Sequence variation in the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus.

Our previous molecular epidemiologic study with gene probes (H. Shirai, H. Ito, T. Hirayama, Y. Nakamoto, N. Nakabayashi, K. Kumagai, Y. Takeda, and M. Nishibuchi, Infect. Immun. 58:3568-3573, 1990) demonstrated that the gene (trh) encoding a thermostable direct hemolysin-related hemolysin was strongly associated with clinical strains of Vibrio parahaemolyticus. Strain-to-strain variation in the intensities of the hybridization signals observed in the above study also suggested that the trh genes in different strains may have significantly divergent nucleotide sequences. To assess the public health significance of the rare environmental strains which exhibited very weak hybridization signals with the trh gene-specific DNA probe, the trh-like sequence was cloned from one of the environmental strains and the nucleotide sequence was determined in this study. A hemolysin gene (trh2) which was 84% homologous to the trh gene (newly named trh1) and 54.8 to 68.8% homologous to the genes (tdh) encoding thermostable direct hemolysins was detected in the cloned sequence. The trh2 gene product showed a profile of hemolytic activities against various animal erythrocytes different from that of the trh1 gene product. The trh2 gene product was antigenically related (partially identical) to the trh1 and tdh gene products. DNA colony blot and Southern blot hybridization analyses with trh1- and trh2-specific DNA probes showed that the trh1 probe-positive strains exhibiting hybridization signals with varying intensities could be clustered into trh1 and trh2 subgroups. In addition, hybridization analysis with oligonucleotide probes demonstrated significant strain-to-strain variation in the trh1 and trh2 gene sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence variation in the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus.

Our previous molecular epidemiologic study with gene probes (H. Shirai, H. Ito, T. Hirayama, Y. Nakamoto, N. Nakabayashi, K. Kumagai, Y. Takeda, and M. Nishibuchi, Infect. Immun. 58:3568-3573, 1990) demonstrated that the gene (trh) encoding a thermostable direct hemolysin-related hemolysin was strongly associated with clinical strains of Vibrio parahaemolyticus. Strain-to-strain variation in the intensities of the hybridization signals observed in the above study also suggested that the trh genes in different strains may have significantly divergent nucleotide sequences. To assess the public health significance of the rare environmental strains which exhibited very weak hybridization signals with the trh gene-specific DNA probe, the trh-like sequence was cloned from one of the environmental strains and the nucleotide sequence was determined in this study. A hemolysin gene (trh2) which was 84% homologous to the trh gene (newly named trh1) and 54.8 to 68.8% homologous to the genes (tdh) encoding thermostable direct hemolysins was detected in the cloned sequence. The trh2 gene product showed a profile of hemolytic activities against various animal erythrocytes different from that of the trh1 gene product. The trh2 gene product was antigenically related (partially identical) to the trh1 and tdh gene products. DNA colony blot and Southern blot hybridization analyses with trh1- and trh2-specific DNA probes showed that the trh1 probe-positive strains exhibiting hybridization signals with varying intensities could be clustered into trh1 and trh2 subgroups. In addition, hybridization analysis with oligonucleotide probes demonstrated significant strain-to-strain variation in the trh1 and trh2 gene sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid detection and enumeration of trh-carrying Vibrio parahaemolyticus with the alkaline phosphatase-labelled oligonucleotide probe

An alkaline phosphatase (AP)-labelled oligonucleotide probe was developed to detect and enumerate trh(+)Vibrio parahaemolyticus in seafood. The probe was evaluated using 40 isolates of V. parahaemolyticus, 45 isolates of other vibrios and 55 non-vibrio isolates. The probe reacted specifically with V. parahaemolyticus possessing either the trh1 or trh2 variant of the trh gene and was found to be 100% specific for trh(+)V. parahaemolyticus. Using the trh probe, V. parahaemolyticus carrying trh gene was targeted in 34 seafood samples by direct plating and colony hybridization procedure. The trh(+)V. parahaemolyticus could be detected in five of 34 (14.7%) samples and the levels ranged from 5.0 x 10(2) to 3.4 x 10(3) cfu g(-1). Colonies of trh(+)V.parahaemolyticus were isolated from the five positive samples. Forty seafood samples were analysed for trh(+)V. parahaemolyticus by colony hybridization following enrichment in alkaline peptone water. 16 samples (40%) were positive for trh gene and trh(+)V. parahaemolyticus was isolated from 15 samples (37.5%). To assess the sensitivity of the trh probe, seafood homogenates spiked with known concentrations of trh-positive V. parahaemolyticus were plated and hybridized. Counts obtained using the probe were similar to those of inocula. The results suggest that the AP-labelled trh probe is useful for the detection and enumeration of trh(+)V. parahaemolyticus in seafood.     

