Study On The Mechanism Of Salmonella SlyA Regulating Host Gene Expression

Salmonella is a foodborne pathogen,which mainly lives in the intestinal cavity of humans and animals,can spread between hosts.It can cause intestinal infection and diarrhea.Severe infection can lead to host death.Statistics show that Salmonella poisoning accounts for the first place among food poisoning cases around the world,and the proportion of bacterial food poisoning caused by Salmonella in China can reach 70% to80%.SlyA protein,a pathogenic transcriptional regulator of Salmonella enterica serovar Typhimurium,is encoded by sly A gene.sly A gene is the intermediate link to initiate the expression of genes related to Salmonella pathogenicity islands 2(SPI-2)of S.Typhimurium.Its transcriptional regulatory protein SlyA is related to virulence,antioxidant stress,antimicrobial peptides and the survival of S.Typhimurium in macrophages.Through the bioinformatics analysis of the amino acid sequence of SlyA in the previous laboratory,it was found that there was an “AKRIKL” sequence with high similarity to nuclear location signal(NLS)between 89 and 94 amino acids.NLS is a short peptide containing basic amino acids,which is a sufficient and necessary signal fragment that mediates the entry of some proteins into the nucleus.It plays an important role in regulating the nuclear import of proteins.When protein transport into the nucleus,NLS can be recognized by the corresponding nuclear transporters,which can interact with nucleoporin to help NLS-containing proteins reach the nucleus through nuclear pore complex(NPC).The entry of NLS-mediated protein into the nucleus not only allows basic replication and transcription,but also plays a key role in regulating the cell cycle,circadian rhythm,and many other processes.Different from the signal peptides of general proteins,the NLS of nuclear localization proteins can be located in almost any part of the protein sequence.Because it participates in the whole process of protein function,it is generally not removed.S.Typhimurium is a kind of pathogenic bacteria that can infect host cells and reproduce in host cells,so whether the NLS sequence in SlyA can mediate the entry of SlyA into the host cell nucleus and regulate host gene expression during Salmonella infection,and then regulate the intracellular survival of Salmonella.To verify whether SlyA can enter the nucleus of host cells,SlyA protein was heterogeneously expressed in He La cells.A laser confocal technique was used to observe the sublocalization of SlyA protein in He La cells.It was found that the heterologous expression of SlyA protein could successfully enter the nucleus.To further explore the way in which SlyA protein reaches the cytoplasm when Salmonella infects host cells,Salmonella secreted protein Sop B is used as a positive control.By detecting the culture medium supernatant of Salmonella,it is concluded that SlyA protein does not belong to secretory protein,that is,SlyA can reach the cytoplasm of host cells only after Salmonella is killed by cells.After that,Western blot analysis was performed on the nucleoplasmic isolation of Henle-407 epithelial cells infected with S.Typhimurium for studying if SlyA can enter the nucleus of host cells infected by Salmonella,SlyA protein was then successfully detected in the nuclear components of intestinal epithelial cells infected with S.Typhimurium.It can be concluded that SlyA can enter the nucleus of host cells in the process of Salmonella infection.At the same time,by testing the intracellular reproductive ability and toxicity of S.Typhimurium and ?sly A strains,it was proved that the deletion of SlyA protein could weaken the intracellular reproduction of S.Typhimurium and reduce the toxicity to cells.In order to explore whether SlyA regulates host cell gene expression after entering the nucleus,three signal pathways that may be regulated by SlyA: MAPK/ERK signal pathway,P13K-Akt signal pathway and cancer pathway were predicted by RNA-Seq technique,and the MAPK/ERK signal pathway was verified.Western blot analysis showed that SlyA protein in Salmonella could activate the phosphorylation of ERK,JNK in MAPK pathway of Henle-407 cells.And SlyA protein has a certain transcriptional regulation on ERK and JNK itself.However,the regulation of SlyA on these two signal pathways needs to be further studied in the process of Salmonella infection.At the same time,the sly A mutant was constructed by ?-Red homologous recombination technology,and the polyclonal antibody of SlyA was successfully prepared,which laid a foundation for further study of the function of SlyA in the process of Salmonella infection.