Methionyl-tRNA synthetase inhibitor has potent in vivo activity in a novel Giardia lamblia luciferase murine infection model

Abstract

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.

TheGiardialamellipodium-like ventrolateral flange supports attachment and rapid cytokinesis

“…Endocytosis markers indicate that internalization is occurring here (10, 57, 58), but whether directed secretion occurs at the bare area has not been studied. Newly developed genetic tools and reporter strains will allow for in vivo virulence studies to be performed in the future (59–61).…”

Stage-specific, morphological and molecular markers of encystation inGiardia lamblia

“…Generation of the native promoter morpholino-sensitive (ms) HALO_C18_ Gl Rac_PuroR (designed for endogenous expression) and PAK promoter_CRIB_N11_mNG_3HA_NeoR (designed for episomal expression) constructs have been described previously (Hardin et al, 2021, submitted). Plasmid backbones used to build constructs in this paper were sourced from Gourguechon & Cande, 2011, Krtková et al, 2016, Michaels et al, 2020 and Paredez et al, 2014.…”

“…Generation of the native promoter morpholino-sensitive (ms) HALO_C18_GlRac_PuroR (designed for endogenous expression) and PAK promoter_CRIB_N11_mNG_3HA_NeoR (designed for episomal expression) constructs have been described previously (Hardin et al, 2021, submitted). Plasmid backbones used to build constructs in this paper were sourced from Gourguechon & Cande, 2011, Krtková et al, 2016, Michaels et al, 2020and Paredez et al, 2014 For transfection, 5 to µg of DNA was electroporated (375 V, 1,000 µF, 750 Ω; GenePulser Xcell; BioRad, Hercules, CA) into trophozoites. Following electroporation, the cells were added to 13 ml pre-warmed, fresh TYDK medium and allowed to recover at 37°C overnight before beginning selection with G418 or Puromycin for 4-7 days.…”

Staging Encystation Progression in Giardia lamblia Using Encystation-Specific Vesicle Morphology and Associating Molecular Markers

“…Generation of the native promoter morpholino-sensitive (ms) HALO_C18_GlRac_PuroR (designed for endogenous expression) and PAK promoter_CRIB_N11_mNG_3HA_NeoR (designed for episomal expression) constructs have been described previously (Hardin et al, 2021). Plasmid backbones used to build constructs in this paper were sourced from Gourguechon and Cande (2011); Krtková et al (2016), Michaels et al (2020), andParedez et al (2014).…”