IADR Abstract Archives
Involvement of Rab4A and VAMP2 in intracellular Porphyromonas gingivalis in gingival epithelial cells
Objectives: Porphyromonas gingivalis (Pg) can pass through gingival epithelial barrier via intracellular route. We previously showed that some of the intracellular pathogens are sorted to lysosomes and autolysosomes, while the others locates recycling endosomes (REs) and exit the host cells by the recycling pathway regulated by Rab11A. In general, specific membrane fusion is achieved by Rab proteins and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. Regarding the recycling pathway, Rab4A, a member of Ras oncogene family, and vesicle associated membrane protein (VAMP2) have been suggested to be a key regulator for recycling of membrane proteins. However, the effect of Rab4A and VAMP2 on infection mechanism of bacteria is unknown. In this study, we examined the involvement of Rab4A and VAMP2 in intracellular trafficking of Pg.
Methods: Human immortalized gingival epithelial cells (HIGECs) were transfected with mCherry-Rab4A or either of enhanced green fluorescent protein (EGFP)- or mCherry-VAMP2. Overexpression of EGFP-2xFYVE was used for a marker of early endosomes (EEs). Knockdown of Rab4A or VAMP2 was performed by transfection of siRab4A or siVAMP2. HIGECs were infected with Pg ATCC 33277 and its intracellular localization was examined using confocal microscopy. Bacterial viability in HIGECs was determined by colony formation unit assay.
Results: At 1 hour after infection, intracellular Pg was co-localized with mCherry-Rab4A in HIGECs, and the ratio of colocalization reached a peak at 3 hour after infection. Pg was also colocalized with EGFP-2xFYVE and either of mCherry-Rab4A or VAMP2, respectively. Knockdown of Rab4A or VAMP2 caused continuous accumulation of FYVE-positive Pg for 5 hours after infection, which elicited the increase of intracellular viable bacteria and the decrease of extracellular viable bacteria.
Conclusions: Rab4A and VAMP2 are suggested to be involved in the intracellular trafficking of Pg from EEs to REs.
Methods: Human immortalized gingival epithelial cells (HIGECs) were transfected with mCherry-Rab4A or either of enhanced green fluorescent protein (EGFP)- or mCherry-VAMP2. Overexpression of EGFP-2xFYVE was used for a marker of early endosomes (EEs). Knockdown of Rab4A or VAMP2 was performed by transfection of siRab4A or siVAMP2. HIGECs were infected with Pg ATCC 33277 and its intracellular localization was examined using confocal microscopy. Bacterial viability in HIGECs was determined by colony formation unit assay.
Results: At 1 hour after infection, intracellular Pg was co-localized with mCherry-Rab4A in HIGECs, and the ratio of colocalization reached a peak at 3 hour after infection. Pg was also colocalized with EGFP-2xFYVE and either of mCherry-Rab4A or VAMP2, respectively. Knockdown of Rab4A or VAMP2 caused continuous accumulation of FYVE-positive Pg for 5 hours after infection, which elicited the increase of intracellular viable bacteria and the decrease of extracellular viable bacteria.
Conclusions: Rab4A and VAMP2 are suggested to be involved in the intracellular trafficking of Pg from EEs to REs.