Study On The Effect Of APEC Virulence Protein Hcp2 On Mitochondrial Damage In DF-1 Cells

Avian pathogenic Escherichia coli（APEC）,a widely spread extraintestinal pathogen,is a serious threat to the health of poultry industry worldwide.TypeⅥsecretion systems（T6SS）are widely found in Gram-negative bacteria and are involved in different pathogenic processes in bacteria.Among them,Hemolysin-coregulated protein（Hcp）is abundantly secreted in a T6SS-dependent manner and is involved in the anti-serum bactericidal,adhesion and environmental tolerance of bacteria,playing an important role in their pathogenic processes.Previous studies in our laboratory have shown that Hcp2 proteins are functionally diverse and involved in a variety of pathogenic processes such as bacterial motility,adhesion and intracellular colonization of target cells.However,the regulatory functions played by the effector proteins Hcp2a and Hcp2b during their interaction with eukaryotic cells remain to be revealed in depth.In this study,the T6SS gene cluster of 26 APEC strains was identified to analyze the distribution and genetic evolutionary characteristics of the T6SS gene cluster in APEC;the transcript levels of T6SS structural genes in APEC under serum bactericidal resistance were analyzed by serum culture assay combined with RNA-seq.To investigate the effect of Hcp2a and Hcp2b proteins on DF-1 cells,mitochondrial membrane potential,mitochondrial calcium ion concentration and cellular ROS levels were examined.In addition,the candidate reciprocal proteins in DF-1 cells were enriched by streptavidin-biotin Pull Down combined with LC-MS/MS;the binding ability of Hcp2a and Hcp2b proteins to the candidate reciprocal proteins was analyzed by homology modeling and protein-protein docking.The details and results of the study are as follows:1.APEC-T6SS gene cluster identification and transcriptomic analysis under serum culture In this study,the whole genome sequences of 26 avian pathogenic E.coli strains were analyzed,and the results showed that among the 26 APCE strains,10 strains carried complete T6SS1（38%）,15 strains carried T6SS2（58%）,and 7 strains carried unknown T6SS（27%）.Evolutionary characterization of the T6SS gene cluster of APEC strains showed that 10 strains APEC-T6SS1 were divided into three evolutionary branches and 15strains of APEC-T6SS2 were located in the same evolutionary branch,among which AE17-T6SS2 gene cluster had lower genetic variation.The differentially expressed genes of APEC AE17 after serum and LB culture were analyzed by transcriptomic sequencing.The results showed that a total of 1462 differentially expressed genes were screened,of which 961 genes were up-regulated and 465 genes were down-regulated.KEGG enrichment analysis showed that the differentially expressed genes were mainly enriched in Bacterial chemotaxis,Sulfur metabolism and Pantothenate and Co A biosynthesis and other signaling pathways.Among the differentially expressed genes,nine T6SS2 structural genes were down-regulated and one T6SS2 structural gene was up-regulated.2.Damaging effects of effector protein Hcp2 on mitochondria of DF-1 cellsIn this study,after transfection of DF-1 cells by electroporation using eukaryoticexpression vectors already constructed in the laboratory and overexpression of Hcp2a and Hcp2b proteins,a significant decrease in the number of mitochondria was observed by laser confocal microscopy（P<0.01）.The Hcp2a and Hcp2b fusion proteins were induced in prokaryotic expression and purified,and indirect immunofluorescence assay showed that Hcp2a and Hcp2b proteins were transduced into DF-1 cells after incubation with DF-1 cells for 6 h.In this study,we used JC-1,Rhod-2,AM and DCFH-DA fluorescent probes to label mitochondrial membrane potential,mitochondrial Ca²⁺and cellular ROS,respectively,and observed the fluorescence intensity by fluorescence inverted microscopy,and Image J performed semi-quantitative analysis of fluorescence area and total fluorescence intensity.The results showed that Hcp2a protein incubated with DF-1 for 6 h significantly decreased mitochondrial membrane potential（P<0.01）;significantly increased mitochondrial Ca²⁺level（P<0.01）;and enhanced cellular ROS fluorescence area area and total fluorescence intensity（P<0.05）.After 6 h incubation of effector protein Hcp2b with DF-1,the mitochondrial membrane potential was significantly reduced（P<0.01）;the mitochondrial Ca2+level was significantly increased（P<0.01）;the area of cellular ROS fluorescence region and total fluorescence intensity were significantly enhanced（P<0.01）.3.Interacting protein screening of effector protein Hcp2 in DF-1 cellsIn this study,the protein complexes obtained from the enrichment were subjected to SDS-PAGE separation and liquid chromatography-tandem mass spectrometry（LC-MS/MS）identification and analysis using the streptavidin-biotin system with Pull-down assay.The mass spectrometry results showed that 52 interacting proteins were screened for biotinylated Hcp2a;68 interacting proteins were screened for biotinylated Hcp2b.Functional analysis of these proteins was performed and four mitochondria-associated proteins（GOT1,LETM1,TRMU and SLC25A4）were screened.To explore the binding ability of Hcp2 protein to the candidate interacting proteins,the proteins were modeled in three dimensions by Alpha Fold and the model quality was assessed,which showed that the model quality met the requirements for subsequent analysis.Protein-protein docking was performed using Clus Pro,PEBe PISA to analyze the free energy of solvation（PISAΔⁱG）generated by binding,and DIMPLOT to analyze the interaction residues of Hcp2a and Hcp2b with candidate proteins.The results showed that Hcp2a protein had better binding ability with LETM1 protein,while Hcp2b protein had better binding ability with LETM1 and SLC25A4 protein.In summary,58%APEC strains were found to carry the T6SS2 gene cluster in this study.Transcriptomic analysis showed that the AE17-T6SS2 partial structure gene,which varied in transcript levels under serum culture conditions.Fluorescent probe specific labeling assays showed that the effector proteins Hcp2a and Hcp2b were able to transduce into DF-1cells and cause damage to mitochondria.Streptavidin-biotin Pull down combined mass spectrometry technique successfully screened several candidate reciprocal proteins,and protein-protein docking analysis showed that Hcp2a protein had better binding ability with LETM1 protein and Hcp2b protein with LETM1 and SLC25A4 protein.