Biological Characteristics And Molecular Mechanism Of Inducing Inflammation Of Avian Pasteurella Multocida PM0222 Protein

Avian Pasteurella multocida(P.multocida),as the main representative bacteria of Pasteuraceae,and Pasteurella,which can cause avian Pasteurella disease(avian cholera)in poultry,mainly characterized by acute death and hemorrhagic septicemia.Avian cholera is a serious threat to poultry worldwide and causes significant economic losses.The virulence factors of P.multocida mainly include capsular,lipopolysaccharide,outer membrane protein,fimbriae and flagella,toxin protein,sialidase and hepcidin,which are closely related to the biological characteristics of P.multocida,such as hyaluronic acid synthesis,serum bactericidal activity,cell adhesion and pathogenicity.Previously,our laboratory found a lysogenic phage gene cluster encoding 55 proteins(44.8 kb,Pmorf0179-Pmorf0233)in avian P.multocida virulent strain C48-1 by whole gene sequencing analysis.In addition,the isogenic gene of Pmorf0181,Pmorf0186 and Pmorf0194 in lysogenic phage gene cluster were deleted and the virulence of mutants were significantly reduced.The Pmorf0222 gene is located in the lysogenic phage gene cluster and encodes PM0222,which is a phage non-structural protein and belongs to the SPFH superfamily protein Hfl K/C-like protein.The biological characteristics of PM0222 protein and the molecular mechanism of inflammation have not been resolved so far.In this study,the Pmorf0222 gene isogenic deletion mutant was constructed by Ng Ago homologous recombination technology,and its biological characteristics of animal pathogenicity,colonization ability,cell adhesion ability,hyaluronic acid synthesis and complement mediated bactericidal activity in serum were identified.RNA-Seq technology and bioinformatics technology were used to analyze the differentially expressed genes between the mutant strain and the wild type strain to identify the biological process of PM0222 protein.To determine the molecular mechanism of PM0222 protein response to host inflammatory signaling pathway of innate immune,PM0222 protein was expressed and purified prokaryotic system.This study provided a basis for the pathogenic mechanism of avian P.multocida and laid a foundation for prevention and control of avian cholera.In order to analyse the biological characteristics of the PM0222 protein of the avian P.multocida virulent C48-1 strain,the isogenic Pmorf0222 mutant C48-ΔPmorf0222 mutant was constructed based on the Ng Ago technology,and the complementary plasmid p AL99-Pmorf0222 was electro-transformed into the mutant to obtain the complementary strain.The animal pathogenicity test showed that the pathogenicity of the mutant strain to mice was significantly reduced by comparison to the wild-type strain.Then,the bacterial loads of the mutant were found to decrease by 3.35-fold in the liver and by2.39-fold in the spleen further reducing the adhesion to the DF-1 and He La host cells by 8.11-fold and6.48-fold respectively.Subsequently,the PM0222 promoted the synthesis of hyaluronic acid,inhibited biofilm formation and complement-mediated bactericidal activity of avian P.multocida virulent C48-1strain.The significantly differentially expressed genes between the avian P.multocida virulent C48-1strain and C48-ΔPmorf0222 were compared by the RNA-seq analysis and bioinformatics analysis to determine the effects of the Pmorf0222 on P.multocida.A total of 314 significantly differentially expressed genes were enriched containing 174 upregulated and 140 downregulated genes.13 significantly differentially expressed genes were selected for real-time PCR and compared with RNA-Seq results,which were consistent.Subsequently,bioinformatics analysis showed that the significantly differentially expressed genes were enriched hyaluronic acid synthesis,biofilm formation and iron metabolism-related pathways of P.multocida,and similar to that evident in the in vitro detection.To further evaluate the inflammatory molecular mechanism of PM0222 protein of avian P.multocida virulent C48-1 strain.The soluble recombinant PM0222 protein were purified by procaryotic expression system,and further experiments showed that PM0222 protein can be secreted to the outside of cells by the outer membrane vesicles(OMVs)of P.multocida.The purified recombinant PM0222 was used for treating macrophages and can induce the release of TNF-α and IL-1β pro-inflammatory cytokines through the TLR1/2-MAPK and TLR1/2-MAPK signaling and inflammasome activation.The Pmorf0222 gene isogenic deletion mutant and complemented strain were successfully constructed in this study.The pathogenicity of C48-ΔPmorf0222 deletion mutant to mice was significantly reduced.The biological characteristics of PM0222 protein were elucidated,which was involved in hyaluronic acid synthesis,biofilm formation and complement mediated bactericidal activity in serum of avian P.multocida virulent C48-1 strain.RNA-Seq and bioinformatics analysis showed that significantly differentially expressed genes were enriched in hyaluronic acid synthesis,biofilm formation and iron metabolism of avian P.multocida virulent C48-1 strain.The recombinant PM0222 protein can induce innate immune response in host cells through TLR1/2-NF-κB/MAPK inflammatory signaling pathway.This study provides explaining for the molecular pathogenic mechanism of avian P.multocida,and provides a new strategy for the prevention and control of avian cholera.