Screening And Functional Analysis Of Key Genes Of Salmonella Enteritidis Affecting The Expression Of α-Defensin In Paneth Cells

Salmonella is an important foodborne zoonotic pathogen with more than 2600 serotypes.In recent years,Salmonella Enteritidis has replaced Salmonella Typhimurium as one of the most dominant serotypes in livestock and especially poultry and human infections in many countries including China.Intestinal epithelial cells are the first important barrier that need to breakthrough for intestinal pathogens such as Salmonella to achieve systemic infection.The mechanism of Salmonella Enteritidis invading the host through M cells has been well understood.However,the interaction between Salmonella Enteritidis and other important intestinal epithelial cells,such as Panes cells,remains to be further elucidated.Paneth cells(PCs)are a group of highly differentiated secretory epithelial cells that secrete large numbers of antimicrobial peptides(AMPs),which are the molecular basis for mediating the important functions of PCs in maintaining intestinal homeostasis and participating in intestinal innate immunity.In mouse small intestine,α-defensins are the main AMPs,among which DEFA5 and DEFA6 are the main types of α-defensins of concern because of their high content and antibacterial activity.After infection with Salmonella Typhimurium in an alpha-defensindeficient mouse model,the killing effect of the host on Salmonella is reduced.PCs also play an important role in host resistance to Salmonella infection.However,most of the current studies have focused on the effects of Salmonella infection on PCs,and have not yet delved into the molecular mechanisms of their interaction.In this study,the interaction model between Salmonella Enteritidis and PCs was established by comparing the effects of different Salmonella Enteritidis isolates Z11 and C50041 on the expression of AMPs in organoids.Then the Salmonella Enteritidis mutant library was used to screen the key genes affecting the expression of α-defensins in PCs by qRT-PCR,and the corresponding gene deletion strains were constructed.By analyzing the biological characteristics of its growth,biochemical characteristics adhesion and invasion,as well as its effect on the expression of AMPs in organoids,the effect of the key genes on the expression of α-defensin was clarified.The deletion strain was orally infected with C57BL/6 mice,and the expression of AMPs in small intestinal cryps and PCs,as well as the distribution and colonization of Salmonella in vivo,were analyzed to further clarify the effects of key genes on the expression of AMPs and Salmonella enteritidis infection in mice.This study lays the foundation for further understanding the pathogenic mechanism of Salmonella Enteritidis infection and the development of novel anti-infective agents.1 Screening of the key genes of Salmonella Enteritidis affecting the expression of α-defensin in Paneth cellsSmall intestinal organoids were prepared by isolating mouse intestinal crypts and using them as a model to compare the effects of Salmonella Enterititis Z11 and C50041 strains on the expression of AMPs in PCs.The results showed that Z11 had a more significant effect on the expression of AMPs in PCs.Due to the high cost of organoid preparation and low screening efficiency,intestinal crypts containing PCs were prepared and co-cultured with Z11.The effects of the number of intestinal cryps and co-culture time on the expression of AMPs were compared.An infection model and screening system were established with 2000 intestinal cryps per well,co-cultured for 0.5 h,and α-defensin expression was detected by qRT-PCR.The expression of DEFA5 and DEFA6 in α-defensin(DEFA)of PCs was detected by qRT-PCR after co-culturing the mouse intestinal cryps with different mutant strains of transposons(MOI=10)for 0.5 h.High-throughput screening of key genes affecting the expression of AMPs in PCs was performed by Z-score statistics.The mutant strains with large differences(Z score≥2 or ≤-2)were verified twice to determine the key genes.A total of 920 mutant strains were screened in this study.Among them,31 strains significantly up-regulated the expression of DEFA5,and 14 strains significantly down-regulated the expression of DEFA5.24 strains significantly up-regulated DEFA6 expression and 11 strains significantly down-regulated DEFA6 expression.Among them,17 strains significantly upregulated the expression of both DEFA5 and DEFA6,and 7 strains significantly downregulated the expression of both DEFA5 and