Trichomonas vaginalis inhibits autophagy in VK2/E6E7 cells via the AMPK-ULK1 pathway

Trichomonas vaginalis ( T. vaginalis ) is an infectious protozoan parasite responsible for trichomoniasis, which is one of the world's most pervasive non-viral sexually transmitted infections globally. This parasite primarily causes vaginitis and urethritis and is also linked to complications in premature delivery. Autophagy, a cellular process that regulates epithelial cell permeability by modulating tight junctions, plays an essential role in maintaining optimal cell health. However, whether T. vaginalis infection affects vaginal epithelial cell autophagy and the specific signaling pathways involved, remain poorly understood. Here, we found that the expression of p62 in VK2/E6E7 cells infected with T. vaginalis significantly decreased by 50% at 4 h ( P < 0.000 1), and the LC3II/LC3I ratios were reduced by approximately 20% at 8 h post-infection ( P < 0.05). Indirect immunofluorescence and transmission electron microscopy showed that T. vaginalis stimulation significantly decreased the fluorescence intensity of the LC3 protein ( P < 0.01) and the number of autophagolysosomes in VK2/E6E7 cells. These preliminary results showed that autophagy was significantly inhibited. LC3II levels continued to decrease following infection with T. vaginalis ( P < 0.000 1), even when autophagosome degradation was inhibited by chloroquine (CQ). This suggests that the decrease in LC3II expression in T. vaginalis -infected VK2/E6E7 cells resulted from reduced autophagosome production rather than excessive autophagosome degradation. Further analysis indicated that T. vaginalis infection might disrupt autophagy through specific signaling pathways. T. vaginalis infection can significantly reduce the ratio of LC3II/LC3I in rapamycin (Rapa) pretreated cells ( P < 0.001), reduce the fluorescence intensity of LC3 and reduce the red fluorescence spots of LC3 ( P < 0.000 1), suggesting that mTOR may be involved in the inhibition of autophagy of VK2/E6E7 cells by T. vaginalis . After T. vaginalis infected VK2/E6E7 cells 4 h, Western blotting detection showed that PI3K expression increased by 7-fold compared to controls ( P < 0.000 1), while the protein levels of p-AKT, p-AMPK, p-ULK1 levels were reduced by 25%, 70%, and 80% ( P < 0.01), respectively. p-mTOR and mTOR were all down-regulated ( P < 0.05), indicating that the inhibition of autophagy of VK2/E6E7 cells by T. vaginalis may be mediated by the AMPK-ULK1 signal pathway rather than the classical PI3K-AKT-mTOR pathway. Following the activation of autophagy using the AMPK agonist GSK621, T. vaginalis infection significantly decreased the expression of LC3II, p-AMPK, and p-ULK1 in cells treated with GSK621 ( P < 0.001). It is further confirmed that the AMPK-ULK1 signaling pathway plays an integral role in T. vaginalis inhibiting VK2/E6E7 cells autophagy. In conclusion, T. vaginalis infection inhibits the autophagy of VK2/E6E7 cells through AMPK-ULK1 signaling pathways involved in mTOR.