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.     

Molecular characterization of thermostable direct haemolysin-related haemolysin (TRH)-positive Vibrio parahaemolyticus from oysters in Mangalore, India

Pathogenic Vibrio parahaemolyticus strains producing either or both of a thermostable direct haemolysin (TDH) and a TDH-related haemolysin (TRH) encoded by tdh and trh genes, respectively, are isolated at a low rate from the environment. However, recently we observed that a considerable percentage of APW (alkaline peptone water) enrichment broths of oysters collected off Mangalore India, were trh(+), rather than tdh(+) by PCR. In order to further investigate the prevalence and genetic diversity of trh bearing V. parahaemolyticus in our coast, we attempted to isolate and characterize trh(+)V. parahaemolyticus from oysters. A total of 27 trh(+) strains were isolated during the period between March 2002 and February 2004, of which nine were also tdh(+). All the trh(+) isolates were positive for urease phenotype. The isolates belonged to diverse phenotypes. In order to explore the possible presence of heterogeneity in the trh gene region among trh(+)V. parahaemolyticus, a 1.5 kb region around trh gene was PCR amplified and restriction digested using selected restriction enzymes. The whole genome comparison of strains was performed by randomly amplified polymorphic DNA PCR (RAPD PCR). The PCR-RFLP results revealed fairly well conserved nature of the trh gene region studied in different serotypes. Though 11 strains were positive by PCR for a genomic fragment that has been reported to be amplified in pandemic strains, all strains were negative by group-specific PCR (GS-PCR), orf8 PCR and showed a different RAPD pattern compared with pandemic strains. The results suggest that genetically diverse V. parahaemolyticus carrying virulence genes are associated with the aquatic environment in this region.     

Gene involved in transcriptional activation of the hrp regulatory gene hrpG in Xanthomonas oryzae pv. oryzae

A novel regulatory gene, trh, which is involved in hrp gene expression, is identified in the plant pathogen Xanthomonas oryzae pv. oryzae. In the trh mutant, expression of HrpG, which is a key regulator for hrp gene expression, is reduced both under the in vitro hrp-inducing condition and in planta.     

Soft-agar-coated filter method for early detection of viable and thermostable direct hemolysin (TDH)- or TDH-related hemolysin-producing Vibrio parahaemolyticus in seafood

A novel method for detecting viable and thermostable direct hemolysin (TDH)-producing or TDH-related hemolysin (TRH)-producing Vibrio parahaemolyticus in seafood was developed. The method involved (i) enrichment culture, selective for viable, motile cells penetrating a soft-agar-coated filter paper, and (ii) a multiplex PCR assay targeting both the TDH gene (tdh) and TRH gene (trh) following DNase pretreatment on the test culture to eradicate any incidental DNAs that might have been released from dead cells of tdh- or trh-positive (tdh+ trh+) strains and penetrated the agar-coated filter. A set of preliminary laboratory tests performed on 190 ml of enrichment culture that had been inoculated simultaneously with ca. 100 viable cells of a strain of tdh+ trh+ V. parahaemolyticus and dense populations of a viable strain of tdh- and trh-negative V. parahaemolyticus or Vibrio alginolyticus indicated that the method detected the presence of viable tdh+ trh+ strains. Another set of preliminary tests on 190 ml of enrichment culture that had been initially inoculated with a large number of dead cells of the tdh+ trh+ strain together with dense populations of the tdh- and trh-negative strains confirmed that the method did not yield any false-positive results. Subsequent quasi-field tests using various seafood samples (ca. 20 g), each of which was experimentally contaminated with either or both hemolysin-producing strains at an initial density of ca. 5 to 10 viable cells per gram, demonstrated that contamination could be detected within 2 working days.     

Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh.

