Identification Of Protein Propionylation Modification Of Salmonella Enterica Serovar Typhi And Preliminary Shudy On Its Function

ObjectiveProtein post-translational modification is an important part of epigenetics research,while protein post-translational modification is widespread in bacteria and participates in almost all major physiological processes in bacterial cells,such as virulence,cell-cell interaction,transcription level regulation,and biofilm formation.In recent years,the research results on the regulation of post-translational modifications such as methylation and acetylation in prokaryotes are changing with each passing day,while the regulation of new post-translational modifications such as propionylation and butyrylation is weak.The degree of propionylation modification of protein group depends to a great extent on environmental conditions.For example,the short-chain fatty acids in the intestine can affect the propionylation of bacteria.In this study,the propionylation modification of Salmonella enterica serovar Typhi,an intestinal pathogen,was studied,and the functional changes of the corresponding protein after propionylation modification were deeply explored.Further research on propionylation modification will also promote the understanding of bacterial physiology at present,and provide basic theoretical support for the treatment and application research of related diseases caused by bacteria.Methods1.Western-blot detection of bacterial protein propionylation level:Bacteria were cultured under different culture conditions,and the total protein was extracted by collecting bacteria.The overall propionylation level of bacteria was detected by Western blot with propionylation antibody,and the effects of different culture conditions on the propionylation of S.Typhi were compared.2.Extraction of bacterial RNA and real-time fluorescence quantitative PCR and analysis:Total RNA of bacteria in LB culture and propionate culture were extracted respectively,and the expression differences of pat and cob B genes were compared by q RT-PCR after reverse transcription.3.Construction of mutant with gene deletion:Based on the homologous recombination mediated by p GMB151 suicide plasmid,the gene deletion strainsΔpat andΔcob B of Pat and Cob B were constructed respectively,and the corresponding gene double-defect strainsΔompRΔpat andΔompRΔcob B were constructed based onΔompR preserved in the laboratory.4.Construction of overexpression strain and empty vector control strain:The p BAD33 plasmid was used to construct p BAD33-pat overexpression vector,and the recombinant vector and p BAD33 empty vector were electrically transferred to WT,respectively,to construct overexpression strain WT-pat and its empty vector control strain WT-p BAD33.5.Expression and purification of protein in vitro:BL21 expression strain expressing OmpR protein was constructed based on p ET28a,and OmpR protein was induced by IPTG.The supernatant was collected by high-speed centrifugation after ultrasonic disruption,and His-OmpR recombinant protein was separated and purified by Ni column.S.Typhi expression strain of OmpR was constructed based on p CDSS plasmid,and the expression of OmpR protein was induced by arabinose.After ultrasonic disruption,the supernatant was collected by high-speed centrifugation,and the recombinant protein of His-OmpR was separated and purified by Ni column.6.In vitro acylation test:The purified protein was incubated in acylation buffer without or with a certain concentration of propionyl-Co A for 6 hours,followed by Western blot analysis by SDS-PAGE polyacrylamide gel electrophoresis and pan-propionyl antibody.7.Construction of site mutation strain:using p CDSS-ompR recombinant plasmid as a template,and constructing ompR K184Q and ompR K184R mutation plasmids according to the method of the site mutation kit,and respectively introducing into the corresponding S.Typhi for protein expression.The p BAD33-ompR recombinant plasmid was used as the template,and the ompR K184Q and ompR K184R mutant plasmids were constructed according to the method of site mutation kit and introduced into the corresponding S.Typhi strains for subsequent bacterial phenotyping tests,respectively.8.Electrophoretic mobility experiment:The purified protein OmpR and the corresponding site mutant protein were co-incubated with the ompR promoter fragment in a binding buffer,and the non-denatured polyacrylamide gel was used to conduct electrophoretic mobility experiment on the binding substances.The binding of OmpR and the OmpR promoter fragment was observed