Gl Rac Regulates a Noncanonical, ATG8-Independent Autophagy-Like Pathway in Giardia lamblia

Abstract Autophagy is a conserved catabolic process essential for cellular homeostasis and stress adaptation. The protozoan parasite Giardia lamblia lacks most canonical autophagy-related (ATG) genes, including the hallmark ATG8, raising longstanding questions about whether it can perform autophagy. Here, we show that Giardia mounts a regulated autophagic-like response. Double-membrane compartments resembling autophagosomes are induced in up to 30% of encysting cells and 91% of starved trophozoites, supporting roles in differentiation and survival under nutrient stress. Their clearance is triggered by amino acid replenishment but not by glucose, indicating a nutrient-specific sensing mechanism. Gl Rac, the parasite’s sole Rho family GTPase, labels these structures and regulates their formation, as evidenced by a threefold increase in compartment levels upon constitutive activation and a significant reduction after knockdown. This extends the conserved role of Rho GTPases in regulating autophagy to an evolutionarily early-branching eukaryote. Of nine putative ATG orthologs tested, none localized as clearly as Gl Rac to autophagic structures. These organelles acidify and recruit cathepsin proteases, consistent with degradative capacity. A newly developed live-cell actin marker reveals robust recruitment to these structures, implicating actin-driven remodeling. Finally, quinacrine, an FDA-approved antigiardial drug, promotes the accumulation of autophagic structures, consistent with its known effects on mammalian autophagy. Together, our findings establish Gl Rac as a regulator of an ATG8-independent autophagic response in Giardia , demonstrate that this parasite retains key features of autophagy despite its streamlined genome, and highlight this pathway as a potential therapeutic target. Significance Autophagy is a self-degradative process essential for stress adaptation and cellular recycling. Although considered ancient and broadly conserved, this pathway was long thought to be absent in the protozoan parasite Giardia lamblia , which lacks many canonical autophagy genes, including the classic marker ATG8. Here, we demonstrate that Giardia performs a regulated autophagic-like response. We identify Gl Rac, the parasite’s sole Rho family GTPase, as a regulator that marks autophagosome-like organelles, underscoring the conserved role of Rho GTPases in autophagy across eukaryotes. We also show that quinacrine, a clinically used antigiardial drug, perturbs the autophagic response in this organism. These findings reveal an unrecognized aspect of Giardia biology and support autophagy as a promising therapeutic target for this widespread parasite.