Clathrin-dependent entry of a gingipain adhesin peptide and Porphyromonas gingivalis into host cells.
Porphyromonas gingivalis, a Gram-negative oral anaerobe, is associated with periodontitis, a disease that in some form affects up to 80% of the adult population in the USA. The organism interacts with gingival epithelium and surrounding tissue, and in this study we analysed interactions initiated by P. gingivalis and by a peptide derived from the adhesin domain of arg-gingipain A, a member of a family of surface cysteine proteinases. Recombinant peptide A44 blocked adherence of bacteria to host cell monolayers, and bound to components of the cell membrane fraction. In pull-down assays A44 associated with proteins involved in a clathrin-dependent endocytosis pathway. Inhibitor studies confirmed a role for clathrin, and confocal microscopy demonstrated that both A44-coated beads and intact bacteria colocalized with GFP-clathrin in host cells. Finally, we used siRNA to determine whether clathrin or caveolin-1 was involved in association of peptide and intact bacteria with host cells. Again, the results of these assays indicated that association of both A44 and P. gingivalis depended on the presence of clathrin, and support a working model in which A44 initiates a clathrin-dependent pathway that potentially leads to internalization of peptide or bacteria by host epithelial cells.
Boisvert
Heike
H
Department of Molecular Genetics, The Forsyth Institute, Boston, MA 02115, USA.
Duncan
Margaret J
MJ
D018829:Adhesins, Bacterial / Q000378:metabolism*; D000328:Adult; D001422:Bacterial Adhesion; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004705:Endocytosis; D004847:Epithelial Cells / Q000382:microbiology; D020868:Gene Silencing; D000080867:Gingipain Cysteine Endopeptidases; D006801:Humans; D018613:Microscopy, Confocal; D016966:Porphyromonas gingivalis / Q000502:physiology*; D011485:Protein Binding
D018829:Adhesins, Bacterial; D002966:Clathrin; D000080867:Gingipain Cysteine Endopeptidases; D003546:Cysteine Endopeptidases