There is need for a more efficient cell-based assay amenable to high-throughput drug screening against Giardia lamblia. Here, we report the development of a screening method utilizing G. lamblia engineered to express red-shifted firefly luciferase. Parasite growth and replication were quantified using D-luciferin as a substrate in a bioluminescent read-out plateform. This assay was validated for reproducibility and reliability against the Medicines for Malaria Venture (MMV) Pathogen Box compounds. For G. lamblia, forty-three compounds showed ≥ 75% inhibition of parasite growth in the initial screen (16 μM), with fifteen showing ≥ 95% inhibition. The Pathogen Box was also screened against Nanoluciferase expressing (Nluc) C. parvum, yielding 85 compounds with ≥ 75% parasite growth inhibition at 10 μM, with six showing ≥ 95% inhibition. A representative set of seven compounds with activity against both parasites were further analyzed to determine the effective concentration that causes 50% growth inhibition (EC50) and cytotoxicity against mammalian HepG2 cells. Four of the seven compounds were previously known to be effective in treating either Giardia or Cryptosporidium. The remaining three shared no obvious chemical similarity with any previously characterized anti-parasite diarrheal drugs and offer new medicinal chemistry opportunities for therapeutic development. These results suggest that the bioluminescent assays are suitable for large-scale screening of chemical libraries against both C. parvum and G. lamblia.

Background Methionyl-tRNA synthetase (MetRS) inhibitors are under investigation for the treatment of intestinal infections caused by Giardia lamblia. Objectives To properly analyse 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 Compound-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 non-invasive whole-animal imaging and faecal analysis. Compound-1717 was effective in clearing mice of Giardia infection in 3 days at varying doses, which was 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 Compound-1717, have promising anti-giardiasis properties that merit further study as alternative therapeutics.

Differentiation into environmentally resistant cysts is required for transmission of the ubiquitous intestinal parasite Giardia lamblia. Encystation in Giardia requires the production, processing and transport of Cyst Wall Proteins (CWPs) in developmentally induced, Golgi-like, Encystation Specific Vesicles (ESVs). Progress through this trafficking pathway can be followed by tracking CWP localization over time. However, there is no recognized system to distinguish the advancing stages of this process which can complete at variable rates depending on how encystation is induced. Here, we propose a staging system for encysting Giardia based on the morphology of CWP1-stained ESVs. We demonstrate the molecular distinctiveness of maturing ESVs at these stages by following GlRab GTPases through encystation. Previously, we established that Giardia’s sole Rho family GTPase, GlRac, associates with ESVs and has a role in regulating their maturation and the secretion of their cargo. As a proof of principle, we delineate the relationship between GlRac and ESV stages. Through proteomic studies, we identify putative interactors of GlRac that could be used as additional ESV stage markers. This staging system provides a common descriptor of ESV maturation regardless of the source of encysting cells. Furthermore, the identified set of molecular markers for ESV stages will be a powerful tool for characterizing trafficking mutants that impair ESV maturation and morphology.

FERONIA (FER), a receptor-like kinase involved in plant immunity, cell expansion, and mechanical signal transduction, is known to be endocytosed and degraded in response to treatment with its peptide ligand RAPID ALKALINIZATION FACTOR 1 (RALF1). Using confocal fluorescence microscopy and biochemical assays, we have found that full length FER-eGFP abundance at the plasma membrane is also regulated by mechanical stimulation, but through a separate, cysteine protease-dependent pathway. Like RALF1 treatment, both mechanical bending and mechanical wounding trigger a reduction in plasma membrane-localized, native promoter-driven FER-eGFP in Arabidopsis roots, hypocotyls, and cotyledons. However, pharmacological inhibition of protein trafficking and degradation suggests that while RALF1 induces clathrin-mediated endocytosis and subsequent degradation of FER-eGFP, mechanical stimulation triggers cleavage and/or degradation of FER-eGFP in a cysteine protease-dependent, clathrin-independent manner. Despite the stimulus-dependent differences in these two pathways, we found that both require early FER signaling components, including Ca2+ signaling, FER kinase activity, and the presence of LLG1, a FER-interacting protein with an essential role in FER-dependent signal transduction.

Transcriptional regulation of differentiation is critical for parasitic pathogens to adapt to environmental changes and regulate transmission. How early signaling transcription factors (TF) activate signal transduction to initiate encystation remains an open question in Giardia. Here, we generate a CasRX-mediated knockdown system, together with an established CRISPRi system to screen early signaling TFs in Giardia lamblia. We identified an early response TF, GARP4 that regulates cyst wall protein (CWP) levels during encystation. Depletion of GARP4 increases encystation efficiency resulting in increased cyst production. Interestingly, cyst viability and CWP1 trafficking are not altered in GARP4 knockdowns, suggesting GARP4 regulates the restriction point controlling the portion of cells that terminally differentiate into cysts. Consistent with previous studies, we find that stimulation of encystation shifts the distribution of cells to the G2/M phase and these cells exhibits higher levels of CWP1, indication that entry into the encystation pathway is cell cycle regulated. Key to this increase of CWP1 in G2/M cells is activation of MYB2, a TF commonly observed during the early phase of encystation in Giardia. Remarkably, activated GARP4 only exhibits in G1/S cells, suggesting it has a role in preventing encystation until G2/M. Furthermore, we demonstrate that depletion of GARP4 activates MYB2 and overexpression of GARP4 represses MYB2. Our findings provide the first molecular mechanism underlying the restriction point regulating differentiation during early signaling of encystation in Giardia lamblia.