogen causing food-borne gastroenteritis or septicemia. Recent genome sequencing of the RIMD2210633 strain (a Kanagawa phenomenon-positive clinical isolate of serotype O3:K6) revealed that the strain has two sets of gene clusters that encode the type III secretion system (TTSS) apparatus. The first cluster, TTSS1, is located on the large chromosome, and the second, TTSS2, is on the small chromosome. Previously, we reported that TTSS1 is involved in the cytotoxicity of the RIMD2210633 strain against HeLa cells. Here, we analyzed proteins secreted via the TTSS apparatus encoded by TTSS1 by using two-dimensional gel electrophoresis and identified the proteins encoded by genes VP1680, VP1686, and VPA450. To investigate the roles of those secreted proteins, we constructed and analyzed a series of deletion mutants. Flow cytometry analysis using fluorescence-activated cell sorting with fluorescein isothiocyanate-labeled annexin V demonstrated that the TTSS1-dependent cell death was by apoptosis. The cytotoxicity to HeLa cells was related to one of the newly identified secreted proteins encoded by VP1680. Adenylate cyclase fusion protein studies proved that the newly identified secreted proteins were translocated into HeLa cells. Thus, these appear to be the TTSS effector proteins in V. parahaemolyticus.     

Non-O1 Vibrio cholerae hemolysin: purification, partial characterization, and immunological relatedness to El Tor hemolysin.

Hemolysin of a non-O1 Vibrio cholerae strain was purified and characterized. The purified hemolysin gave a single protein band on conventional and sodium dodecyl sulfate-gel electrophoresis. Its molecular weight was estimated as 60,000 by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. It had a pI of 5.7. The purified hemolysin caused increased vascular permeability of rabbit skin and rapid death of mice on intravenous injection and also lysed erythrocytes of various animal species. An Ouchterlony double gel diffusion test using antiserum against the purified hemolysin indicated that the hemolysin from non-O1 V. cholerae was immunologically related, but not identical, to the hemolysin from El Tor V. cholerae. Antiserum against the purified hemolysin neutralized the hemolytic activity of the hemolysins from not only non-O1 but also El Tor V. cholerae.     

Effect of D-tryptophan on hemolysin production in Vibrio parahaemolyticus

Production of the Kanagawa hemolysin by a strain of Vibrio parahaemolyticus isolated from a gastroenteritis patient was found to correlate with the presence in cell lysates of two unidentified compounds, designated X and Y. The two compounds were present in cell lysates of the organism grown in peptone at the optimal pH for hemolysin synthesis but were not present when cell lysates were grown in peptone at a constant pH of 8.0. They were also absent in cells grown in synthetic medium at pH 6.2 without the addition of D-tryptophan, a condition under which hemolysin is not produced. Both X and Y were present intracellularly only from the time that D-tryptophan was added to synthetic medium, a known method of inducing hemolysin synthesis.     

Production of the Kanagawa hemolysin by Vibrio parahaemolyticus in a synthetic medium.

A synthetic medium capable of supporting growth of Vibrio parahaemolyticus is described. Growth yields and generation times were comparable to growth in a complex medium, although Kanagawa hemolysin was undetectable in the synthetic medium. Upon the addition of single amino acids to this synthetic medium, only D-tryptophan induced production of the hemolysin. L-Tryptophan was found to inhibit the action of the D-stereoisomer. The response to D-tryptophan was pH dependent; the greatest hemolysin expression occurred at pH values below 6.5. The addition of 100 micrometers D-tryptophan to early-logarithmic-phase cultures caused an inhibition of growth and of substrate utilization, both of which lasted 7 h. During this time, hemolysin was produced only intracellularly. The hemolysin appeared in the supernatant only when growth recommenced. The hemolysins within the cell and in the supernatant were both inactivated by antiserum raised against the standard Kanagawa hemolysin.     

Pili of a Vibrio parahaemolyticus strain as a possible colonization factor.

Pili of Vibrio parahaemolyticus were purified from a Kanagawa phenomenon-positive strain (Ha7) that belongs to serogroup O2:K3 and is adhesive to rabbit intestine. The organisms treated with the Fab fraction of antipilus antibody failed to adhere to the intestine. Purified pili had the ability to adhere to the intestine, but the pretreatment of the intestine with purified pili did not allow adherence of the organisms to the intestine. These results suggest that pili of this V. parahaemolyticus strain play an important role in colonization.     

