The Outcome of Intracellular <i>Selenomonas sputigena</i> and its Impact on Gingival Keratinocytes

Selenomonas sputigena is an understudied oral pathobiont associated with periodontitis and dental caries. We recently demonstrated that S. sputigena binds to gingival epithelial cells (GECs), where afterwards the bacterium causes robust in vitro pro-inflammatory cytokine production and migration of monocytes and neutrophils. Here, we report that extracellular adherent S. sputigena are subsequently internalized by GECs. Fluorescently labeled intracellular S. sputigena were observed in two gingival keratinocyte cell lines and primary cells. Oxygen-killed S. sputigena was found within GECs at a similar rate to living bacteria, suggesting active protein synthesis is not required for internalization. Host-cell actin polymerization was essential for the internalization of S. sputigena. Electron microscopy revealed that intracellular S. sputigena is located within a single membrane vesicle tightly wrapped around the bacterium which is decorated with LAMP1, indicating that S. sputigena is ultimately trafficked to a lysosome-like vesicle. Although S. sputigena mRNA was detectable within GECs, it rapidly decreased by 24 h post-inoculation. This differed from bacterial morphology, which remained intact up to 48 h. Induction of CXCL8 expression was concurrent with intracellular S. sputigena morphological integrity, suggesting that, while the bacteria are likely dead, bacterial macromolecules perpetuate inflammation in GECs. Collectively, this study demonstrates S. sputigena can be taken into gingival keratinocytes, where the bacterium induces inflammatory responses, but S. sputigena is ultimately processed by lysosomal machinery and is cleared from the intracellular niche in GECs.