Nitric oxide initiates early differentiation events in Leishmania infantum stage transition

Leishmaniasis, caused by Leishmania parasites, remains a major global health burden. The promastigote-to-amastigote transition is critical for establishing infection within mammalian macrophages, where parasites encounter intense oxidative stress. While reactive oxygen species (ROS) are traditionally viewed as antimicrobial effectors, emerging evidence suggests they may also function as differentiation signals. We investigated the effects of three distinct ROS species, superoxide (via menadione), hydrogen peroxide (H₂O₂), and nitric oxide (via SNAP), on L. infantum differentiation. Following dose optimization, we assessed morphological changes, stage-specific gene expression, amastin protein accumulation, and redox homeostasis. Nitric oxide emerged as the most potent initiator of early differentiation events, inducing significant upregulation of ama34, a gene coding for the amastin protein, characteristic of the amastigote stage, and downregulation of par4, a gene coding for the parasite's flagellum, characteristic of the promastigote stage. These gene expressions are similar to those observed in axenic amastigotes. SNAP treatment also increased amastin protein levels and modulated genes involved in nuclear transport (ntf2), mitochondrial function (afg1), and trypanothione metabolism (tr). H₂O₂ showed moderate effects, while menadione-derived superoxide had minimal impact. Notably, antioxidant enzyme genes remained largely unaffected by ROS treatments despite their upregulation in amastigotes. Our findings demonstrate that ROS, particularly nitric oxide, serve as environmental cues initiating early differentiation events in Leishmania. However, ROS exposure alone is insufficient for complete stage conversion, as evidenced by modest stress-response activation and incomplete antioxidant upregulation compared to fully differentiated amastigotes. Complete conversion requires integration of multiple signals including temperature shift, acidification, and prolonged host exposure. Our data support a model wherein NO^• acts as an early trigger rather than a comprehensive driver of full stage transition.