Structural Characteristics Of Escherichia Coli Nissle 1917 Flagellin And Its Stimulatory Effect On Caco-2 Cell

Flagellin is a novel adjuvant with structural and functional diversity.Our research has been studying on flagellin of E.coli Nissle 1917(Fli C_{Ec N}),including flagellin targeting foreign antigens and Fli C_{Ec N}adjuvant activity,but the structure and functional properties of Fli C_{Ec N}have not been successfully analyzed.This study combines various technologies such as bioinformatics,spectral analysis,and immune detection to analyze the differential genes induced by flagellin based on the Gene Expression Omnibus（GEO）database,predict and analyze the structure and function of Fli C_{Ec N},analyze the structure of Fli C_{Ec N}and its mutant proteins through surface enhanced Raman spectroscopy（SERS）and circular dichroism（CD）,and detect the immune stimulation effect of Fli C_{Ec N}and its mutant proteins in vitro.This study aims to provide data reference for the development of more effective flagellin adjuvants.1.Analyzing the differential genes induced by flagellin based on the GEO databaseTo explore differentially expressed genes（DEGs）stimulated by flagellin and provide more accurate detection indicators for further research on its interaction with the body.Method:（1）Mining flagellin related datasets and screening DEGs induced by flagellin in the body;（2）Conduct GO functional enrichment analysis on DEGs;（3）Conduct KEGG signaling pathway analysis on DEGs;（4）Construct protein-protein interaction（PPI）networks of DEGs and screen high connectivity PPI submodules and Hub genes.The results showed that:（1）a total of 282 DEGs were screened;（2）The biological processes of GO enrichment mainly include signal transduction,immune response,inflammatory reaction,cell apoptosis,etc;The cell components of GO enrichment mainly include cell membrane,nucleus,plasma membrane,membrane bound organelle,and pericytoplasmic nuclear area;The molecular functions of GO enrichment mainly include kinase activation,receptor binding,cytokine activation,membrane signal transduction,etc;（3）DEGs participate in the KEGG pathway,covering cytokine-cytokine receptor interaction,TNF signaling pathway,NF-κB signaling pathway,Toll like receptor signaling pathway,NOD like receptor signaling pathway,etc;（4）The PPI network mainly consists of 2 high connectivity PPI submodules and 10 Hub genes（IL-10,IL-6,CXCL2,CCL2,NFKBIA,My D88,CCL7,ICAM1,JAK2,CCR5）.Conclusions:282DEGs induced by flagellin were screened,which participated in 56 GO biological processes,12 GO cell components,12 GO molecular functions,14 KEGG pathways,and PPI network including 2 high connectivity PPI submodules and 10 Hub genes.2.Prediction and analysis of the structure and function of Fli C_{Ec N}To analyze the structure of Fli C_{Ec N}and its interaction with TLR5 using artificial intelligence algorithms and molecular dynamics simulations.Methods:（1）Nucleotide and amino acid sequences of different serotypes of E.coli flagellin were obtained,multiple sequence alignment was performed and two-dimensional phylogenetic tree was constructed;（2）Predict and analyze the sequence characteristics of Fli C_{Ec N}using various tools,predict and analyze the tertiary structure of Fli C_{Ec N}and analyze key amino acids in the structure of Fli C_{Ec N};（3）Simulate the interaction between Fli C_{Ec N}and TLR5.The results showed that:（1）Fli C_{Ec N}is mainly in the same evolutionary branch as H1,H12,H45,and H49,with identical N-terminal and C-terminal sequences,and part of different amino acid compositions in the middle of the sequence;（2）The Fli C_{Ec N}D0-D1 domain is highly conserved,mainly composed of 40.34%ofα-helix,is the main region of interaction with TLR5;The D2-D3 domain is highly variable,mainly composed of 22.69%ofβ-sheet,β-turn and randomcoil（total proportion of 36.97%）,containing major antigenic epitopes,with strong hydrophobic fragments;β-sheet of Fli C_{Ec N}has more stable residues than other secondary structures;（3）The amino acid residues of Fli C_{Ec N}involved in TLR5 interactions are mainly located at theα-helix area and theβ-sheet area of Fli C_{Ec N};The key forces that make up the Fli C_{Ec N}-TLR5complex include hydrogen bonding,van der Waals forces,polar contact,and hydrophobic interactions,among which the energy contributed by hydrophobic groups accounts for the major part of the total free energy contribution of several interaction energy.Conclusions:The regions ofα-helix,β-sheet and hydrophobic peptide of Fli C_{Ec N}play an important role in promoting the correct conformation folding of Fli C_{Ec N},maintaining its three-dimensional structural stability,and interacting with TLR5.3.Analyzing the the structure of Fli C_{Ec N}and its mutant proteins by Surface enhanced Raman spectroscopy（SERS）and circular dichroism（CD）To analyze the structural characteristics of Fli C_{Ec N},Fli C_△174-506（deleting D2-D3 domain）,and Fli C_△274-406（deleting D3 domain）,laying a foundation for further exploring the correlation between flagellin structure and immune stimulation in vitro.Method:（1）SERS was used to analyze the structural characteristics of Fli C_{Ec N}and its mutant proteins;（2）CD was used to analyze the secondary structural composition of Fli C_{Ec N}and its mutant proteins.The results showed that:（1）Fli C_{Ec N}mainly contains characteristic amino acid peaks,main and side chain peaks,and does not contain disulfide bonds.Fli C_△174-506and Fli C_△274-406basically maintain the peak position of the main chain,with some side chains and characteristic amino acid peak positions lost.The loss area of Fli C_△274-406is relatively large.