Primary Study Of Molecular Mechanism On RGD-binding Integrins Mediated The Phagocytosis Of Edwardsiella Tarda Into Mudskipper MO/MФ

The versatile fish pathogen Edwardsiella tarda is a gram-negative bacteria,which can invade and replicate in host phagocytes and has the characteristics of a wide range of infections.However,the mechanism mediating the uptake of E.tarda in mudskipper(Boleophthalmus pectinirostri)kidney-derived monocytes/macrophages(MO/MΦ)is not yet understood.Understanding of this mechanism will help to find suitable anti-disease strategies.Generating mudskipper kidney-derived MO/MФ transcriptomic resources from mudskipper challenged by E.tarda is crucial for understanding the molecular mechanisms underlying the mudskipper invasion process.In the present study,a total of 2 070 differentially expressed genes(DEGs)were identified using RNA-sequence(RNA-seq)after E.tarda infection for 8 h with mudskipper kidney-derived MO/MΦ,including 1185 up-regulated and 885 down-regulated DEGs.The Gene Ontology(GO)function enrichment and Kyoto Encyclopedia of Genes and Genome(KEGG)pathway analysis of DEGs revealed the centrality of the phagosome and regulation of actin cytoskeleton pathways in the process of E.tarda infection with mudskipper kidney-derived MO/MΦ.In these two pathway DEGs,the receptors associated with phagocytosis are mainly rich in integrin(ITGs)subunit genes,including ITG α5,β1,and β2,which reveal the role of these receptors in the phagocytosis of E.tarda by mudskipper kidney-derived MO/MΦ.The progress of phagosome formation of E.tarda phagocytosed by mudskipper kidney-derived MO/MΦ,especially the formation of the phagosome,was observed by transmission electron microscopy.Additionally,quantitative real-time PCR showed that the mRNA expressions of most ITGs were closely related to the different infection times of E.tarda and the infection of different bacterial pathogens.Further assays demonstrated the phagocytosis of FITC-E.tarda by mudskipper kidney-derived MO/MФ was significantly reduced by the tetrapeptide ArgGly-Asp-Ser(RGDS),indicating that the RGD site of ITGs was involved in mediating phagocytosis.Finally,we simulated the tertiary structure of mudskipper RGD-binding ITGs,including ITG α5β1,αVβ3 and αVβ5,and found that is similar to human ITG α5β1 and ITG αVβ3,and with same conservative RGD binding site.Besides,the outer membrane proteins containing RGD motifs were screened from E.tarda proteome,which may be the potential interaction pathogen protein of RGD binding ITGs and participate in the process of phagocytosis of E.tarda.In summary,phagocytosis is one of the ways E.tarda enters mudskipper kidney-derived MO/MΦ.The interaction between outer membrane proteins on the surface of E.tarda and RGD binding ITGs may be one of the ways to induce host phagocytosis.