Pathogenicity Of Outer Membrane Protein H Of Avian Pasteurella Multocida C48-3and Identification Of Its Receptor On The Surface Of CEF Cells

Our previous study has indicated that the targeted inactivation of ompH genedecreased the lethality of serotype A Pasteurella multocida in mice after intraperitonealinjection. In present study, the ompH gene was amplified by PCR excluding the regioncoding for signal peptide from genomic DNA of P.multocida C48-3, and the PCRproducts was cloned into the pQE-30expression vector to construct recombinantplasmid pQE-ompH. The recombinant protein OmpH (rOmpH) was expressed as afusion protein in E.coli M15cells transformated with recombinant plasmidpQE30-ompH. The expressed protein was purified under denaturing conditions usingNi-IDA agarose columns and the eluted proteins were analysed by SDS-PAGE.Differences of adhesion ability, serum resisnce and antiphagocytic activity between thewild-type strain C48-3, mutant strain ompH and complemented strain C48-3C werecompared by biological experiments. The receptors of OmpH on the surface of chickenembryo fibroblast (CEF) cells was identified by Ligand blot using the purified rOmpHand polyclonal antibodies against rOmpH, while the OmpH receptor was identified byMALDI-TOF Mass spectrometry.SDS-PAGE analysis revealed a single fusion protein band with a molecular weightof38kDa. Western blot analysis demonstrated that the rOmpH and native OmpH ofC48-3were recognized specifically by antibodies against rOmpH, suggesting that therOmpH is an immunogenic protein. Adhesion assay shows that the number of mutantstrain ompH adhered to CEF cells was significantly lower than that of wild-type C48-3and complemented strain C48-3C. Adhesion-inhibition assay showed that the adhesionof wild-type strain and complemented strain to CEF cells was significantly inhibited bytreatment of bacterial cells with antibodies against rOmpH as compared to normal rabbitserum. However, the treatment of mutant strain with the antibodies had no inhibitoryeffect on bacterial adherence to CEF cells. Serum sensitivity assay showed that themutant strain was sensitive to the bactericidal action of chicken serum, whereas thewild-type strain and the complamented strain were both resistant. Phagocytosis assayrevealed that the mutant strain was more phagocytosed than the wild-type strain and thecomplemented strain by mouse peritoneal macrophages. In addition, treatment ofwild-type strain and complemented strain with antibodies against rOmpH significantlyincreased phagocytosis, but does not affect the phagocytosis of macrophages to mutantstrain. Ligand blot assay showed that a single membrane protein of49kDa wassufficient to bind rOmpH in the CEF cells and chicken esophageal mucosal cells.MALDI-TOF spectral analysis indicated that the OmpH-binding protein was β subunitof ATP synthase. In addition, Ligand blot and indirect immune-fluorescence stainingshowed that the OmpH receptor were present in the surfaces of chicken and rabbitmucosal cell, but was not detected in the surfaces of bovine and swine mucosal cell. Inconclusion, the OmpH is an important virulence factor of serotype A P. multocida.