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

Abstract

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.