ToxR represses the synthesis of c-di-GMP in Vibrio parahaemolyticus / 生物工程学报

Vibrio parahaemolyticus, the main pathogen causing seafood related food poisoning worldwide, has strong biofilm formation ability. ToxR is a membrane binding regulatory protein, which has regulatory effect on biofilm formation of V. parahaemolyticus, but the specific mechanism has not been reported. c-di-GMP is an important second messenger in bacteria and is involved in regulating a variety of bacterial behaviors including biofilm formation. In this study, we investigated the regulation of ToxR on c-di-GMP metabolism in V. parahaemolyticus. Intracellular c-di-GMP in the wild type (WT) and toxR mutant (ΔtoxR) strains were extracted by ultrasonication, and the concentrations of c-di-GMP were then determined by enzyme linked immunosorbent assay (ELISA). Three c-di-GMP metabolism-related genes scrA, scrG and vpa0198 were selected as the target genes. Quantitative real-time PCR (q-PCR) was employed to calculate the transcriptional variation of each target gene between WT and ΔtoxR strains. The regulatory DNA region of each target gene was cloned into the pHR309 plasmid harboring a promoterless lacZ gene. The recombinant plasmid was subsequently transferred into WT and ΔtoxR strains to detect the β-galactosidase activity in the cellular extracts. The recombinant lacZ plasmid containing each of the target gene was also transferred into E. coli 100λpir strain harboring the pBAD33 plasmid or the recombinant pBAD33-toxR to test whether ToxR could regulate the expression of the target gene in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and the over-expressed His-ToxR was purified. The electrophoretic mobility shift assay (EMSA) was applied to verify whether His-ToxR directly bound to the target promoter region. ELISA results showed that the intracellular c-di-GMP level significantly enhanced in ΔtoxR strain relative to that in WT strain, suggesting that ToxR inhibited the production of c-di-GMP in V. parahaemolyticus. qPCR results showed that the mRNA levels of scrA, scrG and vpa0198 significantly increased in ΔtoxR strain relative to those in WT strain, suggesting that ToxR repressed the transcription of scrA, scrG and vpa0198. lacZ fusion assay showed that ToxR was able to repress the promoter activities of scrA, scrG and vpa0198 in both V. parahaemolyticus and E. coli 100λpir. EMSA results showed that His-ToxR was able to bind to the regulatory DNA regions of scrA and scrG, but not to the regulatory DNA region of vpa0198. In conclusion, ToxR inhibited the production of c-di-GMP in V. parahaemolyticus via directly regulating the transcription of enzyme genes associated with c-di-GMP metabolism, which would be beneficial for V. parahaemolyticus to precisely control bacterial behaviors including biofilm formation.

Establishment of a Method for the Identification of Vibrio Parahaemolyticus by Taqman-Probe Fluorescence Quantitative PCR Analysis / 现代检验医学杂志

Objective To establish a method for the identification of Vibrio parahaemolyticus based on Taqman-fluorescence probe quantitative PCR method targeting toxR gene.Methods Taking the standard strain of Vibrio parahaemolyticus (VPJS421) and other ommon pathogenic bacteria'standard strain as the research object,using the bio-software to design specific PCR primers and Taqman probe of Vibrio parahaemolyticus toxR gene and detected by fluorescence quantitative PCR instrument.Results ①The designed primers could amplify specific bands.②The amplification efficiency of the 0.5 μl probe in the amplification system was better than that of the 1.0μl probe.③The detection sensitivity of toxR gene of Vibrio parahaemolyticus by Taqman fluorescence quantitative PCR was 10-1 mg/L.④The detection method did not show positive amplification in detection of Enterococcus f aecalis,Staphylococcus aureus,Saprophytic staphylococcus,Enterobacter hormaechei,Pseudomonas aeruginosa,Escheri ch ia coli,Vibrio al ginol yticus,Vibrio vulnficus,Vibrio metschnikovii and Wbrio furnissii 10 other common pathogenic bacteria.The specificity was 100％.Conlusion The fluorescence quantitative PCR method for the identification of Vibrio parahaemolyticus was successfully established.The method was sensitivty and specificity,and it is suitable for rapid detection of Wbrio parahaemolyticus and has a good application value.

