Effects Of Avian Pathogenic Escherichia Coli Outer Membrane Protein OmpA On Autophagy Of Host Cells

Avian pathogenic Escherichia coli(APEC)belongs to extra-intestinal E.coli(ExPEC),which is one of the common pathogens causing extra-intestinal infections in birds(e.g.chickens,geese,turkeys,etc.).APEC can cause local inflammation or systemic septicaemia infections,and is a serious threat to the health development of the poultry industry.A variety of virulence factors play important roles in the host infection process of APEC,such as adhesins,iron uptake systems,protectins,capsule,invasins,etc.As one of the major components of the outer membrane protein complex(OMPs),outer membrane protein A(OmpA)plays an important role in maintaining the stability of the outer membrane structure,participating in metabolism,assisting APEC in the adhesion and invasion,and involving in host immune regulation.Autophagy is a self-protective mechanism that has evolved to degrade misfolded proteins or damaged organelles.Besides maintaining the cellular homeostasis,autophagy also can protect the host against pathogens.At present,very little is known about the effect of the outer membrane protein OmpA of APEC on autophagy in host cells.In addition,it was found that the heat shock protein gp96,as an OmpA-binding receptor,could assist meningitis-causing E.coli to invade human brain micro vascular endothelial cells and disrupt the blood-brain barrier,thus causing meningitis.It has not been verified whether the chicken-derived heat shock protein Hsp108,which is a homolog of gp96,could assist APEC to invade host cells.In this study,we elucidated the pathogenic mechanism of APEC OmpA from the perspective of autophagy,and investigated whether OmpA could help APEC evading the autophagy mediated clearance in host cells.We further verified the interaction between OmpA and Hsp108,and identified whether the autophagy and infection mediated by OmpA was associated with Hsp108,and thus provide a basis for understanding the biological function of OmpA and its role in assisting APEC to escape the clearance from host cells.1 Prokaryotic expression and purification of the outer membrane protein OmpA from avian pathogenic Escherichia coliFirstly,the prokaryotic expression plasmid pET28a-ompA was constructed and transformed into E.coli BL21(DE3)cells for induced expression,and the recombinant protein was purified using Ni-NTA affinity resin.The proliferation ability of APEC E058 strain in OmpA-stimulated chicken macrophages HD11 cells was observed.The effect of OmpA on the production of inflammatory cytokines in HD11 cells was examined using qRT-PCR.As results showed,the prokaryotic expression plasmid pET28a-ompA was successfully constructed,and after induction and purification,a recombinant protein OmpA-His with a molecular weight of 38 KDa was obtained.The HD-11 cell infection results showed that OmpA played an important role in the invasion and intracellular survival of APEC E058.Stimulation of OmpA in HD-11 cells resulted in an increased expression of inflammatory cytokines IL-10,IL-6 and IL-10β.2 Effect of the outer membrane protein OmpA of avian pathogenic Escherichia coli on host cell autophagyTo clarify whether OmpA,an important virulence factor of APEC,could assist in APEC evasion from host cell clearance,this study investigated the effect of OmpA on host cell autophagy.The genomic DNA of APEC E058 was used as a template to recover the ompA gene fragment by PCR amplification,and then cloned into the eukaryotic expression vector pEGFP-N1.The constructed eukaryotic expression plasmid was transfected into chicken embryonic fibroblast cell line DF-1 to endogenous overexpress the OmpA protein,meanwhile,the purified OmpA-His protein was added exogenously to stimulate the HD11 cells.The effect of OmpA on autophagy of DF-1 and HD-11 cells was examined by transmission electron microscopy,immunofluorescence and Western blot.After EcoR I/Xho I enzyme digestion of the sequencing identified recombinant plasmid,two bands with size of 1038 bp and 4700 bp were obtained,which were consistent with the vector and the target fragment,indicating that the eukaryotic expression plasmid pEGFP-N 1-ompA was successfully constructed.After transfection of recombinant plasmid into DF-1 cells,65 KDa OmpA-EGFP fusion protein was successfully expressed as detected by immunofluorescence and western blot.Endogenous overexpression of OmpA in DF-1 cells caused an increased expression of autophagy marker protein LC3 Ⅱ.In addition,after exogenous stimulation of OmpA protein in HD 11 cells,the expression of LC3 Ⅱ protein showed an upregulated level with the enhanced protein concentration and the extended stimulation time.We observed autophagsomes in DF-1 cells by transmission electron microscopy and the increased punctae distrubution of GFP-LC3 in DF-1 cells after OmpA stimulation.These results demonstrated that OmpA could induce autophagy in host cells.Following studies showed that OmpA could inhibit the phosphorylation of AKT/mTOR/p70S6K protein and induce autophagy through this signaling pathway.Furthermore,OmpA affected the degradation of autophagy marker protein p62 and block DF-1 autophagic flux detected by tandem fluorescent-tagged LC3 reporter(RFP-GFP-LC3).LC3 turnover detection showed that OmpA could induce the accumulation of LC3 II protein in HD11 cells.Above results indicate that OmpA can cause autophagy in cells,but it blocks the autophagic flux and induces incomplete autophagy in cells,which will help to better understand the escape mechanism of APEC from the host cells clearance.3 Effects of heat shock protein Hsp108 on APEC invasion and OmpA-induced autophagyTo clarify whether chicken-derived Hsp108 protein can assist APEC in invading host cells and associate with the OmpA-induced autophagy,we firstly verified the interaction between OmpA and Hsp108,and then used CRISPR/Cas9 technology to knockout hsp108 gene and constructed the hsp108 gene knockout cell line.CCK-8 assay was used to detect the knockout of hsp108 gene on the proliferation of DF-1 cells.The APEC strain E058 was inoculated into the wild type DF-1 and hsp108-/-DF-1 cells to detect its invasion and intracellular proliferative capacity.The subcellular localization of Hsp108 protein was observed by immunofluorescence.Finally,the effect of OmpA on autophagy of hsp108-/DF-1 was detected by Western blot.As results showed,we confirmed the interaction between Hsp108 and OmpA by co-immunoprecipitation.Using pX458 gene editing plasmid,we successfully knocked out the hsp108 gene in DF-1 ceils.Western blot results showed that Hspl08 protein expression decreased significantly.The results of CCK-8 proliferation test showed that the deletion of hsp108 gene did not inhibit cell proliferation.Deletion of hsp108 gene did not affect the number of invading bacteria of APEC E058 in DF-1 cells,but significantly increased its intracellular proliferate level.Immunofluorescence results showed that Hsp108 protein was located in the cytoplasm.OmpA also induced autophagy in hsp108-/-DF-1,indicating that OmpA did not induce autophagy through Hsp108 protein.