The Mechanism Analysis Of Shewanella Putrefaciens To Vibrio Parahaemolyticus Virulence Factors Based On Quorum Sensing

Vibrio parahaemolyticus is a leading bacteria of seafood poisoning, it often coexists with Shewanella putrefaciens in coastal seawater and seafood, which is the dominated spoilage bacteria of aquatic product. The hemolytic activity, thermostable direct hemolysin gene(tdh) expression, biofilm-forming ability and virulence of Vibrio parahaemolyticus were significantly increased in the co-culture system with Shewanella putrefaciens, while its mechanism is still unkown. Quorum sensing regulates the virulence factors expression in and between bacteria, so this research analysed the mechanism of Shewanella putrefaciens to Vibrio parahaemolyticus virulence factors based on quorum sensing.1. Using Vibrio parahaemolyticus ATCC33847 as the parent strain, the quorum sensing signal molecule synthase genes lux M, lux S knockout mutants of Vibrio parahaemolyticus were constructed using homologous recombination. The lux M, lux S mutants VPΔLux M, VPΔLux S were verified using chloramphenicol resistance, colony PCR and DNA sequencing. The growth phenotype, hemolytic activity, tdh expression and biofilm-forming ability of VPΔLux M and VPΔLux S were tested and compared with the parent strain. The results showed that the Vibrio parahaemolyticus quorum sensing signal molecule synthase genes lux M, lux S knockout mutants VPΔLux M, VPΔLux S were successfully constructed. The growth of VPΔLux M is weaker than the parent strain, and its hemolytic activity, tdh expression, biofilm-forming ability were significantly increased compared with the parent strain(p<0.05). The hemolytic activity, tdh expression of VPΔLux S were significantly decreased compared with the parent strain(p<0.05), and its biofilm-forming ability was significantly increased compared with the parent strain(p<0.05).The results suggested that, the lux M gene of Vibrio parahaemolyticus negatively regulates its hemolytic activity, tdh expression, biofilm formation and positively regulates its growth. The lux S gene of Vibrio parahaemolyticus positively regulates its hemolytic activity, tdh expression and negatively regulates its biofilm formation.2. To analyse the mechanism of Shewanella putrefaciens to Vibrio parahaemolyticus virulence factors based on quorum sensing, co-culture systems of Shewanella putrefaciens, Shewanella putrefaciens supernatant extract with VPΔLux M, VPΔLux S and the parent strain ATCC33847 were constructed, and its hemolytic activity, tdh expression and biofilm-forming ability were tested and compared.According to the relative hemolytic activity results, the supernatant extract of Shewanella putrefaciens significantly increased the hemolytic activity of ATCC33847, VPΔLux M, and VPΔLux S 1.51, 1.24, 2.46 times, respectively(p<0.05). The hemolytic activity pattern of VPΔLux S and ATCC33847 in the co-culture system is the same.Using real-time PCR to test the tdh expression, the results showed that the tdh expression of VPΔLux M, VPΔLux S and ATCC33847 in the co-culture system with Shewanella putrefaciens were significantly increased 2.46, 7.66, 1.51 times, respectively(p<0.05). And the supernatant extract of Shewanella putrefaciens increased the tdh expression of VPΔLux M, VPΔLux S and ATCC33847 1.60, 17.68, 1.14 times, respectively. The tdh expression pattern of VPΔLux M and ATCC33847 in the co-culture system is the same.The biofilm-forming ability was tested using crystal violet assay, and the results showed that the supernatant extract of Shewanella putrefaciens decreased the biofilm-formation of ATCC33847, VPΔLux M, and VPΔLux S 0.84, 0.53, 0.75 times, respectively(p<0.05). The biofilm formation pattern of VPΔLux M and ATCC33847 in the co-culture system is the same.Using the fluorescence in situ hybridization assay and microscope to observe the biofilm structure, the biofilm structure of VPΔLux M, VPΔLux S is denser and form agglomeration compared with ATCC33847 in the culture system constructed. The supernatant extract of Shewanella putrefaciens significantly enhanced the biofilm structure of ATCC33847, VPΔLux M, and VPΔLux S, while Shewanella putrefaciens significantly slacked the biofilm structure of ATCC33847, VPΔLux M, and VPΔLux S.The results suggested that Shewanella putrefaciens may influence the hemolytic activity, tdh expression and biofilm-forming ability of Vibrio parahaemolyticus by acting on the Lux M quorum sensing system of Vibrio parahaemolyticus through its AHLs signaling molecules combining to the Lux M quorum sensing system of Vibrio parahaemolyticus and some metabolites degrading the AHLs signaling molecules of Vibrio parahaemolyticus, or acting on the Lux S quorum sensing system of Vibrio parahaemolyticus through its AI-2 signaling molecules combining to the Lux S quorum sensing system of Vibrio parahaemolyticus.3. The AHLs signaling molecules, AI-2 signaling molecules, hemolytic activity and biofilm-forming ability in the co-culture system constructed with Shewanella putrefaciens and VPΔLux M, VPΔLux S and ATCC33847 were dynamically tested and compared. The results showed that the AHLs signal molecules in the culture system were accumulated following the growth of bacteria, the AI-2 signal molecules were accumulated till the stable stage and degrade afterwards. The hemolytic activity and biofilm-forming ability of Shewanella putrefaciens and VPΔLux S co-culture system were changing following its AI-2 signal molecules. A rapid and accurate LC-MS/MS method detecting AHLs signal molecules was established and used to evaluate the AHLs signal molecules of Shewanella putrefaciens and Vibrio parahaemolyticus ATCC33847. The results showed that Shewanella putrefaciens produce C6-HSL(23.47μg/L), C12-HSL(3.49μg/L) and 3-oxo-C12-HSL(0.37μg/L), and Vibrio parahaemolyticus produce C4-HSL(11.79μg/L), 3-oxo-C6-HSL(0.46μg/L) and 3-oxo-C14-HSL(0.62μg/L).The results suggested that Shewanella putrefaciens may influence the hemolytic activity, tdh expression and biofilm-forming ability of Vibrio parahaemolyticus through its metabolites and AI-2 signaling molecules, which can degrade and combine to the AHLs signaling molecules of Vibrio parahaemolyticus and Lux S quorum sensing system of Vibrio parahaemolyticus, respectively.In summary, this study analyzed the mechanism of Shewanella putrefaciens acting to Vibrio parahaemolyticus virulence factors based on quorum sensing and the role of quorum sensing system in the regulation of hemolytic activity, tdh expression and biofilm formation of Vibrio parahaemolyticus, which can provide theoretical support and guidance for the Vibrio parahaemolyticus virulence control. It has been proved that Shewanella putrefaciens may influence the virulence factors of Vibrio parahaemolyticus through its metabolites and AI-2 signaling molecules, which can degrade and combine to the AHLs signaling molecules of Vibrio parahaemolyticus and Lux S quorum sensing system of Vibrio parahaemolyticus, respectively. And it can provide theoretical support for the effect and mechanism research of the co-culture system. The LC-MS/MS metod established in this study to detect AHLs signaling molecules and the Vibrio parahaemolyticus quorum sensing signal molecule synthase genes mutant VPΔLux M, VPΔLux S can lay foundation for the following study of Vibrio parahaemolyticus quorum sensing system and other quorum sensing research.