（2）Fli C_△274-406exhibits a negative peak near the wavelength of 192 nm,belonging to the positive band ofα-helix disappears,and the remaining CD bands are consistent with Fli C_{Ec N}and Fli C_△174-506.The secondary structure of Fli C_{Ec N}becomesα-helix（44.4%）,β-sheet（23.4%）,the secondary structure of Fli C_△174-506becomesα-helix（54.5%）,β-sheet（24.5%）,the secondary structure of Fli C_△274-406becomesα-helix（47.6%）,β-sheet（32.4%）.Conclusions:α-helix andβ-sheet is the main secondary structure that constitutes Fli C_{Ec N}and its mutant proteins.Fli C_△174-506maintains SERS peak positions and secondary structure composition similar to Fli C_{Ec N}.Most of the SERS peak positions of Fli C_△274-406and the characteristic band ofα-helix at a wavelength of 192 nm disappears.4.Stimulation effect of Fli C_{Ec N}and its mutant proteins on Caco-2 cellsPreliminary exploration of the changes in cytokine secretion levels of Caco-2 cells in response to Fli C_{Ec N},Fli C_△174-506and Fli C_△274-406.Method:Using Caco-2 cells as an in vitro model,adding a doses of 5μg/m L Fli C_{Ec N},Fli C_△174-506,and Fli C_△274-406to stimulate Caco-2cells separately.after 6 hours and 12 hours of stimulation,the cytokines IL-6,IL-10,and TNF-αsecretting in the cell supernatant were detected.The results showed that:After stimulating Caco-2 cells for 6 hours,compared to the control group,all three experimental groups increased the secretion of IL-6,IL-10,and TNF-αto varying degrees.After stimulating Caco-2 cells for 6 hours,compared to the control group,the secretion level of IL-6 in the Fli C_△174-506group was higher than that in the Fli C_△274-406group and the Fli C_{Ec N}group,but the difference was not significant（P>0.05）;There was no significant difference in the secretion level of IL-10 among the three experimental groups（P>0.05）;The secretion level of TNF-αin the Fli C_△274-406group was higher than that in the Fli C_△174-506group and the Fli C_{Ec N}group,but the difference was not significant（P>0.05）;After stimulating Caco-2 cells for 12 hours,compared to the control group,all three experimental groups increased the secretion of IL-6,IL-10,and TNF-αto varying degrees.The secretion level of IL-6 in the Fli C_△174-506group was higher than that in the Fli C_△274-406group and the Fli C_{Ec N}group,but the difference was not significant（P>0.05）;The secretion level of IL-10 in the Fli C_{Ec N}group was significantly higher than that in the Fli C_△274-406group（P<0.001）and the Fli C_△174-506group（P<0.05）;The secretion level of TNF-αin the Fli C_△274-406group was higher than that in the Fli C_△174-506group and the Fli C_{Ec N}group,but the difference was not significant（P>0.05）;As the stimulation time increased,the secretion levels of IL-6 in all three experimental groups showed a decreasing trend,but the difference was not significant（P>0.05）;The secretion of IL-10 in the Fli C_{Ec N}group and Fli C_△174-506group showed a upward trend,but the difference was not significant（P>0.05）;The secretion of IL-10 in the Fli C_△274-406group showed a downward trend and the difference was not significant（P>0.05）;The secretion levels of TNF-αin all three experimental groups showed a upward trend,but the difference was not significant（P>0.05）;Conclusions:The effect of Fli C_{Ec N},Fli C_△174-506,and Fli C_△274-406stimulating Caco-2 cells to secrete cytokines IL-10,IL-6,and TNF-αis different,with a complete Fli C_{Ec N}structure that can stimulate the secretion of IL-10 more,higher levels of IL-6secretion in the Fli C_△174-506group,and TNF-αin the Fli C_△274-406group is higher.In summary,this study conducted a preliminary analysis of the structure of Fli C_{Ec N}and its mutant proteins,combined with differential gene analysis induced by flagellin expression in the body,and validated the immune stimulation effect of Fli C_{Ec N}and its mutant proteins using an Caco-2 cell model.The results indicate that,α-helix,β-sheet and hydrophobic regions play an important role in maintaining the conformational stability of Fli C_{Ec N}and interacting with TLR5;The spectroscopy properties and secondary structure composition of mutant proteins lacking different domains of Fli C_{Ec N}were changed,and the secretion levels of various cytokines were different after stimulation of Caco-2 cells;The role of different domains of Fli C_{Ec N}in stimulating the secretion of cytokines by Caco-2 cells needs further exploration.