Internalization dynamics and cytoskeletal effects of Gardnerella vaginalis and its extracellular vesicles in vaginal epithelial cells.

Gardnerella vaginalis, an important bacterial pathogen associated with bacterial vaginosis, produces bacterial extracellular vesicles (bEVs), which can contribute to cellular pathogenesis. However, comprehensive host-microbe crosstalk mechanisms such as cellular entry route, after internalization, and host cytoskeleton modifications remain poorly characterized. In this study, we treated female reproductive tract cell lines with G. vaginalis and its vesicles. To evaluate their entry routes, host cells were pretreated with pharmacological inhibitors and treated with labelled bacterial cells or derived vesicles. Next, to delineate the endolysosomal processing routes, vaginal epithelial cells were stained to enable the visualization of endosomal and lysosomal compartments. The results revealed that G. vaginalis and its vesicles were internalized in vaginal epithelial cells and followed nonspecific and caveolin-dependent endocytosis, respectively. Confocal imaging showed that while internalized vesicles trafficked to intracellular compartments, consistent with the endolysosomal pathway, the G. vaginalis underwent noncanonical processing, suggesting a possible escape from cellular degradation. Of note, vesicles mediated F-actin rearrangements consistently in all female genital tract cells while G. vaginalis exhibited this phenomenon only in non-vaginal cells. These novel findings of entry routes, intracellular fates, and G. vaginalis- and vesicle-mediated cytoskeletal changes in host cells advance our understanding of cellular pathogenesis associated with bacterial vaginosis.