Analysis Protein Composition And Inflammatory Effect Of Outer Membrane Vesicles In Haemophilus Parasuis And Construction Of LpxM Gene Deletion Strain

Haemophilus parasuis(H.parasuis)is a conditional pathogenic bacteria that exists in the upper respiratory tract of pigs.When pigs are infected with H.parasuis,the onset is mainly a series of inflammatory reactions with a mortality rate of up to 50%.The aquaculture industry has caused huge losses.Outer membrane vesicles(OMVs)can carry a variety of proteins,lipopolysaccharides and other pathogen associated molecular patterns(PAMPs)into host cells,inducing cellular inflammatory response and even death.H.parasuis can secrete OMVs,but its biological characteristics and effects on host cells have not been studied.In this study,the serum type 5 highly virulent strain H45 was used as the research object,and OMVs were purified by density gradient centrifugation.The structure and particle size of the OMVs were observed,and proteomic analysis was performed.The virulence and inflammatory effects on RAW 264.7 cells,and the effect of caspase-11 gene on OMVs-mediated inflammatory effects of cells;the homologous recombination technology was used to construct the H45 lpx M gene deletion strain,and to study the growth of the gene and its resistance to effects of serum bactericidal ability,biofilm formation ability,cytotoxicity and sensitivity of some antibiotics.Correlation analysis of experimental results using statistical methods,the results are as follows:1.OMVs extracted from H45 is spherical with a particle size distribution between 60-70 nm,with an average particle size of 63.32 nm,containing 478 proteins in H.parasuis,mainly derived from cytoplasm,cytoplasmic membrane,periplasm and outer membrane.Most of the proteins are related to important life activities of the strain.GO functional annotations showed that 47.9% of proteins are involved in biological processes,29.8% of proteins are involved in molecular functions,22.3% of proteins are involved in cell composition.COG functional annotations show that proteins are mainly involved in translation and ribose,body structure and formation,amino acid transport,and metabolism.These results suggest that OMVs may play an important role in the life activities of H.parasuis.2.H45 and its OMVs have a strong toxic effect on mouse monocyte macrophage RAW 264.7,causing cell death,activating inflammsome,and causing cells to secrete a large number of inflammation-related cytokines IL-1? and IL-18.OMVs were used to stimulate caspase-11-reduced cell lines,and IL-1? and IL-18 secretion levels were reduced.These results indicated caspase-11 was assosatied with inflammatory response on RAW264.7 cells stimulated by OMVs.3.The H45 lpx M gene deletion strain was successfully constructed,the growth rate of the deletion strain was slower than that of the wild strain before 8 h,and then remained the same.The biofilm formation ability of the deletion strain was reduced.The resistance of the deletion strain to 50% pig serum complement sterilization was significantly reduced.The deletion strains have reduced virulence on macrophages.The gene deletion strain has the same sensitivity to cefalothiophene and other 11 antibiotics,but to amoxicillin and clavi acid and sulfamethoxazole antibiotics changed from drug resistance to sensitivity,and ampicillin changed from intermediary to sensitivity.