Characterization of Vibrio parahaemolyticus isolated from coastal water in Eastern Province of Saudi Arabia

Aims@#Vibrio parahaemolyticus is a marine and estuarine bacterium that has been documented as the causative agent of food-borne outbreak worldwide. The aim of this study was to confirm the identification of presumptive V. parahaemolyticus isolates to the species level by using PCR targeted to the outer membrane protein regulation operon gene (toxR) and to investigate antibiotic resistance, plasmid profile, and the main core virulence genes of thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh). @*Methodology and results@# A total of 56 presumptive isolates of V. parahaemolyticus were isolated from seawater collected during year a 2010 sampling pilot study performed along the Arabian Gulf coast of the Eastern Province of Saudi Arabia. The purpose of this study was to confirm the identification of presumptive V. parahaemolyticus isolates to the species level by using PCR targeted to the toxR gene and to investigate antibiotic resistance, plasmid profile, and the main core virulence genes of tdh and trh. The toxR-specific PCR assay revealed that a total of 30 out of 56 isolates tested positive for V. parahaemolyticus. None of the 30 strains of the toxR gene were tested positive for tdh and trh genes. All (100%) of isolates were highly resistant to amikacin, cefuroxime, ampicillin, ticarcillin, cefaclor (80%), and tetracycline (70%). The multiple antibiotic resistance (MAR) index was measured for all 16 antimicrobial agents, and the high ranged from 0.25 to 0.56. Among the isolated V. parahaemolyticus, 22 out of 30 strains contained plasmid DNA bands ranging in size from 1.5 to 55 kb and no correlation was observed between the plasmid profiles and antibiotic resistance patterns. @*Conclusion, significance and impact of study@#The results obtained in this study indicate that V. parahaemolyticus is present in the coastal environment of the Eastern Province of Saudi Arabia.

Virulence Factors and Genotyping of Vibrio parahaemolyticus in Sea Water and Clinical Isolates

Six strains of Vibrio parahemolyticus isolated from diarrheal patients and the 12 strains from sea water were serotyped and analyzed for biochemical characteristics, antibiotics sensitivity and detection of toxR, gyrB, tdh, and trh genes. Arbitrarily-primed polymerase chain reaction method were performed on the 6 strains from patients and the following results were obtained. 1. The Vibrio parahemolyticus isolated from patients were belonged to 5 different serotypes: 04:K8, 04:KUT, 06: K18, 010:K71 and 03:K6, but those isolated from sea water were belonged to 5 different serotypes: O1:KUT, 02:KUT, 03:K45, 04:K37 and OUT:KUT. All strains explained have different serotypes depending on the different source, 2. Three serotype (04:K8, 04:KUT, 06:K18) isolated from patients were positive for the urease hydrolysis, whereas only one strain of serotype O1:KUT isolated from sea water was positive to the same. Furthermore, the serotype 06:K18 (1 strain) was positive for the fermentation of dulcito1. Both toxR and gyrB genes were detected from all strains isolated. 3. As for control the 2 strains of serotype 03:K6 and 6 strains isolated from patients, serotype 03:K6 were resistant to oxacillin, penicillin, vancomycin. All strains were sensitive to chloramphenicol and tetracycline yet the antibiogram type showed 6 groups from I to VI. 4. DNA probe hybridization method was used to detect genes. The trh1 was detected both from serotype 04:KUT and 06:K18 isolated from patients and the trh2 was also detected from one strain from each 010:K71 and O1:KUT isolated from patients and sea-water respectively, The tdh gene only was detected from two strains of 03:K6 isolated from patients of 1998. The tdh, trh 1 and trh2 were not detected from 7 strains out of 12 strains isolated from sea water whereas the production titer of TDH isolated from patients showed from 2048 times to 4096 times. 5. Four strains of the serotype 03:K6 isolated from Korea, India and Japan as well as 3 strains from Korean patients were tested by AP-PCR to classify serotypes. As for its result the amplicon showed the same in the 4 strains of the serotype 03:K6 whereas the four strains of different serotype from patients are so difference as to explain no inter- relations at all. The result explains that the serotype 03:K6 is the same genes regardless from where it is isolated.