DEFA6.The 24 mutants that significantly affected the expression of DEFA5 and DEFA6 were screened twice,and 9 mutants were obtained that could stably affect the expression of DEFA5 and DEFA6.The 9 mutant strains screened were gene located by PCR,sequencing and comparative analysis,laying the foundation for subsequent studies.2 Analysis of the effect of key genes of Salmonella enteritidis on the function of Paneth cellsThe in-frame mutation technique was used to successfully construct deletion strain Z11ΔgdhA,Z11ΔSEN1928,Z11ΔpolB,Z11ΔyabL and Z11ΔmdoG,comparing their differences with wild strain Z11 in terms of growth rate,biochemical reaction,adhesion and invasion,and the AMPs resistant ability.The effects of these strains on the biological characteristics of Salmonella Enteritidis were analyzed to explore its role in the pathogenesis of Salmonella Enteritidis infection.Compared with the wild type Z11 strain,the growth curves,biochemical characteristics and the ability to resist the killing of AMPs of the five deletion strains were consistent with the wild type Z11 strain,but the adhesion and invasion abilities of the five deletion strains were decreased to varying degrees.Among them,Z11ΔgdhA and Z11ΔSEN1928 showed significantly decreased adhesion and invasion rates.This suggests that it may have a greater impact on bacterial pathogenicity.To further verify the effects of gdhA,SEN1928,polB,yabL and mdoG genes on the expression of α-defensin in PCs,small intestinal organoids were co-cultured with the gene deletion strains Z11ΔgdhA,Z11ΔSEN1928,Z11ΔpolB,Z11ΔyabL,Z11ΔmdoG and wild type Z11,and the expression of different AMPs was detected by qRT-PCR.The results showed that the above five gene deletion strains could significantly affect the expression of AMPs.The expression of IL-17A,IL-22 and IFN-γ in the organoids of Z11ΔgdhA deletion strains were significantly decreased after 0.5 h of co-culture.The levels of IL-17A and IFN-γ in SEN1928 deletion group were significantly decreased.IL-22 was significantly reduced in the organoids of the Z11ΔpolB deletion strain group.After co-culture for 2 h,the expression of IL-17A,IL-22 and IFN-y in Z11ΔgdhA was significantly increased,while the expression of IL-17A,IL-22 and IFN-y in Z11ΔSEN1928 was not significantly changed,and the expression of IFN-y in Z11ΔpolB was significantly increased.To further verify the effect of the deletion strain on the expression of AMPs in Paneth cells,the Paneth cells sorted by flow cytometry were co-cultured with Z11 and Z11ΔgdhA for 0.5 h,and the expression of AMPs in Paneth cells was significantly affected,which was basically consistent with the results after co-culture with organoids.Furthermore,Z11ΔgdhA and Z11ΔpolB deletion strains were intragastric injected into C57BL/6 mice,and their effects on AMPs and bacterial colonization were analyzed in vivo.The expression of AMPs in the intestinal cryps of mice showed that the expression of DEFA5 and DEFA6 in Z11ΔgdhA group was significantly increased on day 1 after infection,and the expression of DEFA5 in Z11ΔpolB group was significantly increased,but the expression of DEFA6 was not significantly different.On day 3 after infection,the expression of DEFA5 and DEFA6 in each group was significantly lower than that in the Z11 group,indicating that gdhA and polB genes affect α-defensin expression in PCs.The colonisation of the liver,spleen and ileum of mice infected with wild strain Z11,Z11ΔgdhA and Z11ΔpolB deletion strains was analysed by colony counting.Compared with the Z11 strain,the Z11ΔgdhA strain colonized significantly less bacteria in the liver and spleen at 1 day and 3 days after infection.The number of bacteria colonized in the liver,spleen,and ileum in the Z11ΔpolB infected group was lower than that in the Z11 group,but the difference was not significant.It is speculated that Z11ΔgdhA attenuated its invasion of the small intestine due to the induction of high expression of intestinal antimicrobial peptides to be killed.These results suggest that gdhA gene may affect the pathogenicity of Salmonella Enteritidis infection by regulating the expression of antimicrobial peptides in the small intestine of mice.This study provides an entry point for further understanding the molecular mechanism of Salmonella Enteritidis breaking through the intestinal barrier to achieve invasion and pathogenicity,and is expected to provide a basis for the development of new targets for antibacterial drugs and new vaccine candidates for the prevention and control of Salmonella Enteritidis.