| H.parasuis is currently an important bacterial disease that seriously threatens pig farms.The sialidase of H.parasuis is related to bacterial adhesion,virulence and stimulation of the host to produce antibodies,however it interacts with host immune cells and the mechanism regulating the inflammatory response is unclear.The Siglecs receptor that binds sialic acid on the surface of immune cells,can bind to sialic acid ligands on the surface of host cells or pathogenic microorganisms.This project aims to explore whether Haemophilus parasuis sialidase regulates the host inflammatory responses by interfering with the interaction of the sialic acid receptor(Siglecs)and the ligand of the host through cell-level studies by constructing a sialidase gene mutant of H.parasuis and expressing Nan H recombinant protein.It has been found that the sialidase of H.parasuis changes the role of Siglecs receptors on the cell surface and mediates the inflammatory response by MAPK and NF-κB pathways.It provides a theoretical basis for revealing the pathogenic mechanism of H.parasuis.The key research findings were as follows.1.Characterization of the Δnan H::kan mutant and the r Nan H proteinThe Δnan H::kan mutant and its complementary strains were constructed by natural transformation or electrical transformation.The growth curve results showed that the growth of the wild strain was significantly faster than Δnan H::kan mutant strains and the complementary strains.Gram staining showed that the wild strains appeared in a single,short rod-shaped form,and some of the Δnan H::kan strains were clusters,while the complementary strains appeared as long rod-shaped bacteria.In addition,the sialidase activity of the deletion strain was significantly reduced(P<0.0001),and the sialidase activity of the complementary strain was significantly higher than that of the deletion strain(P<0.001),but the sialidase activity of r Nan H at different concentrations(1μg/m L,5μg/m L,10μg/m L)showed an increasing trend with increasing protein concentration.2.H.parasuis sialidase desialylated the surface of 3D4/21 cellsThe JS0135 wild strain,Δnan H::kan strain and Δnan H+p Nan H complementary strain were used to infect 3D4/21 cells for 6h at an MOI of 10,respectively.The results of flow cytometry displayed that both the wild strain and the complementary strain can desialylate 3D4/21 cells,but the Δnan H::kan strain did not.At the same time,we selected different concentrations of r Nan H(1μg/m L,5μg/m L,10μg/m L)to stimulate 3D4/21 cells for 3h.The same method showed that the degree of desialylation on the cells surface showed a dose-dependent effect.3.H.parasuis sialidase affected Siglecs expression in 3D4/21 cellsThe JS0135 and Δnan H::kan strain were used to infect 3D4/21 cells for 0.5h,6h,12 h and 24 h,then the cells were detected for Siglecs reported in pigs in the literature by RT-PCR.At the transcriptional level,Siglec-3 and Siglec-10 were down-regulated,and Siglec-5 and Siglec-14 were up-regulated.Among them,Siglec-5 was tested at the protein level.Western Blot results showed that the expression of Siglec-5 increased,while the expression of Siglec-10 decreased with the extension of infection time,and the trend was consistent with the results at the transcription level.4.H.parasuis sialidase induced production of inflammatory cytokinesJS0135,Δnan H::kan and Δnan H+p Nan H strains were infected 3D4/21 cells,the results showed that the expression levels of pro-inflammatory cytokines IL-1α,IL-6,IL-8,IL-12 and TNF-α were significantly higher during infection of wild and complementary strains than that of deletion strain,while anti-inflammatory cytokines IL-10 was significantly lower than deletion strain.We used r Nan H for subsequent experiments and challenged 3D4/21 cells in the same way,the quantitative results were the up-regulation of pro-inflammatory cytokines and the down-regulations of anti-inflammatory cytokines were both dose-and time-dependent.Detection of changes in inflammatory cytokines at the protein level,Western Blot results showed that when the protein interacted with the cells for 3 hours,as the protein concentration increased,the expression of IL-1α,IL-6,IL-8,IL-12 and TNF-α increased,while the expression of IL-10 decreased.5.H.parasuis sialidase activated inflammatory signaling pathways was mediated by Siglec-53D4/21 cells were stimulated with r Nan H protein(1μg/m L,5μg/m L,10μg/m L)at different concentrations for 3h,and then detected for whether Siglec-5 interacted with SHP2 by Co-IP.The results showed that as the protein concentration increased,the binding amount of SHP2 to Siglec-5 gradually decreased.Then we tested the inflammatory signaling pathways MAPK and NFκB related to the inflammatory response,and the results showed that the degree of phosphorylation of the ERK in the MAPK pathway increased with increasing protein concentration,and the degree of degradation of IκBα also increased. |