Vibrio parahaemolyticus is an important human pathogen which can cause gastroenteritis when consumed in raw or partially-cooked seafood. A multiplex PCR amplification-based detection of total and virulent strains of V. parahaemolyticus was developed by targeting thermolabile hemolysin encoded by tl, thermostable direct hemolysin encoded by tdh, and thermostable direct hemolysin-related trh genes. Following optimization using oligonucleotide primers targeting tl, tdh and trh genes, the multiplex PCR was applied to V. parahaemolyticus from 27 clinical, 43 seafood, 15 environmental, 7 strains obtained from various laboratories and 19 from oyster plants. All 111 V. parahaemolyticus isolates showed PCR amplification of the tl gene; however, only 60 isolates showed amplification of tdh, and 43 isolates showed amplification of the trh gene. Also, 18 strains showed amplification of the tdh gene, but these strains did not show amplification of the trh gene. However, one strain exhibited amplification for the trh but not the tdh gene, suggesting both genes need to be targeted in a PCR amplification reaction to detect all hemolysin-producing strains of this pathogen. The multiplex PCR approach was successfully used to detect various strains of V parahaemolyticus in seeded oyster tissue homogenate. Sensitivity of detection for all three target gene segments was at least between 10(1)-10(2) cfu per 10 g of alkaline peptone water enriched seeded oyster tissue homogenate. This high level of sensitivity of detection of this pathogen within 8 h of pre-enrichment is well within the action level (10(4) cfu per 1 g of shell stock) suggested by the National Seafood Sanitation Program guideline. Compared to conventional microbiological culture methods, this multiplex PCR approach is rapid and reliable for accomplishing a comprehensive detection of V. parahaemolyticus in shellfish.     

Analysis of the tdh gene cloned from a tdh gene- and trh gene-positive strain of Vibrio parahaemolyticus

A variant of the gene (tdh) encoding thermostable direct hemolysin (TDH) was cloned from the chromosome of Vibrio parahaemolyticus AQ3860, which gave positive results in the hybridization tests with the tdh gene probe and the trh (tdh-related hemolysin) gene probe and showed a low level of reaction in an enzyme-linked immunosorbent assay for TDH. Nucleotide sequence analysis of the cloned gene (tdh5) provided no evidence that tdh5 is evolutionally closer to the trh gene than the other tdh genes. The tdh5 gene was flanked by 40 base-pair sequences constituting perfect inverted repeats, which may suggest association of the tdh5 gene with insertion sequence-like structure. These results suggest that the tdh5 gene and the trh gene were not originally produced by gene duplication in AQ3860 but rather that one of the two genes moved into AQ3860 from an external source.     

基于分子马达生物传感器技术的副溶血性弧菌分子分型方法的初步研究

[目的]建立基于分子马达技术的简便快速的分子分型方法,对携带和非携带毒力基因的副溶血性弧菌进行快速分类.[方法]以F0F1-ATPase为核心构建分子马达,以副溶血性弧菌毒力基因tdh、trh和种特异性基因tlh、toxR为靶基因设计4个探针.通过生物素-亲和素系统将探针与分子马达连接构建F0F1-ATPase分子马达生物传感器,对10株副溶血性弧菌分离株进行分类,并与PCR-电泳-凝胶成像结果进行比较;同时对生物传感器的检测灵敏度和特异性进行研究.[结果]10株试验菌株中10株tdh阳性,0株trh阳性,而10株菌都携带tlh和toxR,与PCR-电泳-凝胶成像结果一致;分子马达生物传感器的最低检测限为1 pg/反应体系,且能够对副溶血性弧菌特异性识别,PCR-电泳-凝胶成像方法的最低检测限为10 pg/PCR反应体系.[结论]建立了基于分子马达的分子分型方法,能够对副溶血性弧菌的致病性进行快速诊断,检测灵敏度比PCR-电泳-凝胶成像方法高了10倍,而且特异性非常高.该方法简便、快速、省时、省力,适用于地方疾控部门和口岸检疫部门的基层实验室开展副溶血性弧菌监测和流行病学溯源工作.     

