Preliminary Study On Molecular Mechanism Of Post-transcriptional Regulation Of Vibrio Cholera CAI-1Synthase Gene CqsA By CRP And The Virulence Phenotypes And Molecular Epidemiological Characteristics Of Vibrio Fluvialis

Quorum Sensing (QS) is a process by which bacterial cells communicate with one another by secreting extracellular signaling molecules termed autoinducers. It was first found in Vibrio fischeri in1970. After that, it was proved that QS exists among varieties of bacteria including Vibrio cholera. Cholera is a severe infectious disease caused by V. cholera, which could lead to acute watery diarrhea and spread rapidly. The seventh pandemic of cholera begun in1961and still continues,, and more than100countries from five continents in the world were involved. It has not been effectively controlled yet. Vibrio cholera has three different QS system including CAI-1/CqsS, AI-2/LuxPQ and VarS/A-CsrA/BCD. The system that this study will focus on is CAI-1/CqsS, which uses CAI-1(cholera autoinducerl) as signal molecules, cqsA as synthase gene of CAI-1, CqsS as the membrane bound sensor histidine kinase coded by cqsS. CRP (cAMP receptor protein) is a global transcriptional regulator, and cAMP is an important second signal molecule in cells. The cAMP-CPR complex regulates many transcriptional units through its interaction with RNA polymerase, which is achieved through binding to a special conserved sequence (TGTGA-N6-TCACA) near promoter. These transcription units are associated with various aspects, including the growth metabolism, the expression regulation of virulence genes, and QS system of Vibrio cholera.Our previous studies have showed that cAMP-CRP complex regulates cqsA expression, the synthase gene of CAI-1at post-transcriptional level. The lack of cAMP or CRP decreases the stability of cqsA mRNA. Our current study employed recombinant reporter strain system and transposon random insertion technique to screen regulators which mediate the process. By utilizing the homologous recombinant DNA technologies mediated by suicide plasmid, we constructed the cqsA-lacZ translational fusion recombination reporter strain system. Then we determined the mRNA level of cqsA-lacZ fusion and the quantity of CAI-1molecule under widetype and crp mutated background and found that the established system meets the screening requirement, and the expression of lacZ and the blue and white phenotype of reporter strain faithfully reflect the regulation of CRP on cqsA. Employing blue and white phenotypic transition, we screened the mutation library mediated by transpson randm insertion, and arbitrary PCR was applied to localize the insertion sites in candidate positive clones. Using this strategy, we got a number of candidate genes which need our further identification. In addition, we mapped the transcription start site of the cqsA gene using the5’RACE technique, which locates at41bp upstream the initiation codon ATG. Vibrio fluvialis is considered to be an emerging foodborne pathogen and has been becoming a high human public health hazard all over the world, especially in coastal areas of developing countries and regions with poor sanitation. The distribution of virulence factors, microbiological and molecular epidemiological features of V.jluvialis isolates in China remains to be examined.PCR targeted at the virulence determinants and phenotype tests including metabolism, virulence and antibiotic susceptibility were performed. Pulsed-field gel electrophoresis (PFGE) analysis was used to access the relatedness of isolates. A strain with deletion of the arginine dihydrolase system was first reported and proved in molecular level by PCR. Virulence genes vfh, hupO and vfpA were detected in all strains, the ability to produce hemolysin, cytotxin, protease and biofilm formation varied with strains. High resistance rate to P-lactams, Azithromycin and Sulfamethoxazole were observed. Twenty-seven percent of test strains showed resistant to two and three antibiotics. PFGE analysis demonstrated great genetic heterogeneity of test V. fluvialis strains.This study evaluated firstly the biological characteristics and molecular epidemiological features of V. fluvialis in China. Some uncommon biochemical characteristics were found. Virulence genes were widely distributed in the isolates from patient and seafood sources, and the occurrence of virulence phenotypes varied with strains. Continued and enhanced laboratory based-surveillance is needed in the future together with systematically collection of the epidemiological information of the cases or the outbreaks.