Studies On The Quorum Sensing System Of Vibrio Harveyi And Its Signal Interference

Quorum sensing is a mechanism in which bacteria coordinate the expression of certain genes in response to their population density by producing, releasing and detecting small signal molecules. Quorum sensing is responsible for controlling a plethora of virulence genes in several bacterial pathogens. Vibrio harveyi is a Gram-negative, luminous marine bacterium, which is widely distributed in the marine environment. This organism is a major pathogen of cultured penaeid shrimp, and has also been associated with disease in fish. So far, antibiotics and disinfectants have only had limited success in the prevention or cure of aquatic disease. Moreover, the frequent use of biocides, especially in subtherapeutic doses, is leading to the rapid development of resistance. Therefore, there is an urgent need to develop alternative ways to control infections caused by bacterial pathogens in aquaculture. V. harveyi are found to control virulence factor expression by a cell-to-cell communication system. Hence, disruption of V. harveyi quorum sensing system has been proposed as a new anti-infective strategy.In this study, three bioluminescence strains, i.e. VIB391, VIB295 and E022 were identified among many V. harveyi strains. The bioluminescence change of the three strains on solid and liquid culture media were studied. The results showed that different states of culture media do not affect the relatively bioluminescence intensity. The bioluminescence intensity of VIB391 is the highest, E022 is in the middle and VIB295 is the lowest. The time of bioluminescence last is longest when cultured on the solid plate medium. The bioluminescence phenomenon is convenient for observing when cultured in the liquid culture medium.In the mutants in quorum sensing system of V. harveyi, the activity of the extracellular enzyme such as glutinase, liqase, lecithinase and hemolysin were studied. The results showed that the glutinase was positively controlled by both HAI-1 and AI-2 of the quorum sensing system, and mainly controlled by AI-2. The liqase was positively controlled by HAI and AI-2 to some extent. The lecithinase was negatively controlled by HAI-1, and the hemolysin was negatively controlled by both HAI-1 and AI-2.Special primers of luxS gene were designed from the luxS gene sequence of V. harveyi obtained from the GenBank, and the gene was amplified from a pathogenic strain V. harveyi VIB645 by PCR. The luxS gene was cloned to a cloning vector pUC, and was sequenced. It was found that the open reading frame is 519 bp. The similarity of the luxS gene from V. harveyi VIB645 to the published sequence of V. harveyi strain in GenBank was only 90%. Therefore, the LuxS/AI-2 system in V. harveyi may well be associated with the pathogenicity or bioluminescence of V. harveyi. The luxS gene from V. harveyi VIB645 encodes 172 amino acids, and the molecular weight of the protein was estimated to be 19.08 kDa. The theoretic pI is 5.07 and the molecular formula is C834H1335N229O260S11. This protein is instable with about 20 h half life span. Further phylogenetic analysis indicated that the sequence of luxS gene from V. harveyi VIB645 had great difference with the luxS gene sequences in the GenBank, i.e. V. harveyi, Ruminococus flavefaciens and Prevotella ruminicola. However, the luxS gene sequences among these bacteria have very high similarity, which was not so much associated with evolution. Hence, we speculates that the gene encoding the LuxS/AI-2 system might be obtained through the horizontal transfer.It has been demonstrated that there is a link between quorum sensing system and virulence factor expression in several aquatic pathogens. AiiA protein can block the bacterial quorum sensing by hydrolyzing AHL-lactone, and could greatly attenuate the disease caused by many bacterial pathogens in which quorum sensing regulate the expression of virulence genes. The primers were designed from the conservative sequences of aiiA gene in the genomes of other Bacillus strains, and the gene was amplified from Bacillus thuringensis BF1 by PCR. AiiA gene was cloned to a cloning vector pUC and sequenced. The open reading frame of the aiiA gene was 753 bp. The similarity of aiiA gene from B. thringiensis BF1 to other sequences in GenBank was 99%. The aiiA gene was cloned into an expression vector pET-24d(+), and the recombinant AiiA protein was overexpressed in Escherichia coli overexpression strain BL21(DE3) as a His-tag fused protein. The molecular weight of the expressed AiiA protein was estimated to be 28 kDa by SDS-PAGE. The recombinant AiiA protein could be induced at 37, 25, 17℃, and the best expression condition was at 25℃with 6 h induction. The supernatant of the recombinant E. coli by sonication could not only repress the pigment synthesize of Chromobacterium violaceum ATCC 12472, but also could attenuate the intensity of bioluminescence of V. harveyi VIB391 and V. harveyi BB886 by 68% and 53%, respectively. This study was very important for further research on the disruption of infections caused by V. havreyi in aquaculture.