Pathogenic Effect And Regulation Mechanism Of KdpDE And YgeK In Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli(APEC)is an important bacterial pathogen harmingthe poultry industry,and its pathogenic mechanism is unclear.Two component system(TCS)and Escherichia coli type three secretion system 2(ETT2)play important roles in APEC's pathogenicty.Studies have reported that the two component system KdpDE and the ETT2 transcription regulator YgeK affect the virulence of enterohemorrhagic Escherichia coli.However,there is no report on whether KdpDE and YgeK affect the APEC's pathogenicity.This study is aimed at exploring the role of the two component system KdpDE and the ETT2 transcription factor YgeK in APEC.1.Identification and analysis of TCS and ETT2 gene clusters in APECThe sequencing analysis results showed that 25 APEC strains have 14 ST types,17serotypes,23 virulence factors;TCS is widely in APEC strains and Kdp E is highly conserved in different strains;there are 4 types of ETT2 gene clusters(the strain AE81carries a complete ETT2 gene cluster);the sequence YgeK in AE81 is same as that in E.coli O157:H7(except 66^th)but has an additional 189 bases at the 5'end than that in E.coli K-12.This work provides APEC genome characteristics for next research.2.Study on the role of KdpDE in APECThe KdpDE mutant strain was constructed,and the gene transcription level was detected.KdpDE significantly up-regulated the expression of flagellar assembly genes fli P and fli R,and significantly down-regulated the expression of flagellar transcription repressor regulators yjj Q and bgl J(P<0.05);there were long and curved flagella on the surface of the wild strain,and deletion of KdpDE resulted in loss of flagella and motility ability;in 12.5%,25%and 50%SPF serum concentration,the survival ability of the mutant strain was significantly lower than that of the wild strain(p<0.05),and the LD₅₀of the wild strain was twice as much as that of the wild strain.This work proves that KdpDE participates in APEC's pathogenicity.3.Study on the effect of YgeK in the pathogenicity of APECWithout YgeK the flagella of the mutant strain was damaged,the biofilm structure was sparse,and the sensitive to hydrogen peroxide was lower;the survival ability of the mutant strain in the SPF serum and the adhesion ability of the mutant strain to DF-1 cells were both significantly lower than that of the wild strain(p<0.05);the SDS-PAGE gel analysis showed that the extracellular protein of the wild strain was higher than that of the mutant strain;proteomics sequencing identified 213 differential proteins(Qvalue<0.05 And Foldchange>1.2),using PSORTb software to predict that 188differential proteins are involved in subcellular localization,and 25 proteins are secreted into the extracellular or trapped cytoplasm;the pathogenicity of APEC is significantly reduced after YgeK is deleted.This work proves that the transcription factor YgeK plays an important role in the pathogenicity of APEC.4.Study on the regulation of YgeK in APECIn order to explain the influence of transcription factor YgeK on APEC's multiple biological processes and pathogenicity,two-omics was used to explore the regulatory mechanism of YgeK in APEC.The transcriptome sequencing technology released that YgeK significantly down-regulated 137 genes and up-regulated 138 genes(p<0.05and Fold-change>2.0);The i TRAQ sequencing identified 91 differential proteins in different regulatory pathways,among which the flagellar apparatus pathway and the bacterial chemotaxis pathway are associated with the protein Fli N and Fli G,and the TCS pathway is associated with Fli G and Fli C.The TCS is associated with the bacterial chemotaxis pathway through Che Y,and with the metabolic pathways of different environmental microorganisms through Nar Y and Nar G;30 proteins and genes are differentially expressed,of which 18 proteins and genes have the same change trend.Compared with the STRING database,Fli C,Fli D,Fli F,Fli N,Flg E,Flg L,and Che Z have interactions.This showed that YgeK connects different pathways and plays a regulatory role through flagella-related proteins.In this study,the genome characteristics of 25 APEC strains were analyzed,and the influence of KdpDE in APEC on the pathogenicity of APEC was investigated.The function of YgeK was explored and the regulatory pathways involved in YgeK were analyzed at the gene level and protein level.All of these provide a data basis for in-depth exploration of the pathogenic mechanism of APEC.