Immune Response Mechanism Of Drosophila S2 Cells Infected By Salmonella Enteritidis

Innate immunity is one of the important defense mechanisms of animals against microbial infections.Since insects do not have specific immunity,innate immunity plays a vital role in preventing insects from invading foreign microorganisms.Salmonella enterica,a serotype from chicken enteritis,has a broad host spectrum galliformes,and is able to spread in higher mammals in addition to animals from Galliformes.However,it has a strong pathogenicity in susceptible hosts,which can easily cause pathological changes and even death of the host,which is not conducive to studying the host’s defense mechanism.Therefore,an intermediary model needs to be introduced to overcome the disadvantages,and Drosophila have been proved to be an ideal model for studying such pathogens.In this paper,transcriptome sequencing in Drosophila S2 cells after S.enteritidis infection found that signaling pathway such as Drosophila innate immunity was large-scale upregulated.Moreover,we deeply analyzed immune interaction mechanism between the pathogen and the host S2 cells.The results are listed as below:1.q PCR and fluorescence observation showed that S.enteritidis could successfully infect Drosophila S2 cells and proliferate in Drosophila S2 cells at 3 h and 6 h.Morphological observation showed that S.enteritidis infection within 24 h had no obvious pathological effection on Drosophila S2 cells.2.Transcriptome sequencing analysis revealed that most immune-related genes of Drosophila S2 cells were significantly up-regulated after S.enteritidi infection for 3 h and 6h.Meanwhile,other functional genes were also changed significantly,the number of differential genes gradually increased along with the extension of infection time.q PCR verified that the variation of upregulated antimicrobial peptide genes were consistent with the transcriptome data.3.After Drosophila S2 cells infected by S.enteritidi for 1 h,2 h,3 h,4 h,5 h and 6 h,the protein levels of the autophagy labeling protein Dm Atg8 and autophagy-specific substrate Dm Ref(2)P/Dmp62 was detected,and results showed that Dm Atg8a–PE conjugation was increased gradually from 1 h to 5 h compared;while,Dm Atg8a–PE formation was slightly reduced compared to that at 5 h,but still markedly upregulated compared to 0 h controle.In comparison,the protein levels of Dm Ref(2)P/p62 were decreased post infection,which were opposite to the variation of Dm Atg8a–PE formation.In addition,Lyso Tracker staining showed that the acidification of lysosomes in Drosophila S2 cells was enhanced after S.enteritidi infection for 3 h.4.m RNA leves of Dm PGRP-LB,LC and LD from the peptidoglycan recognition protein PGRP-L family were significantly up-regulated in Drosophila S2 cells after S.enteritidi infection for 3 h.However,only Dm PGRP-LC and Dm PGRP-LD were potential for Drosophila S2 cells against S.enteritidi infection.5.Dm PGRP-LC and Dm PGRP-LD regulated the proliferation of S.enteritidi in S2 cells.The relationship between Dm PGRP-LC or Dm PGRP-LD and autophagy or antimicrobial peptides expression were detected.The results showed that during Drosophila S2 cells protect against S.enteritidi infection,Dm PGRP-LC and Dm PGRP-LD regulated the expression of antimicrobial peptides,which were downstream of the Imd signaling pathway;moreover,Dm PGRP-LD promoted autophagy occurrence.6.Mass-spectrum analysis after Dm PGRP-LD immunoprecipitation revealed that it was suggested to interact with multiple pathways during S.enteritidi infection,mainly including autophagy,ubiquitination,endocytosis and phagocytosis.7.The extracellular polysaccharides(ESP)extracted from S.enteritidis were used to treat Drosophila S2 cells for 3 h,and then the changes of PGRP-L family were detected.The results showed that the variations of PGRP-L family memebers were consistent with theire changes induced by S.enteritidis.The ESP5 was them mainly components of ESPs,and its further treatment induced the expression of Dm PGRP-LC and Dm PGRP-LD.Molecular weight determination and monosaccharide composition analysis show that EPS5 was comprised by mannose,galactose and glucose in a ratio of 7: 2.7: 1.In general,although the pathogenic S.enteritidis can successfully infect and proliferate in the host Drosophila S2 cells,the extracellular polysaccharides secreted by the pathogen will be recognized by the host cells and induce the expression of Dm PGRP-LC and Dm PGRP-LD,which subsequently trigger the innate immune pathways such as antimicrobial peptides and autophagy.The analysis of these key nodes in the pathways after S.enteritidis infection can provide a theoretic basis for immune targeting and prevention of autophagy-related diseases.