Iron triggers <i>Tv</i> PI4P5K proteostasis and Arf-mediated cell membrane trafficking to regulate PIP ₂ signaling crucial for multiple pathogenic activities of the parasitic protozoan <i>Trichomonas vaginalis</i>

<i>Trichomonas vaginalis</i> 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 (PIP₂) signaling in the actin-dependent pathogenicity of <i>T. vaginalis</i>. This study further demonstrated that iron transiently regulated <i>T. vaginalis</i> phosphatidylinositol-4-phosphate 5-kinase (<i>Tv</i>PI4P5K) proteostasis and its complex formation with an active ADP ribosylation factor <i>Tv</i>Arf220, facilitating co-trafficking to the plasma membrane, crucial for PIP₂ production. In dominant-active HA-<i>Tv</i>Arf220 Q71L mutant, <i>Tv</i>PI4P5K plasma membrane trafficking, PIP₂ 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, PIP₂ replenishment reversed these inhibitions in the T31N mutant, suggesting the critical role of <i>Tv</i>Arf220 activation in PIP₂-calcium signaling. Also, T31N mutant and BFA treatment impaired actin dynamics and cytoskeleton-dependent processes in <i>T. vaginalis</i>, further linking the role of <i>Tv</i>Arf220 to PIP₂-calcium-dependent actin dynamics. Beyond cytoadherence, during host-parasite interactions, <i>Tv</i>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 PIP₂ signaling, orchestrating the interplay between <i>Tv</i>Arf220-PIP₂-calcium signaling and downstream actin cytoskeleton-driven pathogenicity in <i>T. vaginalis</i>.IMPORTANCE<i>Trichomonas vaginalis</i> actin cytoskeleton-centric pathogenicity is regulated by the phosphatidylinositol 4,5-bisphosphates (PIP₂)-triggered calcium signaling cascade in response to environmental iron, though the detailed mechanism by which iron modulates PIP₂ signaling remains unclear. Our findings reveal that iron rapidly induces <i>T. vaginalis</i> phosphatidylinositol-4-phosphate 5-kinase (<i>Tv</i>PI4P5K) translation followed by its degradation, while simultaneously activating <i>Tv</i>Arf220 binding, which facilitates <i>Tv</i>PI4P5K localization to the plasma membrane for PIP₂ production. In contrast to the <i>Tv</i>Arf220 Q71L mutant, the reduced PIP₂ production, intracellular calcium, actin assembly, morphogenesis, and cytoadherence in the dominant-negative T31N mutant were recovered by PIP₂ supplementation, indicating the essential role of <i>Tv</i>Arf220 in PIP₂-dependent calcium signaling. Additionally, the contact-dependent or -independent cytotoxicity, along with the phagocytosis, was impaired in the <i>Tv</i>PI4P5K- or <i>Tv</i>Arf220-deficient parasites, as well as in those treated with BAPTA or Latrunculin B. These findings highlight that <i>Tv</i>Arf220-mediated PIP₂-calcium signaling cascade regulates actin cytoskeleton and cytopathogenicity of <i>T. vaginalis</i>. This study uncovers a novel pathogenic mechanism and suggests potential therapeutic targets for parasite control.