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GlRac regulates a noncanonical, ATG8-independent autophagy-like pathway in Giardia lamblia

Klippel, Angélica1; Newman-Boulle, Corryn1; Reed, Grant1; Dule, Dule1; Kedia, Saanya1; Kelty, Martin1; Chan, Wai1; Shih, Han-Wei2; Paredez, Alexander1

Published Sep 28, 2026 on Dryad. https://doi.org/10.5061/dryad.bzkh189sc

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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. GlRac, 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 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.