Regulation Of The Tat System On The Motility Of Extraintestinal Pathogenic Escherichia Coli

Protein secretion system is involved in many important physiological functions in bacteria,and is related to the pathogenicity of pathogens closely.Twin arginine translocation(Tat)system is a special protein transport and secretion system existing in bacteria,which can insert fully folded proteins into cell membrane or transport them to periplasm.Tat system is crucial to the energy metabolism,cell division and pathogenicity of bacteria,and is suggested as a potential new antibiotic drug target.In recent years,porcine extraintestinal pathogenic Escherichia coli(ExPEC)is a kind of pathogenic bacteria which can cause systemic infections such as meningitis and pneumonia in porcine.It causes great economic losses to the porcine industry,and is also an important zoonosis pathogen,threatening public health and safety.Therefore,it is great significant to study the etiology and pathogenic mechanism of ExPEC for the prevention and control of the disease.Previous studies in the laboratory found that the deletion of Tat system led to a significant virulence decrease of the strain ExPEC PCN033.In addition,we found that the absence of Tat system affected the motility of ExPEC significantly,which is one of the important virulence factors of pathogens.In order to reveal the mechanism of the Tat system regulating the motility of ExPEC,we first used m RNA-seq to analyse the differential expressed genes(DEGs)in Tat system deletion strain,then we identified the substrate proteins of the Tat system which is related to the motility,finally we verified the signal pathways regulating the motility by gene deletion and phenotype experiments.The main results are as follow.1.The results of motility phenotype test show that the absence of Tat system lead to the loss of motility in ExPEC PCN033.By comparing the m RNA expression profiles of wild-type strain and ?tat deletion strain,566 genes are significantly up regulated and 218 genes are significantly down regulated(fold change > 2,P < 0.05,).Cluster analysis of DEGs showed that a large number of genes related to bacterial chemotaxis and flagellum assembly are expressed differentially,and the flagellum structure genes fli C and fli D are down regulated 5.3 fold and 4.5 fold in ?tat deletion strains,respectively.These results suggest that the deletion of Tat system causes the differential expression of a large number of genes in ExPEC,which leads to the loss of motility by affecting the expression of genes related to flagellum synthesis.2.We verify the motility of 20 Tat substrates deleted strains,and find that the deletion of substrate Mdo D and the simultaneous deletion of Ami A and Ami C resulted in the significant motility decrease in ExPEC PCN033.It is found that the expression of Fli C and Fli D genes is significantly down regulated after the deletion of Mdo D.3.On the basis of ?tat and ?mdo D strains,we delete the Rcs signal transduction system.Through the several motility experiments,it is found that the double deletion strains PCN033?mdo D?Rcs and PCN033?tat?Rcs complement to the wild-type strain level.By complementing the Rcs system and the spot mutation Rcs(H-G)which could not be phosphorylated,we find that the strain had no motility after the Rcs system is complemented,but it still has motility after the Rcs(H-G)system is complemented.These results suggest that the loss of motility in ExPEC caused by the deletion of Tat system and Mdo D may be due to the activation of Rcs signal transduction system and the inhibition of the expression of fli C and fli D.In this study,we find that Tat system affects the motility of ExPEC by influencing the transcription of flagellum gene.Moreover,we identify the Tat substrate protein Mdo D,Ami A and Ami C related to motility.The phenotype experiments show that the motility of the strains with deletion of Tat system in ExPEC is complemented after deleting the Rcs signal transduction system.Therefore,we believe that the absence of Tat system leads to the inability to transport Mdo D,Ami A and Ami C substrates,which can activate the Rcs envelope stress response,thus inhibiting the expression of flagellum related genes,and affecting the motility.This study reveal the potential relationship between Tat system and bacterial motility for the first time,and preliminarily clarify the signal pathway of Tat system regulating the motility of ExPEC,which can make a foundation for further revealing the function of Tat system and studying the pathogenic mechanism of ExPEC.