Identification Of New Effectors Of Vibrio Parahaemolyticus T3SS2 And Primary Research Of Its Function

Vibrio parahaemolyticus is a bacterial pathogen that causes disease throughout the world,virulent strains can cause acute gastroenteritis,wound infections,even induce septicemia.It’s main pathogenicity is associated with a wide variety of virulence factors,including hemolytic toxin,adhesin,type III secretion system,type VI secretion system,etc.The related effectors of type III secretion system are closely related to the pathogenicity of the bacteria.One of the type III secretion system T3SS1 is encoded in the chromosome one can secrete effectors mainly to exert cytotoxicity by inducing autophagy,cell rounding and pyrolysis,the other,type III secretion system T3SS2 is encoded in the chromosome two can secrete effectors mainly through the destruction of the cytoskeleton and manipulation the signal transduction of cell to cause enterotoxicity.Nowadays,domestic and foreign scholars had pay more attention to the study of T3SS2,due to T3SS2 has 80 related genes encoding on pathogenicity island VP-PAI,only a few proteins have been studied,so we select several new type of effectors for research.Firstly,we use bioinformatics to select two genes VPA1324 and VPA1360 for research from the T3SS2 related VP-PAI,we construct the recombinant expression plasmid of two genes,induce their expression and purify their proteins,then obtain corresponding antiserum by immunization of mice.The results of RT-PCR and Western blot verify VPA1324 and VPA1360 can be transcribed and expressed with the action of bile salts,and show protein VPA1324 and VPA1360 are the related secretory protein of T3SS2.The result of bioinformatics analysis show VPA1324 is a diguanylate phosphodiesterase.Taking c-di-GMP as substrate,the activity detection of VPA1324 by high-performance liquid phase chromatography,indicates VPA1324 can degrade c-di-GMP,has the activity of phosphodiesterase.we construct its recombinant plasmid pEGFP-VPA1324-C1,then the recombinant plasmid transfected into HeLa and Caco-2 cells,the expression of VPA1324 protein can induce the cell shrinkage,annexin V-FITC / PI double stain and Hoechst 33342 stain show that VPA1324 protein can cause cell apoptosis.In conclusion,VPA1324 and VPA1360 are secreted protein related with T3SS2,VPA1324 is a phosphodiesterase,inducing cell apoptosis.The study of new protein will not only help us to understand the mechanism of virulence and infection,but also provide a unique visual angle for the signal functional mechanism of eukaryotic organism.