Study On The Mechanism Of Regulation Of The Mobility Of Salmonella

Salmonella is one of the main foodborne pathegens that is considered as a high risk causing intestinal diseases.Salmonella is capable to transmit through livestock and their faeces,eggs and polluted water etc.,subsequently infects animals and humans.The swimming motility of Salmonella is important for the invasion and adhesion of its potential host cells.Therefore,motility mechanism exploration can bring important theorical and practical significance for the prevention and control of Salmonella infection.It has been reported that Salmonella is swimming mediated by flagella,and its synthesis and assembly are controlled by third-order regulatory elaborately.However,the study on flagellar gene expression regulation and motility mechanism in Salmonella still remains unclear.In this study,our laboratory and collaborators isolated and identified a naturally defective swimming strain named Salmonella A60.The Salmonella A60 strain grew slight slowly and showed poor adhesion capability to mammalian cells(e.g.,Caco-2 cells)compared with a Salmonella strain NCTC10384.The results of electron microscopy showed that the A60 strain had no flagellum and extracellular protein showed the flagellin secretion was abnormal.Quantitative Real-time PCR detection of flagellar gene in A60 strain showed that the transcriptional expression of primary and secondary flagellar genes was normal,but the transcription level of the third-order regulatory factor Fli A and its target genes was in low expression.However,overexpression the Fli A in A60 strain could not restore the swimming motility of A60 strain,suggesting that in addition to the abnormal expression of Fli A and its target genes,other factors caused the loss of swimming motility of A60 strain.We further sequenced the flagellum gene of A60 strain and found there were mutations of Ydi V Q123 X and Mot B V239 M in A60 strain compared with NCTC10384.However,the wildtype Ydi V and Mot B could not be restored in the A60 knockout strain.The mutant of Ydi V Q123 X and Mot B V239 M was completed by NCTC10384 knockout strain,and the swimming was still normal.The above experiments indicated that the swimming defect of Salmonella A60 strain was weakened in cell adhesion,slow growth,and the fli A and its target genes in low transcription are one of the factors contributing to the loss of A60 motility,there are still other factors contributing to the loss of A60 motility to be clarified.Based on the functional analysis of Mot B V239 M,we further analyzed the effects of E97,E147,D217,R238,R260 and S262 of Mot B on the swimming movement in Salmonella were analyzed with point mutation.The results showed that after mutation of E97,E147,D217,R238 and R260 into A,the swimming ability of Salmonella was not affected.After S262 mutation to A,its swimming ability was significantly reduced.Further studies showed that the swimming ability of S262 of Mot B recovered to the same level as the wild-type strain after being mutated into T,D,E and W.After mutation into C,K,F,Y and L,their swimming ability was completely lost.After mutation into N,it shows very weak swimming ability.The mass spectrometry of Mot B showed that there was no phosphorylation at S262.The results of electron microscopy showed that the flagellum morphology of the mutant strain Mot B S262 A was normal.Growth curve analysis showed that the mutant strain of Mot B with swimming defect was slightly slower than the wild-type strain.The complement experiment of Mot B proton plug deletion strain showed that S262 was not the proton binding site.Relevant studies have laid a theoretical foundation for further elucidation of the action mechanism of S262 locus in the regulation of swimming motility of Salmonella,which is of great theoretical significance for the prevention and control of Salmonella.