Function Of Protein VgrG In Type Six Secretion System(T6SS) Of Porcine Extraintestinal Pathogenic Escherichia Coli PCN033 And Mechanism Of Inflammation Induced By T6SS In PCN033

Extraintestinal pathogenic Escherichia coli(ExPEC),a kind of E.coli possessing special virulence factor,causes diseases of multiple extraintestinal organs in humans and veterinary species.ExPEC brings huge economic losses to the pig industry and poses a threat to public health.Type VI secretion system(T6SS)plays an important role in bacterial pathogenesis and virulence.The gene cluster of T6 SS generally exist in Gram-negative bacteria and VgrG is one of the core component of T6SS's functional realization.Inflammation is an acute reaction of the body to the infections and tissue damage.The type 6 secretion system in bacteria has been reported to plays an important role in bacterial infection and inflammation process.The whole genome of ExPEC strain PCN033 has been sequenced by our laboratory,and we found an integrated T6 SS cluster located in the strain's genome.Four putative of vgrG genes are encoded in the genome of ExPEC PCN033.Two of them are located in T6 SS cluster(VgrG1 and 0248)while the other two are quite a distance away from the T6SS(VgrG2 and 1588).This study explored the role of four putative vgrG genes in the function of T6 SS,and initially explored the role of T6 SS in in the process of PCN033 involvement in the inflammatory response.The main results were as follows: 1.Study on the function of VgrG1/0248 and VgrG2/1588 in porcine Extraintestinal pathogenic Escherichia coliFour putative VgrGs were identified in the porcine extraintestinal pathogenic Escherichia coli PCN033.VgrG1 and 0248 were located in the T6 SS gene cluster,while VgrG2 and 1588 were located outside the T6 SS gene cluster.We first explored the function of VgrG inside and outside the gene cluster.The antibacterial activity,cell interaction,mouse pathogenicity,and whole blood killing experiments confirmed that only VgrG1 and/or 0248 inside T6 SS plays an important role in the antibacterial activity and the pathogenecity process of T6 SS in PCN033.While VgrG2 and 1588 outside the gene cluster had no significant effect in the antibacterial,cell interaction and pathogenic process of PCN033.2.VgrG1 plays a major role in the antibacterial and pathogenicity process of PCN033Further analysis of the conserved domains revealed that 0247(VgrG1)and 1587(VgrG2)possessed the conserved VgrG domain,interestingly,VgrG1 and VgrG2 only differed in one amino acid.While 0248 and 1588 had no conserved gp27/gp5 domain,and the sequences of 0248 and 1588 are identical.Further research found that only VgrG1 inside T6 SS plays an important role in the antibacterial activity and the pathogenecity process of T6 SS in PCN033.The reason why 0248 and 1588 have no function could be attributed to the fact that they have no conserved VgrG domain.The difference in one amino acid might not have affected the function of proteins VgrG1 and VgrG2.The reason why VgrG2 has no function most likely to result in its low expression in PCN033.3.T6 SS participates in the inflammatory response of PCN033 by activating NLRP3 inflammasomeInflammation is a defense response of the body to the external infections.Inflammation is usually beneficial,while inappropriate or severe inflammatory reactions are harmful.To investigate the role of T6 SS in the pathogenesis of PCN033,we performed inflammation activation assays using PCN033 and T6 SS mutant.By investigating the role of T6 SS in the inflammatory response induced by PCN033,we found that the inflammatory level caused by PCN033 was significantly decreased after the absence of T6 SS.These results suggested that T6 SS was involved in the inflammatory reaction process caused by PCN033.Further experiments found that T6 SS may promote the inflammatory response induced by PCN033 through activate the NLRP3 inflammasome.In general,our study about the function of the four putative VgrG family proteins in PCN033 reveals that only VgrG1 participates in the antibacterial ability of PCN033 and facilitates its adherence and invasion to host cells,enhancing its pathogenicity in host.Further experiments demonstrated that T6 SS can participate in the pro-inflammatory pathogenesis of PCN033 by activating NLRP3 inflammasome.This study laid the foundation for an in-depth understanding of the mechanism of T6 SS and the pathogenesis of PCN033.