Integrin α5β1-fimbriae binding is essential for P. gingivalis invasion of osteoblasts. A. Confocal imaging demonstration of the colocalization of P. gingivalis fimbriae and osteoblast integrin α5β1 1 h after bacterial inoculation. Osteoblast nuclei, α5β1 integrin, and P. gingivalis fimbriae are labeled in blue, red and green, respectively. Panel A. Control, P. gingivalis was inoculated, but neither primary antibody was included. Panel B. Control, P. gingivalis was not inoculated, and both primary antibodies were included. Panels C, E and G, representative images showing the co-localization of α5β1 and fimbriae. Panels D, F and H, clipped magnified views of panels C, E and G, respectively. In panel D, the top panel shows the red channel only; the bottom panel shows the three merged channels. Panels F and H show the blue, green, and red channels and the three merged channels. Presumed binding sites are shown as yellow where the red and green labels co-localize. Note the increased red intensity at the potential binding sites. B. Demonstration of the physical association between integrin α5β1 and fimbriae by immunoprecipitation. Western blot showing the presence of α5 and β1 in the immunocomplex precipitated with anti-fimbriae antibody, and the presence of fimbriae in the immunocomplex precipitated with anti-α5β1 antibody in the P. gingivalis-infected cultures, but not in the controls. Arrowheads indicate the molecular weights of the target proteins. C. Association between integrin α5β1 and fimbriae is necessary for P. gingivalis entry into osteoblasts. Quantitative confocal imaging demonstrates that P. gingivalis invasion of osteoblasts is partially prevented by addition of anti-α5β1 antibody, and new α5β1 synthesis is not required for invasion. Abbreviations: IP, immunoprecipitation; Fim A, major fimbriae of P. gingivalis; Ctrl, control; OB, osteoblasts; Pg, P. gingivalis; WB, western blot, Prot-inhi, protein synthesis inhibitor; min, minute; h, hour. * denotes P < 0.05.
Zhang et al. BMC Microbiology 2013 13:5 doi:10.1186/1471-2180-13-5