Development of CRISPR/Cas9-mediated gene disruption systems in Giardia lamblia

Lin, Ziqi; Gan, Soo Wah; Tung, Szu-Yu; Ho, Chun-Che; Su, Li‐Hsin; Sun, Chin-Hung

doi:10.1371/journal.pone.0213594

Cited by 19 publications

(74 citation statements)

References 65 publications

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“…We have also identified genes that are regulated by the level of iron in the growth medium. The recent development of CRISPR repression and mutagenesis systems (Lin et al, 2019;McInally et al, 2019) will make it possible to define the role of these genes in Giardia's virulence. Mutant parasites can be tested in vitro in enteroid systems or in vivo in mice or gerbils and this can lead to a more detailed molecular understanding of giardiasis.…”

Section: Discussionmentioning

confidence: 99%

High Cysteine Membrane Proteins (HCMPs) Are Up-Regulated During Giardia-Host Cell Interactions

Peirasmaki

Ma’ayeh

et al. 2020

Front. Genet.

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Giardia intestinalis colonizes the upper small intestine of humans and animals, causing the diarrheal disease giardiasis. This unicellular eukaryotic parasite is not invasive but it attaches to the surface of small intestinal epithelial cells (IECs), disrupting the epithelial barrier. Here, we used an in vitro model of the parasite's interaction with host IECs (differentiated Caco-2 cells) and RNA sequencing (RNAseq) to identify differentially expressed genes (DEGs) in Giardia, which might relate to the establishment of infection and disease induction. Giardia trophozoites interacted with differentiated Caco-2 cells for 1.5, 3, and 4.5 h and at each time point, 61, 89, and 148 parasite genes were up-regulated more than twofold, whereas 209, 265, and 313 parasite genes were down-regulated more than twofold. The most abundant DEGs encode hypothetical proteins and members of the High Cysteine Membrane Protein (HCMP) family. Among the up-regulated genes we also observed proteins associated with proteolysis, cellular redox balance, as well as lipid and nucleic acid metabolic pathways. In contrast, genes encoding kinases, regulators of the cell cycle and arginine metabolism and cytoskeletal proteins were down-regulated. Immunofluorescence imaging of selected, up-regulated HCMPs, using C-terminal HA-tagging, showed localization to the plasma membrane and peripheral vesicles (PVs). The expression of the HCMPs was affected by histone acetylation and free iron-levels. In fact, the latter was shown to regulate the expression of many putative giardial virulence factors in subsequent RNAseq experiments. We suggest that the plasma membrane localized and differentially expressed HCMPs play important roles during Giardia-host cell interactions.

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Section: Discussionmentioning

confidence: 99%

High Cysteine Membrane Proteins (HCMPs) Are Up-Regulated During Giardia-Host Cell Interactions

Peirasmaki

Ma’ayeh

et al. 2020

Front. Genet.

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“…Given that CCVs have not been detected in Giardia , this begs the question of the functional role of a NECAP1 cardiolipin-binding orthologue in G. lamblia which was found to interact with G. lamblia AP2 subunits and Gl FYVE. Recent developments in gene knock-out [63] and CRISPR-Cas9-based knock-down [64, 65] methodologies tailored to G. lamblia will be instrumental towards a full functional characterization of Gl NECAP1’s function(s)…”

Section: Discussionmentioning

confidence: 99%

Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protistGiardia lamblia

Černíková

Faso

Hehl

2019

Preprint

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Phosphorylated derivatives of phosphatidylinositol (PIPs), are key membrane lipid residues involved in clathrin-mediated endocytosis (CME). CME relies on PI(4,5)P2 to mark endocytic sites at the plasma membrane (PM) associated to clathrin-coated vesicle (CCV) formation. The highly diverged parasitic protist Giardia lamblia presents disordered and static clathrin assemblies at PM invaginations, contacting specialized endocytic organelles called peripheral vacuoles (PVs). The role for clathrin assemblies in fluid phase uptake and their link to internal membranes via PIP-binding adaptors is unknown.Here we provide evidence for a robust link between clathrin assemblies and fluid-phase uptake in G. lamblia mediated by proteins carrying predicted PX, FYVE and NECAP1 PIP-binding modules. We show that chemical and genetic perturbation of PIP-residue binding and turnover elicits novel uptake and organelle-morphology phenotypes. A combination of co-immunoprecipitation and in silico annotation techniques expands the initial PIP-binding network with addition of new members. Our data indicate that, despite the partial conservation of lipid markers and protein cohorts known to play important roles in dynamic endocytic events in well-characterized model systems, the Giardia lineage presents a strikingly divergent clathrin-centered network. This includes several PIP-binding modules, often associated to domains of currently unknown function that shape and modulate fluid-phase uptake at PVs.

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“…Given that CCVs have not been detected in Giardia, this begs the question of the functional role of a NECAP1 cardiolipin-binding orthologue in G. lamblia, which was found to interact with G. lamblia AP2 subunits and GlFYVE. Recent developments in gene knock-out [67] and CRISPR-Cas9-based knock-down [68,69] methodologies tailored to G. lamblia will be instrumental towards a full functional characterization of GlNE-CAP1's function(s).…”

Section: An Interactome-based Model For Pip-binding Proteins and Clatmentioning

confidence: 99%

Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia

2020

View full text Add to dashboard Cite

Phosphorylated derivatives of phosphatidylinositol (PIPs) are key membrane lipid residues involved in clathrin-mediated endocytosis (CME). CME relies on PIP species PI(4,5)P 2 to mark endocytic sites at the plasma membrane (PM) associated to clathrin-coated vesicle (CCV) formation. The highly diverged parasitic protist Giardia lamblia presents disordered and static clathrin assemblies at PM invaginations, contacting specialized endocytic organelles called peripheral vacuoles (PVs). The role for clathrin assemblies in fluid phase uptake and their link to internal membranes via PIP-binding adaptors is unknown. Here we provide evidence for a robust link between clathrin assemblies and fluid-phase uptake in G. lamblia mediated by proteins carrying predicted PX, FYVE and NECAP1 PIP-binding modules. We show that chemical and genetic perturbation of PIP-residue binding and turnover elicits novel uptake and organelle-morphology phenotypes. A combination of co-immunoprecipitation and in silico analysis techniques expands the initial PIP-binding network with addition of new members. Our data indicate that, despite the partial conservation of lipid markers and protein cohorts known to play important roles in dynamic endocytic events in well-characterized model systems, the Giardia lineage presents a strikingly divergent clathrin-centered network. This includes several PIP-binding modules, often associated to domains of currently unknown function that shape and modulate fluid-phase uptake at PVs.

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Development of CRISPR/Cas9-mediated gene disruption systems in Giardia lamblia

Cited by 19 publications

References 65 publications

High Cysteine Membrane Proteins (HCMPs) Are Up-Regulated During Giardia-Host Cell Interactions

High Cysteine Membrane Proteins (HCMPs) Are Up-Regulated During Giardia-Host Cell Interactions

Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protistGiardia lamblia

Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia

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