and analyzed by a gel imaging system.9.Hela epithelial cell invasion experiment:cells were inoculated in 24 well plates and equal amounts of the bacteriaΔompR-C,ΔompR-184Q,andΔompR-184R were added to the cultured cells according to the multiplicity of infection(MOI=20).After incubation for another 90 min,the cells were washed three times with PBS,and0.5%(v/v)Triton X-100 was added into a part of the wells to lyse the cells.After lysing for 15 min,the cells were smeared on LB plates and cultured overnight.The counted colonies were recorded as T0.The cells in the other part of the wells were further cultured with 100μg/m L gentamicin for 90 min and then lysed with0.5%(v/v)Triton X-100.After the same incubation for 15 min,the cells were plated on LB plate for overnight culture and the counted colonies were recorded as T90.T90/T0indicated the invasive ability of S.Typhi to He La cells.10.Intracellular survival experiment of THP-1 macrophages:THP-1 cells were added into a 24-well plate at the cell density of 2.5×10~5/m L per well,and PMA was added for induction and culture for 48 hours to induce macrophages.The multiplicity of infection(MOI)=20:1 was used to add the same amount of bacteriaΔompR-C,ΔompR-184Q,andΔompR-184R,respectively,for the cell infection test.A portion of the cells were incubated in a cell incubator for 1 h,and gentamicin with a final concentration of 100μg/m L was added for action for 1 h to kill extracellular bacteria.After the cells were lysed by Triton,they were plated onto LB plates.After overnight incubation,the number of colonies was counted and multiplied by the dilution multiple to obtain T0.The other part was put into the cell incubator for culture for 1 h,and gentamicin with the final concentration of 100μg/m L was added for action for 1h to kill the extracellular bacteria.The culture was continued for 24h,and the above cell lysis steps were repeated.At this time,the bacterial count was T24,and T24/T0indicated the survival and replication ability of S.Typhi in THP-1 macrophages.Results1.There is extensive propionylation in S.Typhi,and the propionylation level of S. Typhi can be significantly improved after adding propionate to the culture.2.Propionate culture can reduce the transcription expression of deacylase cob B but has no obvious effect on the transcription expression of acyltransferase pat.3.In S.Typhi,Pat participated in propionylation and Cob B participated in deacylation.4.OmpR can be propionylated by Pr-Co A in vitro.With the increase of Pr-Co A concentration,the propionylation level of OmpR increased significantly.5.The propionylation modification of OmpR is regulated by Pat and Cob B.6.The propionylation of OmpR 184 decreased the binding ability of OmpR protein to the ompR promoter.7.Compared withΔompR-C andΔompR-184R,ΔompR-184Q,which mimics propionylation of OmpR184 site,has a significantly decreased ability to invade Hela cells.8.Compared withΔompR-C andΔompR-184R,ΔompR-184Q,which mimics propionylation of OmpR184 site,has decreased its survival and replication ability in THP-1 cells.ConclusionsThis study found that there was extensive propionylation modification in S.Typhi,and the metabolism of propionate in bacteria was closely related to the propionylation process.On the whole,it is verified that the regulation mode of acylation and deacylation widely existing in prokaryotes plays an important role in the regulation of propionylation of S.Typhi,that is,acyltransferase Pat and deacetylase Cob B participate in the propionylation and deacetylation of Salmonella typhi.The propionylation mass spectrometry of bacterial protein showed that K184 of OmpR,a very important regulatory protein in bacteria,could be propionylated.Through the analysis of protein expression purification and acylation modification,the propionylation of OmpR protein was not enzymatically regulated by Pr-Co A,but also regulated by Pat and Cob B.When the propionylation of protein 184 was simulated,the EMSA test showed that the binding ability of OmpR to the ompR promoter was weakened,indicating that propionylation on OmpR protein affected the binding ability of the protein to DNA.To observe the effect of this mutation on bacterial phenotype,we constructed mutant strainsΔompR-184Q andΔompR-184R which mimic the propionylation of ompR184 site,and found that the cell invasion ability and intracellular survival and replication ability of ompR-184Q decreased obviously,which further confirmed that the propionylation regulation of S.Typhi protein affected the life activities and pathogenic characteristics of bacteria.