| Vibriosis caused by vibrios brings about huge damages to the worldwide mariculture. Vibrio alginolyticus is the major pathogen to the grouper in the South China. The potential virulence factors, including extracellular protease, motility, siderophore-dependent iron uptake system, and exopolysaccharide, have been identified in the previous work. However, the regulation circuit involved in the virulence factors production has not been well known except for the quorun sensing system. Quorum sensing, a signal transduction circuit according to the concentration of the intracellular or intercellular autoinducers, is widely present in the bacteria and involved in various cell processes and plays pivotal roles in the virulence and pathogenicity regulation in many pathogens.Here, we characterized two elements, Hfq and LuxT which were involved in the quorum sensing system in V. alginolyticus EPGS, and identified their location and roles in quorum sensing system and the virulence regulation.Firstly, the sRNA chaperon Hfq was cloned and characterized. The results showed that Hfq played positive roles in the motility and biofilm formation but negative roles in regulating the extracellular protease production in V. alginolyticus. The results of Western-blot against the main extracellular protease Asp indicated that when Hfq expression was disrupted, the Asp production was largely promoted and the regulation of Asp production by Hfq was realized mainly through the negative modulation of the expression of the core element of quorum sensing, LuxR. Besides, Hfq was involved in the stress response such as temperature,10% ethonal,30% sucrose, and the depletion of iron. Under all the tested stresses, the growth of theΔhfq strain was significantly inhibited, which was overlapped with the detection results inΔrpoS strain. Therefore, the plasmid containing the intact promoter region and the ORF of rpoS was complemented into theΔhfq strain and then theΔhfq strain restored the normal growth under the tested stresses except for the depletion of iron. Besides, the qRT-PCR result showed that the transcription level of rpoS gene significantly decreased inΔhfq strain, which illustrated that Hfq takes effect on stress response through the sigma factor, RpoS. The in-frame deletion of hfq resulted in the 17-fold attenuation of the V. alginolyticus in zebra fish infection, even though the extracellular protease production increased obviously. Furthermore, the viability of theΔhfq strain in zebra fish was impaired and it was eliminated by the host at the low infection dose without proliferation and also cleared at the high dose in spite of its marginal replication in host, while the wild-type strain could survive in the host and cause the death of the fish. The zebra fish and grouper vaccinated with Ahfq strain were well protected against the infection of V. alginolyticus both in the injection and immersion vaccination method, and the relative percent survival reached higher than 70%, indicating the potential of the Ahfq strain as a live attenuated vaccine.LuxT, another novel element involved in quorum sensing system, was characterized. The results showed that the transcription and expression of LuxT was cell-density dependent and positively regulated by the LuxU protein. Moreover, the transcription of LuxT required the cooperation of the three quorum sensing signal synthesis pathways. Meanwhile, LuxT was characterized to locate upstream of the LuxO-LuxR cascade. LuxT might modulate the transcription of LuxO through the direct binding to the promoter region of luxO gene. Yet, LuxT played marginal roles on the transcription of luxR but positively regulated the expression of LuxR as tested by Western-blot against LuxR antibody. In other words, LuxT activated not only the transcription of the post-transcriptional LuxR-repressor LuxO, but also the expression of LuxR, which is thoroughly different from the mode in V. harveyi, V. vulnificus, and V. anguallarum. Besides, LuxT promoted the swarming mobility and extracellular protease production and, however, AluxT displayed no attenuation in zebra fish infection. LuxT repressed the expression of LuxR by the activation of luxO transcription and LuxR expression through another unknown post-transcriptional regulator to finally fine-tune the virulence of V. alginolyticus.This study also constructed a set of plasmids based on Gateway cloning tachnology used for genetic analysis. The constructions of traditional mutant and complemented strain were based on the restriction enzyme digestion and ligation, which relys much on the enzyme digestion activity, the stability of the plasmid and the ligation efficiency. Besides, the limited restriction enzyme sites carried on the plasmid and the versatile enzyme sites contained in the target genes always hampered the cloning process. To avoid the above problems, the plasmids utilized for mutant and complemented strain construction were modified to be Gateway-compatible. Five genes involved in T6SS in V. alginolyticus were selected to evaluate the Gateway-compatible technology. Their mutants and complemented strains were successfully obtained by using pDM4.DEST and pMMB206.DEST. The Gateway cloning technology, based on the lambda phage site-directed recombination way can move the target genes into various destination plasmids rapidly and conveniently without enzyme digestion and the recombination efficiency was high, thus makes the high-throughput characterization of unidentified genes realizable.
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