The Research Of T6SS-2 Effector Protein Hcp On The Biological Characteristics And The Regulatory Of Pathogenicity Of APEC

Avian Enterobacteriaceae caused by avian pathogenic Escherichia coli(APEC)has caused huge economic losses for poultry industry worldwide.To some extent,the numerous serotypes and complex pathogenic mechanism bring some difficulties for its prevention and treatment.Type VI secretion systems(T6SS)is one of many pathogenic factors and plays a vital role in the regulation of its pathogenicity.Hemolysin-coregulated protein(Hcp)is an important effector protein of T6 SS involved in many pathogenic processes,such as bacterial movement,inter-bacteria competition,adhesion,colonization in animal and plant hosts,target cell colonization levels and bacterial biofilm formation.However,the specific regulatory function of the effector protein Hcp in the immune response and resistance mechanism of the APEC infected spleen of the host immune organs and the interaction with the host cells remain unclear.In this study,the hcp2 a and hcp2 b gene deletion strain and complementation strain were constructed by Red recombination technology,and their biological characteristics were analyzed.The effect of hcp2 a and hcp2 b genes on the colonization of vital organs during APEC infection in chicks was investigated through tissue load measurement,then through transcriptomics sequencing,its immune regulation on the spleen during infection was explored.In addition,by constructing a eukaryotic expression vector and transfecting DF-1 cells,transcriptome sequencing was performed on DF-1 cells transfected for 24 h in order to explore the specific regulatory mechanism of effector proteins Hcp2 a and Hcp2 b on DF-1 cells.The research contents and results are as follows: 1.Construction of hcp2 a and hcp2 b gene deletion and complementation strains and analysis of biological characteristicsIn this experiment,AE17 was used as a wild strain,and the hcp2 a and hcp2 b gene deletion strain and complementation strain used by the low copy plasmid p STV-28 were successfully constructed by Red recombination technology.Growth curve measurement results show that hcp2 a and hcp2 b genes have no effect on bacterial growth rate.The results of exercise test showed that the exercise ability of the hcp2 b gene is significantly reduced(P<0.01)while the deletion of hcp2 a gene haven't obvious changed the mobility of AE17.The results of the biofilm formation ability showed that the hcp2 a and hcp2 b mutant strain is significantly enhance compared with the wild strain(P<0.01).The results of the antiserum sterilization ability showed that the anti-serum sterilization ability was significantly reduced after the deletion of hcp2 a and hcp2 b genes(P<0.05).Environmental tolerance test showed that hcp2 a gene mutant strain significantly reduce tolerance to acid and alkali environments(P<0.05),however,there is no change in antioxidant stress than wild strain.Meanwhile,the hcp2 b gene mutant strain is also no significant change in acid and alkali environments tolerance,but its ability to resist oxidative stress was significantly enhanced compared to wild strains(P<0.05).2.Effect of T6SS-2 effector protein Hcp on spleen transcriptome of chicks The wild strain AE17,the deleted strains ?hcp2a and ?hcp2b and the complementation strain Chcp2 a and Chcp2 b were infected with chicks for 24 h,the results of the tissue-borne bacteria test showed that the amount of bacteria in hcp2 a and hcp2 b deficient tissue(spleen,liver,lung,lung)significantly decreased compared to wild strains.Analysis results of transcriptomics sequencing of chick spleen showed that based on the conditions of ?log2foldchange??1 and P?0.05,the hcp2 a gene-deficient strain obtained 512 differentially expressed genes,of which 221 genes were upregulated and 291 genes down-regulated expression.The hcp2 b gene-deficient strain obtained 515 differentially expressed genes,of which 185 genes were upregulated and 330 genes down-regulated expression compared with the wild strain AE17.The differentially expressed genes screened by Real Time quality PCR were verified,which was consistent with the sequencing results,indicating reliability of the sequencing results.The KEGG database showed that the differentially expressed genes of ?hcp2a were enriched in the pathways of cytokine and cytokine receptor interaction,cell adhesion molecules(CAMs)and ECM-receptor interaction.The differentially expressed genes of ?hcp2b were enriched in the pathways of protein processing in endoplasmic reticulum,Cytokine-cytokine receptor interaction,Cell adhesion molecules(CAMs)and MAPK signaling pathways.3.Effect of T6SS-2 effector protein Hcp on the stability of DF-1 cytoskeletonThe recombinant eukaryotic expression vectors p EGFP-N1-Hcp2 a and p EGFP-N1-Hcp2 b were successfully constructed by double digestion and transfected into DF-1 cells.The fluorescence was observed at 24 h,48 h and 72 h after transfection.The results showed significant aggregation of fluorescent particles of effector protein Hcp2 a and Hcp2 b.Transcriptomics sequencing of DF-1 cells revealed that Hcp2 a has 46 differentially expressed genes,Hcp2 b has 59 differentially expressed genes,Real Time quality PCR was used to validate the randomly selected differentially expressed genes,which was consistent with the sequencing results,indicating the reliability of sequencing results.The KEGG database showed that the differentially expressed genes of Hcp2 a mainly enriched in immune-related pathways such as cytokine-cytokine receptor interaction and Toll-like receptor signaling pathway.The differentially expressed genes of Hcp2 b mainly enriched in endocytosis,phosphatidylinositol signaling system and calcium signaling pathway.In addition,the expression level of TPM4 gene related to cytoskeleton was greatly reduced in DF-1 cells overexpressing proteins Hcp2 a and Hcp2 b.The test results show that the effector proteins Hcp2 a and Hcp2 b result in decreased stability of the cytoskeleton after expression in DF-1 cells.In summary,this experiment successfully constructed the T6SS-2 effector protein hcp2 a and hcp2 b gene deletion and complementation strain,and hcp2 a and hcp2 b genes affect biological characteristics.T6SS-2 effector protein hcp2 a and hcp2 b gene deletion have effect on APEC colonization ability of liver,spleen,lung and kidney.hcp2 a and hcp2 b genes mainly affect the pathogenic mechanism of the host by regulating immune related pathways such as cytokines and cytokine receptor interaction and cell adhesion molecules(CAMs).The effector proteins Hcp2 a and Hcp2 b impact on the stability of the cytoskeleton by regulating the expression of TPM4 in DF-1 cells.