Iron triggers PI4P5K proteostasis and Arf-mediated cell membrane trafficking to regulate PIP signaling crucial for multiple pathogenic activities of the parasitic protozoan .

is the etiologic agent of trichomoniasis, one of the most common non-viral sexually transmitted infections globally. Our previous work reported the role of phosphatidylinositol 4,5-bisphosphates (PIP) signaling in the actin-dependent pathogenicity of . This study further demonstrated that iron transiently regulated phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) proteostasis and its complex formation with an active ADP ribosylation factor Arf220, facilitating co-trafficking to the plasma membrane, crucial for PIP production. In dominant-active HA-Arf220 Q71L mutant, PI4P5K plasma membrane trafficking, PIP production, and intracellular calcium levels were increased, while these processes were inhibited in dominant-negative T31N mutant or those by Brefeldin A (BFA) treatment. Additionally, PIP replenishment reversed these inhibitions in the T31N mutant, suggesting the critical role of Arf220 activation in PIP-calcium signaling. Also, T31N mutant and BFA treatment impaired actin dynamics and cytoskeleton-dependent processes in , further linking the role of Arf220 to PIP-calcium-dependent actin dynamics. Beyond cytoadherence, during host-parasite interactions, Arf220 influenced both contact-dependent and -independent cytotoxicity, as well as phagocytotic capacity, contributing to the cytopathogenesis of human vaginal epithelial cells. Our findings underscore the key upstream regulation mechanisms of the PIP signaling, orchestrating the interplay between Arf220-PIP-calcium signaling and downstream actin cytoskeleton-driven pathogenicity in .IMPORTANCE actin cytoskeleton-centric pathogenicity is regulated by the phosphatidylinositol 4,5-bisphosphates (PIP)-triggered calcium signaling cascade in response to environmental iron, though the detailed mechanism by which iron modulates PIP signaling remains unclear. Our findings reveal that iron rapidly induces phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) translation followed by its degradation, while simultaneously activating Arf220 binding, which facilitates PI4P5K localization to the plasma membrane for PIP production. In contrast to the Arf220 Q71L mutant, the reduced PIP production, intracellular calcium, actin assembly, morphogenesis, and cytoadherence in the dominant-negative T31N mutant were recovered by PIP supplementation, indicating the essential role of Arf220 in PIP-dependent calcium signaling. Additionally, the contact-dependent or -independent cytotoxicity, along with the phagocytosis, was impaired in the PI4P5K- or Arf220-deficient parasites, as well as in those treated with BAPTA or Latrunculin B. These findings highlight that Arf220-mediated PIP-calcium signaling cascade regulates actin cytoskeleton and cytopathogenicity of . This study uncovers a novel pathogenic mechanism and suggests potential therapeutic targets for parasite control.