Han-Wei Shih

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.

Objectives: Methionyl-tRNA synthetase (MetRS) inhibitors are under investigation for the treatment of intestinal infections caused by Giardia lamblia . To properly analyze the therapeutic potential of the MetRS inhibitor 1717 , experimental tools including a robust cell-based assay and a murine model of infection were developed based on novel strains of G. lamblia that employ luciferase reporter systems to quantify viable parasites. Methods: Systematic screening of Giardia-specific promoters and luciferase variants led to the development of a strain expressing the click beetle green luciferase. Further modifying this strain to express NanoLuc created a dual reporter strain capable of quantifying parasites in both the trophozoite and cyst stages. These strains were used to develop a high throughput cell assay and a mouse infection model. A library of MetRS inhibitors was screened in the cell assay and 1717 was tested for efficacy in the mouse infection model. Results: Cell viability in in vitro compound screens was quantified via bioluminescence readouts while infection loads in mice were monitored with noninvasive whole-animal imaging and fecal analysis. Compound 1717 was effective in clearing mice of Giardia infection in 3 days at varying doses, which is supported by data from enzymatic and phenotypic cell assays. Conclusions: The new in vitro and in vivo assays based on luciferase expression by engineered G. lamblia strains are useful for the discovery and development of new therapeutics for giardiasis. MetRS inhibitors, as validated by 1717 , have promising anti-giardiasis properties that merit further study as alternative therapeutics.