Mechanisms By Propionate Affect The Virulence Of Salmonella Enterica Serovar Typhi By Promoting Propionylation Of PhoP K102 And Fis K32

Propionate(PA)is one of the major components of intestinal short-chain fatty acids and plays an important role in inhibiting infection by enteropathogenic bacteria,but the exact mechanism of action is not fully understood.Salmonella enterica serotype Typhi(S.Typhi)is a human-specific enteropathogenic bacterium in the Enterobacteriaceae family,S.Typhi infection remains a major public health problem worldwide.Propionylation,a novel posttranslational modification of proteins,can affect protein activity by altering the ability of proteins to bind DNA,enzymatic activity,and protein structural stability.It plays an essential role in gene expression,protein function,and other aspects.In addition,the level of propionylation modification is influenced by propionate.Therefore,there is a need to investigate in depth the mechanism of action of propionate through propionylation affecting the pathogenicity of S.Typhi.Objective:In this study,we investigated the relationship between propionate modification of S.Typhi and its pathogenicity by altering the level of propionylation modification.We explored the key virulence regulators that differed significantly in propionylation modification after propionate treatment.The exact role and regulatory mechanism of S.Typhi pathogenicity after propanoylation modification were investigated.Methods:1.Exploring the effect of propionate on global propionylation modifications in S.TyphiBacteria were cultured under different culture conditions,total proteins were collected,and the overall level of bacterial propionylation was measured by Western blot using propionylation antibodies to compare the effect of different culture conditions on propionylation in S.Typhi.The peptides were detected by liquid chromatography-mass spectrometry(LC-MS)and compared with the database for global differential analysis of S.Typhi propionylation modifications and identification of differential propionylation proteins and loci.2.Validation of the propionyl CoA synthase Prp EA homologous recombination method mediated by the suicide plasmid p GMB151 was used to construct the ΔprpE mutant strain,and the propionyl CoA concentration was measured by ELISA between the WT and the ΔprpE under different propionate conditions.The differences in the overall propionylation levels between the WT and the ΔprpE under different propionate conditions were also observed by Western blot3.Validation of the PhoP K102 propionylation modification siteThe in vivo expression strain ΔphoP::p CDSSphoP was constructed from the p CDSS plasmid,and the presence of propionylation modification at the K102 site was confirmed by mass spectrometry analysis after the expression of purified PhoP.Analysis of the PhoP protein structure through the Uniprot database and amino acid sequence alignment of the PhoP K102 locus using Bio Edit 7.0 software to analyse the conservation of the protein locus;Q and R mutations at the K102 site were constructed to mimic full and non-propionylated modifications,and the purified wild-type PhoP(WT)and mutant PhoP(K102Q and K102R)recombinant target proteins were expressed in E.coli BL21;the PhoP protein was propionylated in vitro using different concentrations of propionyl coenzyme A,and propionylation antibodies were used.The difference in propionylation between PhoP K102 Q and PhoP(WT)was observed by Western blot.4.Exploring the effect of PhoP K102 propionylation on the pathogenicity of S.TyphiIn vitro phosphorylation of wild-type PhoP(WT),mutant PhoP(K102Q and K102R)proteins using ACP was followed by Phos-tag gel analysis of protein phosphorylation levels;in vitro phosphorylated PhoP proteins were observed by gel migration assay(EMSA)for altered DNA binding capacity;differential phoP back-complement strains were constructed by p BAD plasmids and electrically The p FPV25.1 fluorescent plasmid was transferred into the S.Typhi intracellular survival assay to observe the changes in the intracellular survival of S.Typhi.q RT-PCR was performed to detect the expression levels of ssr A,ssr B,and spi C.5.Validation of the Fis K32 propionylation modification siteThe Fis protein was purified by expression of the p ET28 a plasmid in E.coli BL21,and the presence of the propionylation modification at the K32 site was confirmed by mass spectrometry analysis.Analysis of the Fis protein structure through the Uniprot database and amino acid sequence alignment of the Fis K32 locus using Bio Edit 7.0 software to analyse the conservation of the protein locus;Q and R mutations at the K32 site were constructed to mimic full and non-propionylated modifications,and the purified wild-type Fis(WT)and mutant Fis(K32Q and K32R)recombinant target proteins were expressed in E.coli BL21;the Fis proteins were propionylated in vitro using different concentrations of propionyl CoA,and the differences in propionylation between Fis K32 Q and Fis(WT)were observed by