The Role Of 43K OMP In The Adhesion Of Fusobacterium Necrophorum To Host Cells

Fusobacterium necrophorum is a Gram-negative anaerobic bacterium that causes hepatic abscesses,foot rot,and necrotic laryngitis.In addition,it plays an important role in the development of cow mastitis and endometritis,and it is responsible for the severe necrotic infection of tissues and cells.In recent years,F.necrophorum infection is widespread distributed in cattle industry,and provides significant theoretical basis and practical significance to explore the molecular mechanism involved in the pathogenesis of F.necrophorum.As an important outer membrane protein(OMP)of F.necrophorum,the 43 k Da outer membrane protein(43K OMP)may play an important role in F.necrophorum infection.however,the relevant molecular mechanisms by which this protein mediates adhesion remain unclear.Therefore,clarifing the role of 43 K OMP in F.necrophorum adhesion to host cells,and screening and identification of host cells protein that interacts with the 43 K OMP of F.necrophorum,and then exploring the inflammatory response mediated by 43 K OMP after the adhesion of F.necrophorum to host cells,will help elucidate the role of 43 K OMP in the development of F.necrophorum adhesion,and provides a theoretical basis for pathogenic mechanism of F.necrophorum and the design of antimicrobial drugs against F.necrophorum.In order to clarified the role of 43 K OMP in the adhesion of F.necrophorum to host cells,bovine endometrium epithelial cells,bovine mammary epithelial cells,mouse mammary epithelial cells and mouse liver cells were selected as cell models.Protein adhesion assay and adhesion inhibition assay with antibodies or 43 K OMP protein were used to explore the effects of 43 K OMP in F.necrophorum on the adhesion of the bacteria to host cells.The 43 K OMP gene of F.necrophorum was cloned into the expression vector PET-32 a,the plasmid was transformed into E.coli BL21DE3 cells,and then the recombinant protein was expressed.The attachment assay,attachment inhibition assay with antibody or 43 K OMP protein and proteinase treatment assay were used to analyse the adhesion characterization of 43 K OMP in F.necrophorum.Our results indicated that the 43 K OMP could bind to the cell membrane of BEECs,MAC-T cells and MMECs.When F.necrophorum was preincubated with antibodies against the recombinant43 K OMP,the adhesion of F.necrophorum to host cells significantly decreased compared with the negative serum control and the no antibody control(P<0.05).When the host cells were preincubated with native 43 K OMP,the adhesion of F.necrophorum decreased significantly(P<0.05).When E.coli carrying the recombinant plasmid(p ET-32a-43 K OMP)was induced by IPTG,there was significantly enhancement in the binding to host cells compared to the E.coli carrying control vector only(P<0.05).Preincubation of induced E.coli carrying the recombinant plasmid with antibodies against the recombinant 43 K OMP reduced the binding amount to host cells(P<0.05).When host cells were incubated with native 43 K OMP,E.coli carrying the recombinant plasmid showed lower levels of binding(P<0.05).The adhesion of E.coli carrying the recombinant plasmid was significantly decreased after proteinase K treatment(P<0.05),and dose-dependent relationship was shown between the decrease degree of E.coli adhesion to host cells and the concentrations of proteinase K.To confirm the relationship between the 43 K OMP gene of F.necrophorum and bacterial adhesion,the homologous recombinant fragment and the thiamphenicol resistance fragment were amplification and fused to construct the target fragments by fusion PCR.Then,the target fragment was digested with Sma I and then ligated into the suicide vector p CVD442.The resultant plasmid was electrotransformed into E.coli ?2155,then conjugated from the donor strain into F.necrophorum A25 strain.The deletion mutant was subsequently isolated on thiamphenicol-containing agar plates,and confirmed by PCR amplification and sequencing.We verified the effect of the 43 K OMP gene on the adhesion of F.necrophorum to host cells by the adhesion assay using deletion mutants.These results showed that the length of the fusion target fragment was 1423 bp,and the target plasmid was constructed successfully.The PCR amplification and sequencing results showed that the fragment lengths obtained with 5? insertion and 3? insertion were 1159 bp and 808 bp,the plasmid stability inserted into the 43 K OMP internal gene,and then a 43 K OMP-deficient mutant of F.necrophorum(A25?43K OMP)were constructed successfully.The bacterial attachment to host cells was significantly higher with the F.necrophorum A25 strain than with mutant strain A25?43K OMP(P<0.01),and the deletion of43 K OMP reduced the binding of F.necrophorum to host cells by 90.4%–94.9%.In order to screening and identified the host cells protein that interacts with 43 K OMP of F.necrophorum,recombinant 43 K OMP were incubated with MAC-T cell lysates,the host cell protein that interacts with the recombinant 43 K OMP was screened by immunoprecipitation–mass spectrometry.The suspected differential proteins were analysed by subcellular localization and single-molecule functional classification,and then the Co-IP assays were performed to identified the interaction between the 43 K OMP and the target differential proteins.The results revealed that 127 proteins and 280 peptides were successfully detected by immunoprecipitation-mass spectrometry,and 39 potential differential proteins were successfully screened.Fibronectin,collagen and myosin were selected as the target protein by subcellular localization and bacterial adhesion function analysis,and the Co-IP results s revealed a direct interaction between the 43 K OMP of F.necrophorum and fibronectinIn order to investigated the role of the 43 K OMP mediated the inflammatory response of host cells after F.necrophorum adhesion,quantitative proteomic analysis was used to analyse and screen the differential expressed proteins and bacterial infection related pathway of MAC-T cells by treatment with the 43 K OMP of F.necrophorum.The relative m RNA levels of IL-1?,IL-6,TNF-?,IL-4 and IL-10 of RAW264.7 cells by treatment with the 43 K OMP were detected by q RT-PCR,the concentration of IL-1?,IL-6 and TNF-? in cells supernatant were measured by ELISA,and the I?B?,P65,IL-6,TNF-?,p-S536 and MYD88 protein expression were analysed by Western blot.The results showed that 224 differential expressed proteins were screened out in43 K OMP treatment group,including 118 up-regulated proteins and 106 down-regulated proteins.Differential proteins were mainly concentrated in NF-?B pathway,bacterial invasion,cell adhesion molecules pathways,and were mainly involved in biological processes such as bacterial adhesion and inflammatory response.The relative m RNA levels of IL-1?,IL-6 and TNF-? in RAW264.7 cells were significantly increased(P<0.01),the relative m RNA levels of IL-4 in RAW264.7 cells were significantly decreased in 43 K OMP treatment group(P<0.01),and the concentration of IL-1?,IL-6 and TNF-? in cells supernatant were significantly increased(P<0.05).The I?B? protein expression decreased(P<0.05),and the p-S536/P65 protein expression increased(P<0.01).These results indicate that 43 K OMP mediates the adhesion of F.necrophorum to bovine endometrium epithelial cells,bovine mammary epithelial cells,mouse mammary epithelial cells and mouse liver cells,the deletion of 43 K OMP gene inhibits the adhesion ability of F.necrophorum to host cells,and the 43 K OMP mediates the adhesion of F.necrophorum to host cells through interacting with fibronectin.At the same time,43 K OMP mediates the inflammatory response after F.necrophorum adhesion by promoting the secretion of the inflammatory cytokines such as IL-6,IL-1? and TNF-? and the NF-?B activation.Our study confirms that 43 K OMP plays a crucial role in F.necrophorum adhesion,these findings increase our understanding of the mechanism of F.necrophorum infection and provide new insights into the design of adhesin-based vaccines and adhesin inhibitor therapy.