Entamoeba gingivalis induces gingival cell death, collagen breakdown, and host immune response via VAMP8/-3-driven exocytosis pathways.

The protozoan Entamoeba gingivalis commonly colonizes anaerobic periodontal pockets, induces a severe innate immune response, invades gingival mucosa, and kills epithelial cells. E. gingivalis infection is associated with the common oral inflammatory disease periodontitis. DNA variants in vesicle-associated membrane proteins (VAMP) -3 and -8 genes are linked to increased periodontitis risk. These genes mediate host-pathogen interactions, including mucin exocytosis to form protective barriers and matrix metalloproteinase (MMP) secretion in intestinal amoebiasis caused by Entamoeba histolytica. This study aimed to investigate the roles of VAMP3/8 in gingival defense and E. gingivalis infection mechanisms. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing was used to create VAMP3/8-deficient gingival epithelial cells and fibroblasts. Functional analyses included immunofluorescence, enzyme-linked immunosorbent assay (ELISA), cytotoxicity, and collagenase assays. VAMP8 co-localized with mucins in gingival epithelial cells (gECs), and VAMP3 with MMPs in gingival fibroblasts. In gECs, E. gingivalis infection increased mucin (MUC1: 3.6×, MUC21: 14.4×) and interleukin secretion (IL-8, IL-1B: >6×, P = 0.019). VAMP8 deficiency in gECs caused higher cell death (35% vs 4% in controls) with reduced exocytosis of mucins and interleukins. Likewise, E. gingivalis-induced VAMP8 translocation into lipid rafts was lost in VAMP8 knockout cells, validating the participation of VAMP8 in exocytosis. In wild-type but not VAMP3-deficient gingival fibroblasts, E. gingivalis strongly activated collagenases. E. gingivalis effects were more pathogenic than those of the oral anaerobic bacterium Porphyromonas gingivalis. E. gingivalis exploits VAMP8/3-driven exocytosis pathways, driving inflammation and tissue destruction, underscoring its role as a significant periodontal pathogen.