The Role Of Type Vi Secretion System In The Porcine Extraintestinal Pathogenic Escherichia Coli Induced Host Innate Immunity

Gram-negative bacteria have evolved a variety of protein secretion systems to export or import macromolecules across membranes for survival and fitness.In 2006,the Type ? secretion system(T6SS)was first defined as a novel protein secretion system originally in Vibrio Choerae and Pseudomonas aeruginosa,and was proved to be widely distributed among bacterial pathogens and may play a general role in mediating host interactions.Putative T6SS-encoding gene clusters have now been identified in over one fourth of sequenced Gram-negative bacterial genomes.T6 SS is a versatile injection machine that can deliver effector molecules into the environment,eukaryotic hosts and prokaryotic competitors.The T6 SS apparatus can be divided into two distinct assemblies: a transmembrane complex and a phage tail-like needle structure.In the study of structural model of T6 SS,it is generally believed that Hcp is a key structural component of the T6 SS.Hcp hexamers stack rings in head-to-tail or head-to-tail mode and form long nanotubes of T6 SS structures that is thrust towards the target cell to deliver effectors upon sheath contraction.Hcp is not only an important part of T6 SS structure,but also the secretion of Hcp serves as a hallmark of functional T6 SS.There has been significant progress in understanding the structural and mechanistic aspects of bacterial secretion systems.In recent years,the complex roles of secretion systems in the host-pathogen interaction,particularly as they pertain to immune responses,have been a research hot topic.Molecules delivered by bacterial secretion systems with the design to manipulate host cell homeostasis,in one way or another,influence inflammasome pathways.In this study,PCN033 is one of the porcine extraintestinal pathogenic Escherichia Coli strains,which was isolated from a sick swine.The presence of the T6 SS gene cluster was found by whole genome sequencing.As with many other pathogenic Escherichia coli,three copies of hcps are encoded in the chromosome of PCN033 T6 SS,two of which are located in T6 SS cluster while the third one keeps quite a distance,named hcp1,hcp2,hcp3,separately.Gene deletion strain ?hcp1?hcp2?hcp3 has been constructed and conserved in our laboratory.As a marker for functional T6 SS,the deletion of hcp causing T6 SS cannot be assembled normally to function.Therefore,in this study,PCN033 and ?hcp1?hcp2?hcp3 strains were used to explore the host response to PCN033 infection and the role of T6 SS in activating inflammation.The main contents are as follows: 1.Transcriptome changes of RAW264.7 infected with PCN033.We used mouse macrophage RAW264.7 as host cell model for PCN033,Transcriptome changes of RAW264.7 infected with PCN033 were analyzed,508 differentially expressed genes were found,and the differential genes were analyzed for their function.In the GO database,differential genes involve 2052 biological processes,133 cellular components and 211 molecular functions,and 52 KEGG metabolic pathways related and statistically significant.Among the 52 KEGG pathways,there are signaling pathways related to innate immunity such as NF-?B,TNF,Apoptosis,TLR,and NLR.2.PCN033 activates NF-?B signaling pathway and NLRP3,NLRC4 inflammasome.We next investigated the activation of NF-?B signaling pathway and intracellular proteins expression of NLRP3,NLRC4,and ASC in RAW264.7 in response to PCN033 infection by Western-blot,and observed phosphorylation of p65,I?B? degradation along with infection,and ASC,NLRP3,NLRC4 expression are increased obviously.These suggested that PCN033 infection contributes to activation of NF-?B signaling pathway and NLRP3,NLRC4 inflammasome.3.The role of T6 SS in the interaction between PCN033 and host cell.It has been reported that T6 SS contributes to cytotoxicity of murine macrophages.LDH in cell culture mediums were detected and we found that compared with PCN033 infection,cytotoxicity of RAW264.7 infected with ?hcp1?hcp2?hcp3 was significantly decreased,indicating that T6 SS has an enhanced effect on mouse macrophage toxicity.We intravenous injected PCN033 and ?hcp1?hcp2?hcp3 at the same dose into mice,IL-1? in the serum of mice was measured by ELISA.We found that compared with the wild strain PCN033,deletion of hcp resulted in reduced ability of mice to produce IL-1?,indicating that T6 SS may play a role in the release of IL-1?.We next investigated the transcription of inflammatory factors by q RT-PCR,and found that T6 SS unregulate expression of pro-inflammatory cytokines IL-1?,IL-18,CXCL3,and TNF-?,indicating that T6 SS have pro-inflammatory effection.By Western-blot,found that PCN033 contributes to caspase1 activation and pro-IL-1? processing in RAW264.7,and T6 SS plays a role in this process.In addition,we also found that T6 SS is involed in activating of NF-?B signaling pathway and NLRP3,NLRC4 inflammasomes of host cell infected with PCN033.