TRH1 encodes a potassium transporter required for tip growth in Arabidopsis root hairs

Root hair initiation involves the formation of a bulge at the basal end of the trichoblast by localized diffuse growth. Tip growth occurs subsequently at this initiation site and is accompanied by the establishment of a polarized cytoplasmic organization. Arabidopsis plants homozygous for a complete loss-of-function tiny root hair 1 (trh1) mutation were generated by means of the T-DNA-tagging method. Trichoblasts of trh1 plants form initiation sites but fail to undergo tip growth. A predicted primary structure of TRH1 indicates that it belongs to the AtKT/AtKUP/HAK K(+) transporter family. The proposed function of TRH1 as a K(+) transporter was confirmed in (86)Rb uptake experiments, which demonstrated that trh1 plants are partially impaired in K(+) transport. In line with these results, TRH1 was able to complement the trk1 potassium transporter mutant of Saccharomyces, which is defective in high-affinity K(+) uptake. Surprisingly, the trh1 phenotype was not restored when mutant seedlings were grown at high external potassium concentrations. These data demonstrate that TRH1 mediates K(+) transport in Arabidopsis roots and is responsible for specific K(+) translocation, which is essential for root hair elongation.     

Construction and characterization of an isogenic mutant of Vibrio parahaemolyticus having a deletion in the thermostable direct hemolysin-related hemolysin gene (trh)

A mutant, Vp-MX02, having a deletion in the gene (trh) for the thermostable direct hemolysin-related hemolysin (TRH) was constructed by double-crossover gene conversion from a TRH-producing Vibrio parahaemolyticus strain, TH3996. The deleted region was the upstream half of trh. Hemolysis was completely lost, and TRH antigen was undetectable in the mutant. Administration of the mutant into ligated rabbit small intestines elicited partial, but apparent, fluid accumulation. These results suggest that the enterotoxicity of TRH-producing V. parahaemolyticus TH3996 may be attributed to the remaining C-terminal of TRH or, more likely, to an unknown virulence factor(s) in addition to TRH.     

Detection of Vibrio parahaemolyticus in cockle (Anadara granosa) by PCR

This study aimed to determine the occurrence of Vibrio parahaemolyticus in cockles (Anadara granosa) at a harvesting area and to detect the presence of virulent strains carrying the thermostable direct hemolysin (tdh) and TDH-related hemolysin genes (trh) using PCR. Of 100 samples, 62 were positive for the presence of V. parahaemolyticus with an MPN (most probable number) value greater than 3.0 (>1100 MPN per g). The PCR analysis revealed 2 samples to be positive for the tdh gene and 11 to be positive for the trh gene. Hence, these results demonstrate the presence of pathogenic V. parahaemolyticus in cockles harvested in the study area and reveal the potential risk of illness associated with their consumption.     

Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters

Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >10(4) CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh(+) and trh(+) strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.     

Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan

Seawater and organic material (live and/or dead matter deposited on any substratum submersed in seawater) were collected during the cool weather season from a coast of the Seto-Inland Sea, Japan, and analyzed to determine Vibrio parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by the polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples. About 95% of the samples were positive for V. parahaemolyticus (with densities of 3 to >1400 cells per 100 ml water or 10 g organic samples) by the most-probable-number (MPN)-PCR technique with species-specific toxR primers, but only 40% were positive by the conventional MPN-culture technique (with densities ranging from 3 to 240 cells per 100 ml water or 10 g organics). Furthermore, the tdh and trh genes were positive in 55% and 20% of samples, respectively, by the MPN-PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN-culture procedure. The difference in detection between the MPN-culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors.     

Potassium carrier TRH1 is required for auxin transport in Arabidopsis roots

Disruption of the TRH1 potassium transporter impairs root hair development in Arabidopsis, and also affects root gravitropic behaviour. Rescue of these morphological defects by exogenous auxin indicates a link between TRH1 activity and auxin transport. In agreement with this hypothesis, the rate of auxin translocation from shoots to roots and efflux of [3H]IAA in isolated root segments were reduced in the trh1 mutant, but efflux of radiolabelled auxin was accelerated in yeast cells transformed with the TRH1 gene. In roots, Pro(TRH1):GUS expression was localized to the root cap cells which are known to be the sites of gravity perception and are central for the redistribution of auxin fluxes. Consistent with these findings, auxin-dependent DR5:GUS promoter-reporter construct was misexpressed in the trh1 mutant indicating that partial block of auxin transport through the root cap is associated with upstream accumulation of the phytohormone in protoxylem cells. When [K+] in the medium was reduced from 20 to 0.1 mm, wild type roots showed mild agravitropic phenotype and DR5:GUS misexpression in stelar cells. This pattern of response to low external [K+] was also affected by trh1 mutation. We conclude that the TRH1 carrier is an important part of auxin transport system in Arabidopsis roots.