Knockout typeface Activator#

Close examination of the encystation process using deep RNA-seq led to the categorization of encystation proteins according to the time points showing their maximal expressions. lamblia demonstrated that this pathogen colonizes the gut in high-density foci where the expressions of encystation-related proteins are upregulated. An animal infection model using bioluminescence-tagged G. The encystation process was reinforced to make cysts more efficient, and the related transcriptomes at various time-points were investigated using RNA-seq. Giardia proteomes at various time points (early, middle, and late stages of encystation) have been examined and documented. The result showed only 18 overlapping genes with upregulated expression, including genes for cyst wall proteins, glycan synthesis, and a transcription factor, G. RNA samples were prepared from Giardia cells treated with two different in vitro encystation protocols and were subsequently used for a comparative microarray analysis. Encystation-induced genes have been investigated at transcriptomic or proteomic levels from the cells in the encystation process induced in vitro or occurring in vivo. It has been investigated more extensively than other processes, such as excystation, owing to its higher efficiency for in vitro reconstitution. Before being released from the human body, they differentiate into cysts, non-dividing but infective forms, via the encystation process triggered by different physiological elements, including alkaline pH, bile concentration, and lipid components, and unidentified factors.Įncystation is one of the two key differentiation processes in the Giardia life cycle (see for a review). Trophozoites inhabit the human small intestine, where they adhere, divide, and cause diseases. It has a simple life cycle, comprising trophozoites (vegetative stage) and cysts (dormant stage). Giardia lamblia, a pathogen that causes giardiasis in humans, is an interesting protozoan because it has simplified and degenerated organelles, such as the mitosome, and has two or four nuclei with multiple genomes. Further investigation using glmyb2 null mutant will provide knowledge regarding transcriptional apparatus required for the encystation process of G.

Knockout typeface Activator#

Our data confirm that GlMyb2 acts as a transcription activator especially during encystation by comparing the glmyb2 knockout mutant with the wild type. Detailed analysis on the promoter region of glcwp1 defined three GlMyb2 binding sites important in its encystation-induced expression. Nineteen genes were confirmed as GlMyb2 regulons, which include the glmyb2 gene, six for cyst wall proteins, five for signal transduction, two for transporter, two for metabolic enzymes, and three with unknown functions. Chromatin immunoprecipitation experiments revealed dozens of target genes. The addition of the wild-type glmyb2 gene to the null mutant restored the defects in encystation.

cyst formation, and expression of GlCWP1.

ResultsĬharacterization of the null glmyb2 mutant indicated loss of functions related to encystation, i.e. The promoter region of glcwp1 was analyzed via deletion and point mutagenesis of the putative GlMyb2 binding sites in luciferase reporters. Quantitative real-time PCR was performed to confirm an expression of candidate GlMyb2-regulated genes by comparing the transcript level for each target candidate in wild-type and knockout mutant Giardia. Chromatin immunoprecipitation experiments using anti-GlMyb2 antibodies were performed on the encysting G. lamblia cyst wall protein 1 (GlCWP1), a well-known target gene of GlMyb2, was measured via western blotting and immunofluorescence assays. Two sequential CRISPR/Cas9 experiments were performed to remove four glmyb2 alleles. Giardia lamblia Myb2 (GlMyb2) is a distinct encystation-induced transcription factor whose binding sites are found in the promoter regions of many encystation-induced genes, including its own.

Encystation is one of the two processes comprising the life cycle of Giardia lamblia, a protozoan pathogen with tetraploid genome.