Purification and some properties of Aeromonas hydrophila hemolysin

A hemolysin produced by a strain of Aeromonas hydrophila isolated from a patient with diarrhea was purified by acid precipitation and quarternary aminoethyl-Sephadex chromatography. The molecular weight of the hemolysin was estimated at 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at 48,000 by Sephadex G-100 gel filtration. In polyacrylamide gel electrophoresis at pH 4.0 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the hemolysin migrated as a single band, whereas electrophoresis at pH 9.4 and thin-layer isoelectric focusing demonstrated multiple bands. The results may indicate charge isomers of the hemolysin. The purified hemolysin had a hemolytic activity of 134 hemolytic units per microgram of protein on rabbit erythrocytes. It caused fluid accumulation in infant mouse intestines and rabbit ligated ileal loops. Purified hemolysin also elicited cytotoxicity to Vero cells and lethal toxicity to mice. All these biological activities were lost on heating for 5 min at 56 degrees C. These findings support the notion that A. hydrophila hemolysin is a cytotoxic enterotoxin.     

Purification and characterization of a cell-associated hemagglutinin of Vibrio parahaemolyticus.

We found a positive correlation between cell-associated mannose-sensitive hemagglutination and adherence of Vibrio parahaemolyticus to rabbit enterocytes by investigating 35 strains of V. parahaemolyticus for cell-associated hemagglutinin (cHA) and for the ability to adhere to the enterocytes. We purified a mannose-sensitive cHA from a Kanagawa phenomenon-positive clinical strain of V. parahaemolyticus that exhibited a high level of mannose-sensitive hemagglutination and strongly adhered to the enterocytes. The purified cHA is a heat-labile, tetrameric protein consisting of four identical subunits of approximately 26 kDa each. The adherence to rabbit enterocytes was inhibited in a dose-dependent manner by pretreatment of the bacterial cells with D-mannose and with the Fab fraction of immunoglobulin G against the purified cHA. Furthermore, pretreatment of the enterocytes with the purified cHA inhibited the adherence of V. parahaemolyticus. Immunogold electron microscopy revealed that the cHA is located on the bacterial cell surface and is not associated with pili. These results suggest that cHA is involved in the adherence mechanisms of V. parahaemolyticus to the enterocytes and that the receptors for cHA on the enterocyte appear to be a D-mannose-containing compound.     

Comparison of the nucleotide sequences of the genes for the thermostable direct hemolysin and the thermolabile hemolysin from Vibrio parahaemolyticus

The nucleotide sequences of genes encoding the thermostable direct (TSD) hemolysin and the thermolabile (TL) hemolysin of Vibrio parahaemolyticus were determined. From the nucleotide sequence of the TSD hemolysin gene, it was revealed that the preprotein and the mature protein consisted of 189 amino acids and 165 amino acids, and that the molecular weights were 21.1 kDa or 18.5 kDa, respectively. Our data regarding TSD hemolysin were in complete agreement with previously published data. From the nucleotide sequence of the TL hemolysin gene, it was revealed that the preprotein and the mature protein consisted of 418 amino acids and 398 amino acids, and that the molecular weights were 47.5 kDa and 45.3 kDa, respectively. The GC content of the TSD hemolysin gene was 35.6%, while that of the TL hemolysin gene was 47.6% which is almost the same as that of V. parahaemolyticus genome. Maxicell analysis revealed that the molecular weights of the proteins encoded by the TSD hemolysin gene were 22.0 and 19.5 kDa, and that of the protein encoded by the TL hemolysin gene was 45.5 kDa, and that the promoters of these two hemolysin genes of V. parahaemolyticus were functional in Escherichia coli.     

Purification and characterization of a hemolysin produced by Vibrio cholerae biotype El Tor: another toxic substance produced by cholera vibrios.

A thermolabile direct hemolysin from an El Tor cholera vibrio strain has been isolated and partially characterized as a simple protein of ca. 20,000 molecular weight. In addition to its hemolytic activity, the hemolysin is cytotoxic, cardiotoxic, and rapidly lethal. In these respects it resembles the thermostable direct hemolysin/cytotoxin/cardiotoxin/lethal toxin of Vibrio parahaemolyticus and certain other bacterial hemolysins, although there are other significant differences. Because identical diseases are produced by both hemolytic and nonhemolytic cholera vibrios, the El Tor hemolysin may be presumed to be pathogenetically irrelevant. These observations raise the question of "When is a toxic substance also a toxin?"