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

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. GlRac, the parasites 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 GlRac 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 GlRac 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. SignificanceAutophagy 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 GlRac, the parasites 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.