| As a member of the family Pasteurellaceae,Glaesserella parasuis(GPS)can colonize the upper respiratory tract of healthy pigs and invade the organism and cause Gl(?)sser’s disease under certain conditions,which can cause great damage to the pig industry.PilA is the major pilin subunit of GPS and also serves as an important adhesion factor.However,the specific adhesion mechanism remains unclear.In this study,newborn pig tracheal epithelial(NPTr)cells were taken as the research objects.By constructing the pilA deletion strain,complementary strain and expressing recombinant PilA,CD147-Fc and CD44-Fc proteins,the adhesion of GPS PilA to tracheal epithelial cells and its specific mechanism were explored at the cellular and protein levels using immunofluorescence,pull-down,bi-directional immunoprecipitation,Western blot and so on.These findings provide a certain theoretical basis for understanding the adhesion mechanism of GPS.The research results can be summarized into three main aspects:1.The deletion of GPS pilA inhibited bacterial adhesion to epithelial cells in vivo and in vitroThe hematoxylin-eosin(HE)staining showed that deletion of pilA reduced the pathological damage to the respiratory tract of piglets by GPS.Bacteria staining suggested that very few ΔpilA bacteria were found on the respiratory tract,indicating that PilA can mediate bacterial adhesion.WT,ΔpilA and CΔpilA were compared for cell adhesion using the newborn pig tracheal(NPTr)cell line.The ΔpilA strain exhibited substantial adhesion defects compared to the WT strain.Complementation of the ΔpilA strain with PilA restored adhesion to epithelial cells,indicating that PilA can affect bacterial adhesion both in vivo and in vitro.2.Screening of proteins interacting with PilA using Pull down-MSPull down-MS technique was performed to identify proteins that potentially interact with PilA.952 proteins were identified in the experimental group and 356 proteins in the control group.Through comparison and screening of the differential proteins,we finally identified two suspected interactions: CD147 and CD44.3.Direct interaction of PilA with CD147 and CD44 promotes adhesion of GPS to epithelial cellsConfocal microscopy analysis revealed that CD147 and CD44 accumulated in the vicinity of attached GPS on the surface of NPTr cells.This aggregation of membrane proteins was found to be closely linked to the expression of PilA in the context of GPS.To investigate the cellular candidate receptors,we overexpressed or knocked down CD147 and CD44 and then performed adhesion assays.The results showed that the expression of CD147 and CD44 on NPTr cells affected the adhesion of PilA-expressing GPS,but not pilA-deficient strains.Adhesion inhibition assays using anti-CD147 and anti-CD44 monoclonal antibodies,CD147-Fc,CD44-Fc,and rPilA demonstrated that all of these inhibited bacterial adhesion.Finally,the direct interaction between PilA and CD147/CD44 was confirmed through Pull down and ELISA experiments.In summary,our study revealed that PilA can mediate adhesion in GPS and explored the receptors that interact with PilA.These findings not only enrich our understanding of the pathogenic mechanism of GPS but also provide new ideas for its control. |
