The fate of Treponema denticola within human gingival epithelial cells

Abstract

Treponema denticola is one of the major pathogens associated with chronic periodontitis. Bacterial invasion into gingival tissues is a critical process in the pathogenesis of periodontal disease. We recently reported that T. denticola can invade gingival epithelial cells. The aim of this study is to determine the fate of internalized T. denticola in gingival epithelial cells. Immortalized human gingival epithelial HOK-16B cells were infected with 5- (and 6-) carboxy-fluorescein diacetate succinimidyl ester (CFSE)-labeled live or heat-killed T. denticola for 24 h, and the presence of bacteria inside the cells was confirmed by confocal microscopy. Live T. denticola, but not heat-killed bacteria, invaded HOK-16B cells. Confocal microscopy also revealed that internalized T. denticola rarely colocalized with either endosomes or lysosomes. Transmission electron microscopy of infected cells showed that intracellular T. denticola was localized inside endosome-like structures. Although a culture-based antibiotics protection assay could not detect viable intracellular T. denticola 12 h after infection, a substantial number of bacteria were observed by confocal microscopy and weak expression of bacterial 16S ribosomal RNA was detected 48 h after infection. In addition, flow cytometric analysis of HOK-16B cells infected with CFSE-labeled T. denticola showed no loss of fluorescence over 48 h. Collectively, T. denticola invades gingival epithelial cells and remains within the host cells for many hours by resisting endolysosomal degradation. These findings may provide new insight into the role of T. denticola in the pathogenesis of periodontitis.