Study On The Regulation Of T6SS2 By The Density Sensing System Of Vibrio Fluvialis

V.fluvialis(Vibrio fluvialis)is a kind of common halophilic enteric pathogenic bacteria in the coastal environment,and it easily causes sporadic or outbreak diarrhea as well as extra-intestinal diseases,and caused aquaculture animal diseases,resulting in serious economic losses.The biochemical phenotypic features of V.fluvialis are similar to these of Vibrio cholerae and Aerotrionas spp.In spite of successful identification of some of its virulence factors,the pathogenesis and environmental survival mechanism of V.fluvialis still need to be further investigated.The type VI secretion system(T6SS)is a newly discovered secretion system in gram-negative bacteria,and it injects effector protein into target cells through contact-dependent manner and kills the cells,and this system plays a critical role in competitive environmental survival among bacteria.V.fluvialis contains two gene clusters coding T6SS,known as T6SS1 and T6SS2,and the later shows functional expression and mediates inter-bacteria killing capacity.Quorum sensing system(QS)is a communication system employed by bacteria to sense and density-dependent respond to single or a variety of signal molecules secreted by itself or surrounding bacteria to synchronize gene expressions,thus participating in many physiological processes,including the regulation of virulence and pathogenecity.In our previous study,we investigated the effects of temperature,salinity,osmolarity and growth density on functional expressions of T6SS2 in V.fluvialis.In the following study,we further explored the regulatory role of QS system on V.fluvialis T6SS2.There are two QS systems in V.fluvialis:the VflI-VflR system which utilizes acyl-homoserine lactone(AHL)as a signal molecule;the CqsA/luxS-vflS system which uses cholerae autoinducer 1(CAI-1)and autoinducer 2(AI-2)as molecular signals.In addition,it has been identified that vflI is the gene which codes the synthetase for AHL,and cqsA and luxS are genes encoding synthetase for CAI-1 and AI-2 respectively.Furthermore,signal transduction pathways downstream CAI-1 and AI-2 converge on the following genes:IuxU,luxO and vflS(referred to as hapR in V.cholerae).Our study found that both of the two QS systems in V.fluvialis take part in the modulation of T6SS2 activity,and CqsA/LuxS QS activates the expression and function of T6SS2 through VflS,while VflI-VflR system positively regulates the expression and function of T6SS2 via VflR.Further investigations demonstrated that VflS and VflR up-regulate the expressions of the T6SS2 core gene cluster and three unique hcp-vgrG gene clusters.Specifically,employing wild-type V.fluvialis 85003 as initial strain,we constructed a series of deletion mutants for cqsA,luxU,ZuxO,luxS and vflS.Subsequently,we cultured these mutants together with its wild type in the following growth conditions:1%Nacl,30℃ and OD=1.5.We then collected the bacterial pellets and their supermatants,and analyzed target protein levels by Western blotting.Our results showed that,comparing to its wild type,△vflS strain has a significantly reduced expression and secretion of Hep.Opposite to the effect observed for △vflS strain,both △luxO and △luxU strains display increased Hep expression and secretion in contrast to its wild type,and the induction effect in △luxO strain is much more prominent,suggesting that vflS is required for the expression and secretion of Hep,whereas both luxO and luxU produce opposite modulatory effects.Consistent with Western blot results,the mRNA level of hcp in △vflS strain is greatly diminished,while it is slightly elevated in △luxO than the wild type.As it has been reported that luxO is inactive under high growth density,we assayed the expression and secretion of Hcp in 85003,△luxO and △luxU under different cell growth densities(OD600=0.2,0.5 and 1.0),and found that at low cell density,the mRNA and protein levels of Hep vastly increased in △luxO strain than its wild type,indicating that luxO is a negative regulator of Hep under low cell growth density,and this regulator effect probably ceases with increasing cell density.VflS is the terminal regulator of CqsA/LuxS QS system,the repression of Hep expression by luxO and luxU may be actually achieved through affecting the VflS level.So we evaluated the VflS level by transforming pBB1,a cosmid containing the luxCDABE operon from V.harveyi,into WT,△luxO and △luxU and measured the luminescence activity.The luminescence activities showed that △luxO has the highest level of VflS,△luxU the second,then the WT,These results are consistent with the Hcp expression and secretion in these strains.Besides,deletion of vflS in △luxO background completely abolished the expression of Hcp,further illustrating that luxO excutes its regulation through affecting the VflS.Due to the great decrease of Hcp expression in △vflS,we believe that VflS is the major positive regulator of CqsA/LuxS QS in regulating T6SS2.After validating VflS is the major regulator of CqsA/LuxS QS in regulating T6SS2,we try to find out whether it works at the transcriptional level.For this purpose,we constructed recombinant reporter plasmids by cloning the promoter regions of T6SS2 core cluster and three hcp-vgrG clusters in PBBRlux and measured their luminesce activities in WT and △vflS.The results showed that the luminesce activities of reporter plasmids are vastly higher in WT than in △vflS,indicating that VflS activates the transcription of T6SS2 core cluster and three orphan hcp-vgrG clusters.Bioinformatics analysis revealed the existence of binding sites similar to that of HapR binding in V.cholerae in the promoter regions of T6SS2 core cluster and hcpA and hcpB.To determine the direct binding interaction between VflS and promoter DNA,we purified VflS,amplified the promoter regions of hcpA and hcpB and performed EMSA assay.The result showed that retardation of hcpA and hcpB probes by VflS protein display does dependent manner,indicating that VflS might directly bind to the promoters of hcpA and hcpB to produce the regulatory effect.We also investigated the regulation of T6SS2 by AHL based QS through creating△vflI and △vflR mutants.qRT-PCR and WB analysis showed that Hep expression and secretion was greatly decreased in △vflR,but not in △vflI.This result suggests that Vfll-VflR QS activates T6SS2 through regulator VflR.To make clear whether the regulation functions at transcriptional level,we introduced the recombinant reporter plasmids containing the promoter regions of T6SS2 core cluster and there hcp-vgrG clusters into the WT and △vflR mutant and measured their promoter activities.Our results show that the promoter activities of reporter plasmids are much higher in WT than in △vflR,suggesting that VflR transcriptionally activates the T6SS2 core cluster and three orphan hcp-vgrG clusters.In conclusion,our study reveals that both of CAI-1/AI-2 and AHL QSs positively regulate the expression and function of T6SS2 in V.fluvialis through the regulator VflS and VflR,respectively.The regulatory effects occur at the promoter level of T6SS2 core cluster and three hcp-vgrG clusters.