Pathogenic Effects Of ClpV And Hcp On Tracheal Mucosal Epithelial Cells Of Chicks Infected With APEC

Avian pathogenic Escherichia coli(APEC),as a common gram-negative bacterium,which often causes chickens,ducks,geese and other birds disease.As APEC has the potential of zoonosis,it is of great public health significance to strengthen the prevention and control of APEC.There are many kinds of pathogenic factors in APEC.Type VI secretion systems(T6SS),as one of pathogenic factors in APEC,which plays an important role in the pathogenic process of APEC.Clp V protein is an energy-related protein in T6SS-2 which plays a role in providing energy in the process of T6SS-2 secreting effector protein.Hemolysin-coregulated protein is an important effector protein of T6SS-2,which not only participates in the pathogenic process of APEC,but also plays a role in bacterial motility,biofilm formation and bacterial competition.An ordinary way of APEC infection is through the respiratory tract.Clp V and Hcp are key proteins of T6SS-2,but the pathogenic mechanism of Clp V and Hcp in tracheal mucosal epithelial cells of APEC infected chicks is not clear.In order to explore the effect of T6SS-2 on tracheal mucosal epithelial cells,chicken trachea was infected with APEC strain AE17,and the pathogenic effect of AE17 on chicken tracheal mucosal epithelial cells was studied by transcriptome sequencing;The clp V gene mutant strain and complementation strain were constructed by Red recombination technology.And the clp V gene mutant strains were infected with chicks by chick tracheal and m RNA sequencing was performed.In addition,the△hcp2a and△hcp2b strains were infected with chicks by chick tracheal infection model and m RNA sequencing and proteomic sequencingwas performed.The specific research contents and results are as follows:1.Pathogenic effect of AE17 on tracheal mucosal epithelial cells of chicksThe chicks were inoculated with APEC strain AE17(50μL、1×10~8cfu/m L)by tracheal injection.Result showed that chicks became apathetic,loss of appetite,excreted yellow and green feces.Pathological results suggested that the trachea,liver,lung and spleen had edema,hyperemia and heteroneutrophil infiltration.The results of tissue-borne bacteria test showed that the colonization of bacteria in the trachea,liver,spleen,and lung at 4 h,8h,12 h,and 24 h postinfection,indicating that AE17 can infect other organs of chicks by tracheal injection.The m RNA sequencing results showed that the differently expression gene were analysed to be 436 and 340 at 12 h and 24 h.To confirm the results of RNA sequencing analyses,some differently expressed gene were verified by Real time PCR,and the results were consistent with the sequencing results,indicating the sequencing results were reliable.The KEGG database showed that cytokine-cytokine receptor interaction and the toll-like receptor signalling pathway were enriched at 12 h post infection,cell cycle and DNA replication pathway were enriched at 24 h post infection.1.Construction of clp V gene mutant strain and its effection on transcriptome of chick tracheal epithelial cellsIn this study,AE17 was used as a wild strain,the clp V gene mutant strain and complementation strain were constructed by Red recombination technology and the low copy plasmid p STV-28.Growth curve measurement results suggested that clp V had no effect in the growth performance.The motility test showed that the△clp V strain had lower motility than AE17 strain(P<0.05).The results of the biofilm formation ability showed that the clp V mutant strain was enhanced significantly compared with the wild strain AE17(P<0.05).Compared with wild strain AE17,the DF-1 cell adhesion ability of△clp V strain were increased(P<0.01),while declined in invasion capacity(P<0.01).The m RNA sequencing results showed that the differently expression gene were analysed to be 108and 59 at 12 h and 24 h.The KEGG database showed that cytokine-cytokine receptor interaction and protein processing in endoplasmic reticulum pathway were enriched at 12h post infection,tight junction and endocytosis pathway were enriched at 24 h post infection.3.Effects of mutant strains△hcp2a and△hcp2b on transcriptomics and proteomic sequencing of tracheal epithelial cells in chicks.The△hcp2a,△hcp2b mutant strains were infected with chicks by chick tracheal infection model.m RNA sequencing results showed that the different expressed genes were analysed to be 268 and 138 at 12 and 24 h when infected by△hcp2a strain,144 and 135 at12 and 24 h when infected by△hcp2b strain.The KEGG database showed that cytokine-cytokine receptor interaction and the toll-like receptor signalling pathway were enriched at 12 h and 24 h infected by△hcp2a strain.Toll-like receptor signalling pathway and protein processing in endoplasmic reticulum pathway were enriched at 12 h and 24 h infected by△hcp2b strain.Similarly,proteomic sequencing was performed on epithelial cells of chick tracheal after 12 h of infection.The result suggested that different expressed proteins were analysed to be 79 and 107 in the two groups.KEGG database showed that TGF-βsignalling pathway,and PPAR signalling pathway and ECM-receptor interation pathway were enriched at 12 h infected by△hcp2a strain and△hcp2b strain.The different expressed protein STING was verified by Western blot,and the results were consistent with sequencing.In summary,this test found that AE17 can infect other organs of the chicks by tracheal injection,The m RNA profile of chick tracheal epithelium was dynamic at different times.The different expressed genes were found to be involved in cytokine–cytokine receptor interaction and cell cycle pathway.T6SS-2 clp V mutant strain and complementation strain were constructed by Red recombination technology,and it’s found that clp V affected the biological characteristics of APEC.clp V gene mainly affected the pathogenic mechanism of the host by cytokine-cytokine receptor interaction pathway.The different expressed genes were enriched in cytokine-cytokine receptor interaction pathway when the chick tracheal infected by△hcp2a and△hcp2b strains.Proteomic sequencing was showed that hcp2a and hcp2b genes mainly affected the pathogenic mechanism of the host by TGF-βsignalling pathway and ECM-receptor interation pathway.