Screening And Identification Of Persistent Infection Related Genes In Salmonella Enteritidis

Salmonella Enteritidis is an important zoonotic pathogen that can spread between different hosts through various pathways,causing gastrointestinal diseases such as abdominal pain and diarrhea.In severe cases,it can lead to host shock or even death.Salmonella Enteritidis can evade the clearance of host immune system by complex immune escape mechanisms,allowing for long-term colonization and causing persistent infection.The counterbalance between the pathogenicity of Salmonella and the immune response ability of the host constantly affects the process of persistent infection.However,the key factors of Salmonella Enteritidis affecting persistent infection in the escape of host immune response by different virulence factors remain unclear.In this study,Tn-seq technology was used to screen genes related to persistent infection of Salmonella Enteritidis Z11 strain.The effects of these genes on the persistent infection of Salmonella were explored in both cell and animal models,providing key targets for further exploring the mechanisms of Salmonella Enteritidis evading host immune response and establishing persistent infection.1 Screening related genes of persistent infection causing by Salmonella Enteritidis.The study used the Salmonella Enteritidis Z11 strain as the research object and orally infected 129 S2/SvE mice with a dose of 1×108 CFU to simulate a persistent infection state.The colonization of Salmonella Enteritidis in five organs,including liver,spleen,gallbladder,cecum,and Peyer’s patches was observed at different time points.The results showed that Z11 strain mainly colonized in the cecum and Peyer’s patches,and a small proportion of the bacteria could break through the intestinal epithelial cell barrier and spread to liver,spleen,gallbladder,and other parts.Therefore,the cecum and Peyer’s patches were selected as research objects for the subsequent experiments,and Tn-seq technology was used to screen genes related to persistent infection of Z11.A high abundance transposon insertion mutant library of Z11 strain was constructed as the initial library,and the Salmonella genome from the cecum and Peyer’s patches was extracted at different time points after oral infection of mice with a dose of 1×108 CFU as a recovery library.A total of 37 effective samples were obtained for genome fragmentation,end repair,phosphorylation plus A,and PCR amplification to construct a high-throughput sequencing library.According to the Tn-seq results,11 genes,such as KMIPBBJO₀1249 and fadB,may play an important role in the colonization of Z11 strain in the cecum during the late stage of persistent infection,while no genes closely related to the late stage of infection were found in Peyer’s patches.Furthermore,43 genes,including csrA,may be closely related to the early colonization of Z11 strain in the cecum and Peyer’s patches.2 Biological identification of candidate genes for persistent infection.To investigate the role of these genes in persistent infection of Z11 strain,we constructed 9 umarked in-frame deletion mutant strains of genes,such as KMIPBBJO₀1249 and fadB,and researched the biological function.The results showed that all deletion mutant strains had no significant in growth ability compared to wild strain（WT）.Motility results showed that the swimming ability of ΔKMIPBBJO₀1249,ΔfadB,ΔassT₄ and ΔnrdE was significantly decreased compared to WT,indicating that these genes could affect the motility of the Z11 strain.This study also measured the cytotoxicity,adhesion,invasion,and intracellular replication ability of these deletion mutant strains on RAW 264.7.The results showed that the cytotoxicity of ΔfadB to macrophages was significantly lower than that of WT,and the invasion ability of ΔKMIPBBJO₀1249 and ΔfadB was significantly lower than that of WT,indicating that KMIPBBJO₀1249 and fadB could affect invasion and cytotoxicity to host cells.The fadB gene encodes a fatty acids β oxidative multi enzyme complex which can participate in the utilization of fatty acids by Salmonella.Therefore,this study investigated the growth of different deletion mutant strains of Salmonella under the condition of different fatty acid as the sole carbon source.The results showed that the growth ability of ΔfadB is significantly inhibited when sodium octanoate was the sole carbon source.FadB may affect the utilization of fatty acid salts such as sodium octanoate by Salmonella in the host,providing favorable conditions for the colonization of Salmonella Enteritidis in the intestine.129 S2/SvE mice were used as a model to investigate the persistent infection ability of Z11,ΔKMIPBBJO₀1249 and ΔfadB.The results showed in the early stage of infection,the bacterial load of ΔfadB was significantly upregulated compared to WT in the cecum of mice,and in the late stage of infection,the bacterial load of ΔfadB was significantly lower than that of WT,indicating that FadB can participate in the process of persistent infection by affecting the intestinal colonization ability of Z11 strain.In the early stage of infection,the bacterial load of ΔKMIPBBJO₀1249 in the cecum of mice was lower than that of WT,and the bacterial load in the Peyer’s patches was significantly higher than that in Z11 strain,but there was no significant difference in the late stage of infection.Histopathological observations of the cecum and Peyer’s patches showed that the cecum of the mice had severe tissue damage,with a large number of ulcers,disappearance of intestinal gland necrosis,and infiltration of lymphocytes and granulocytes in the submucosa and mucosa.The degree of lesion in Peyer’s patches increased with the infection,but there was no significant difference between the infection groups of different strains.