Investigate The Mechanism Of Cinnamaldehyde Inhibiting Salmonella Typhimurium Based On ITRAQ Technology

Salmonella typhimurium?S.typhimurium?,a common pathogenic bacterium,could induce various infectious disease in poultry,domestic animals and human,possessing important public health significance.Cinnamaldehyde,one of the effective components in cassia twig,has certain inhibitory effect on S.typhimurium.However,the inhibitory mechanism of cinnamaldehyde against S.typhimurium is still not clear.Therefore,this thesis explored the inhibitory mechanism of cinnamaldehyde against S.typhimurium in vitro,and discovered the targets for cinnamaldehyde.The research was consisted of three parts:1.Study on the antibacterial mechanism of cinnamaldehyde against S.typhimurium in vitro The minimum inhibitory concentration?MIC?of cinnamaldehyde against S.typhimurium ATCC14028 was tested using broth microdilution method.The effects of cinnamaldehyde on S.typhimurium proteins metabolism was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis?SDS-PAGE?.Besides,electric conductivity,DNA exosmosis and scanning electron microscope were also conduct to study on the antibacterial mechanism of cinnamaldehyde.The results showed that the MIC of cinnamaldehyde agaisnt S.typhimurium was 128?g/mL.At sub-inhibitory concentrations,cinnamaledehyde had no effect on growth of S.typhimurium.The peotein bands of treated with 64?g/mL cinnamaldehyde were faded compared with negative control.64?g/mL cinnamaldehyde can destroy the cell membrane and wall of S.typhimurium,and lead to increased conductivity and DNA leakage.Results indicated that cinnamaledehyde can restrain S.typhimurium growth,alter the permeability of cell membrane and cell wall,and suppress protein metabolism.2.Investigate the differently expressed proteins in S.typhimurium treated with cinnamaldehyde by iTRAQ-LC-MS/MSThe function and pathway of differently expressed proteins were defined by iTRAQ-LC-MS/MS,and functions and cell pathways related with these proteins were researched according to bioinformatics.2212 proteins were detected.We found that 73proteins were up-regulated and 82 proteins were down-regulated when S.typhimurium treated with 64?g/mL cinnamaldehyde.Gene Ontology showed that differently expressed proteins involved 10 cellular components,13 biological processes and 9 molecular functions.These differently expressed proteins related 57 pathways by KEGG pathway functional annotation.These pathways mainly concentrate in pantothenate and CoA biosynthesis,cyanoamino acid metabolism and nitrotoluene degradation pathways.Results showed that cinnamaldehyde had an impact on the biosynthesis and metabolism process of amino acids in S.typhimurium,and then inhibit protein biosynthesis process.Meanwhile,cinnamaldehyde influenced translation levels of redox proteins,energy metabolism,drug-resistant related proteins,and some virulence factors.3.The regulatory effects of cinnamaldehyde on the virulence factors of S.typhimuriumThe transcriptional level of virulence genes were verified by reverse transcription fluorescent quantitative PCR?RT-PCR?,using 16s as house-keeping gene.Data was analyzed according to 2^-??Ct.Transmission electron microscopy was used to detect effect on fimbria.Results showed that after co-cultured with different concentrations of cinnamaldehyde,transcriptional levels of invB,CR079-01855 and fimA were decreased,while transcriptional levels of sipA,prgI and fimW were up-regulated.Under transmission electron microscopy,we found that numbers of fimbria were sharply decreased after co-cultured with cinnamaldehyde.The results indicated that inhibitory mechanism of cinnamaldehyde against type I fimbria of S.typhimurium could be increasing transcriptional expression of nagetive regulator fimW.In conclusion,cinnamaldehyde could inhibit the growth of S.typhimurium in vitro.The inhibitory mechanism of cinnamaldehyde against S.typhimurium may be the following ways:?1?cinnamaldehyde suppressed the synthesis and metabolism of amino acids,inhibit the synthesis of proteins,thus affected the physiological processes of S.typhimurium;?2?cinnamaldehyde destroyed cell membrane of S.typhimurium,revealing cell contents,causing physiological and metabolic disorders of bacteria,and eventually leading to death;?3?cinnamaldehyde could decreased the pathogenicity of S.typhimurium by down-regulating virulence factors of S.typhimurium.