Western blot using propionylation antibodies.6.Exploring the effect of Fis K32 propionylation on the pathogenicity of S.TyphiIn vitro purified differential Fis proteins were observed for changes in DNA binding capacity by gel migration assay(EMSA);differential phoP back-complement strains were constructed by p BAD plasmid and electrotransferred into p FPV25.1 fluorescent plasmid to observe changes in intracellular viability of S.Typhi using THP-1 intracellular survival assay,q RT-PCR to detect ssr A,ssr B,and spi C expression levels;S.Typhi invasion ability was observed using the T84 invasion assay in intestinal epithelial cells,and iagA,inv F,and inv H expression levels were detected by q RT-PCR.Results:1.S.Typhi was extensively propionylated,and the propionylation modifications were significantly enhanced by the addition of 20 mM propionate to the culture.Analysis of the propionylation proteomic expression profile resulted in the identification of 4858 propionylation sites on 1382 proteins.A total of 491 proteins with 1370 sites elevated and 2proteins with 2 sites decreased after propionate incubation were identified based on the expression abundance ratio of propionylated proteins WT+PA/WT > 1.5 or WT+PA/WT <1.5 as a criterion for differential propionylated proteins.Bioinformatic analysis showed that the differential prolylated proteins had a major impact on the activation of enzymatic activity and RNA binding capacity in molecular function,and enrichment of the KEGG pathway showed that the differential prolylated proteins were involved in 14 different metabolic pathways,mainly at the ribosome,TCA cycle,and fatty acid synthesis nodes.Structural domain enrichment analysis of differential prolylated proteins showed that the structural domains of proteins that undergo prolylation are mostly bacterial DNA-binding regions and S1 RNA-binding regions.2.The viability of S.Typhi was significantly reduced in THP-1 macrophages after propionate incubation(P<0.0001),and the expression of associated ssr A,ssr B,and spi C was significantly reduced(P<0.001).In vivo expression of PhoP protein propionylation modifications was enhanced with increasing propionate concentration.The mass spectrometry results verified the presence of propionylation at the PhoP K102 site,which is highly conserved.PhoP can be propionylated by co-incubation with propionyl CoA in vitro,and the Q mutation at the K102 site reduced the propionylation of PhoP.The DNA binding capacity of PhoP K102 Q was significantly lower than that of wild-type PhoP and slightly lower than that of PhoP K102 R,and PhoP K102 Q The expression of ssr A,ssr B,and spi C was significantly lower(P<0.0001),and the viability in THP-1 cells was significantly lower(P<0.05)than that of the wild-type phoP back-complemented strain(P<0.01)and lower than that of the K102 R mutant phoP back-complemented strain(P<0.01)and the wild-type phoP back-complemented strain(P<0.01)and the wild strain of the empty plasmid control strain(P<0.0001).3.The concentration of propionyl CoA in WT increased with increasing propionate concentration in the medium at sodium propionate concentrations below 10 mM(P<0.001).Prp E could inhibit ssr B and spi C expression by affecting propionylation levels(P<0.01).4.The Fis protein expressed in E.coli BL21 can be propionylated by propionyl CoA in vitro.Mass spectrometry results verified the presence of propionylation at the Fis K32 site,which is highly conserved.The Q mutation at the K32 site reduced the propionylation of Fis.The DNA binding capacity of Fis K32 Q was significantly lower than that of wild-type Fis and Fis K32 R.The K32 Q mutant K32 Q mutant fis back-complemented strains were significantly less able to invade intestinal epithelial T84 cells than wild-type fis back-complemented strains(P<0.01)and K32 R mutant fis back-complemented strains(P<0.05),and the expression of iagA,inv F,and inv H was significantly lower than that of wild-type fis back-complemented strains(P<0.001).The viability of the K32 Q mutant fis-complemented strain was also significantly lower than that of the wild-type fis-complemented strain in THP-1 cells(P<0.01),and the expression of ssr A,ssr B,and spi C was also significantly lower than that of the wildtype fis-complemented strain(P<0.001).Conclusion:Propionate entry into bacteria is activated by the propionyl CoA synthase Prp E to produce propionyl CoA,which significantly enhances the propionylation level of S.Typhi.The K102 site of the PhoP protein was modified by propionylation of propionyl CoA,which resulted in a loss of phosphorylation and reduced protein activity,inhibiting the expression of the corresponding genes of SPI-2 and reducing the survival of S.Typhi in macrophages.Expression of the corresponding genes reduced the ability of S.Typhi to invade intestinal epithelial cells and to survive in macrophages.Our study suggests that propionate acts as an environmental cue that can influence the activity of S.Typhi virulence regulators and influence the pathogenicity of S.Typhi.