Construction And Functional Analysis Of LuxS Deletion Strains Of Edwardsiella Tarda

Edwardsiella tarda(E.tarda)disease is the Class III aquatic animal disease in China.Its pathogenic strains,Edward Edwards,has extensive infection hosts including fish,reptiles,birds and mammals.In particular,fish which were infected has some symptoms such as skin ulceration,organ necrosis,and systemic hemorrhage.It caused a lot of economic losses in the aquaculture industry.Quorum sensing(QS)is an important communication system in bacterial species.Signal molecules in this system are the common language for bacteria to communicate within and between species.Gram-positive bacteria and Gram-negative bacteria have their own quorum-sensing systems,and they also share a unique quorum-sensing system which is named the LuxS/AI-2 quorum-sensing system.In this quorum-sensing system,autoinducer-2(AI-2)is a signaling molecule.The synthetic enzyme Lux S of the AI-2signaling molecule is the key to the synthesis of AI-2 and an important component of the methyl cycle.LuxS can not only participate in quorum sensing,but also can influence the induction of bacterial bioluminescence,the formation of biofilms,and the phenomenon of group migration.This study successfully cloned the Lux S gene of E.tarda,which has a length of 516 bp and encodes 172 amino acids.Its molecular weight is estimated to be approximately 22 kDa.Analysis of the LuxS gene sequence revealed that the Lux S gene is highly conserved and has high homology with various bacteria.In this study,the LuxS gene of E.tarda was successfully knocked down and named as △Lux S strain.This study provided a key basis for the subsequent research on the biological function of Lux S,which is a synthetase of AI-2molecules.And compared with methods such as RNA interference which foucused on the function of gene,it has more advantages in avoiding some problems like affecting bacterial growth,interfering with other gene expressions and so on.Since LuxS is closely related to the growth rate and density of bacteria,this study measured the effect of luxS gene deletion on the growth rate of E.tarda.The results showed that after the deletion of luxS gene delayed the logarithmic growth phase of bacteria,the highest concentration of bacteria was decreased sharply(P<0.05)which revealed that luxS gene expression influenced both the growth rate and bacterial concentration of E.tarda.It has already been confirmed that LuxS affects the pathogenicity of bacteria,so thisstudy established the mouse macrophage RAW264.7 infection model and zebrafish infection model to study its influence on pathogenicity.It was found that the deletion of the luxS gene reduced the pathogenicity of the E.tarda to a certain extent,and the LD50 of the deleted strain was increased by 20 times.At the same time,we also measured the movement and colonization ability of E.tarda by constructing an in vitro mucin model.The results showed that the deletion of the luxS gene significantly(P<0.05)reduced the ability of the slow Edwards to penetrate mucin,and the deletion strain could only penetrate into the fourth layer and the number of bacteria per layer was significantly lower than that of the wild strain,which revealed that deletion of luxS gene significantly reduced the movement,colonization,and virulence of the E.tarda.LuxS can not only participate in density sensing,but also can influence the induction of bacterial bioluminescence.In this study,by testing the bioluminescence of Vibrio harveyi BB170 induced by the production of exogenous AI-2.It is showed that the luminescence intensity of BB170 induced by the wild strain was 56 times that of the negative control.Which proved that the E.t EIB202 could produce the signal molecule AI-2.Simultaneously,according to the results that the deletion strain could not induce the luminescence of BB170,it was founded out that LuxS and AI-2 were closely linked,indicating that the luxS gene influences the synthesis of AI-2 and thus further influences the function of bacterial bioluminescence.LuxS is associated with the formation of bacterial biofilm formation.In this study,crystal violet staining and laser confocal microscopy were used to detect the biofilm formation ability.The results showed that the OD value of the deletion strain group was significantly lower than the wild strain group,and the formation of biofilm could not be observed by microscopy which suggested that the deletion of the luxS gene significantly reduced the biofilm formation ability of the E.tarda(P<0.05),indicating that the luxS gene has the ability to inhibit the formation of biofilms of E.tarda.In conclusion,Lux S is one of the most important pathogenic factors of E.tarda,and the deletion of Lux S protein gene significantly reduces the ability of growth,colonization,movement,virulence,bioluminescence of bacteria and biofilm formation of bacteria.It provides a theoretical basis for the study of the E.tarda quorum sensing system and the pathogenic mechanism research,as well as a new method for the prevention and control of E.tarda disease.