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Research on Giardia lamblia has accumulated large information about its molecular cell biology and infection biology. However, giardiasis is still one of the commonest parasitic diarrheal diseases affecting humans. Additionally, an alarming increase in cases refractory to conventional treatment has been reported in low prevalence settings. Consequently, efforts directed toward supporting the efficient use of alternative drugs, and the study of their molecular targets appears promising. Repurposing of proton pump inhibitors is effective in vitro against the parasite and the toxic activity is associated with the inhibition of the G. lamblia triosephosphate isomerase ( Gl TIM) via the formation of covalent adducts with cysteine residue at position 222. Herein, we evaluate the effectiveness of omeprazole in vitro and in situ on Gl TIM mutants lacking the most superficial cysteines. We studied the influence on the glycolysis of Giardia trophozoites treated with omeprazole and characterized, for the first time, the morphological effect caused by this drug on the parasite. Our results support the effectiveness of omeprazole against Gl TIM despite of the possibility to mutate the druggable amino acid targets as an adaptive response. Also, we further characterized the effect of omeprazole on trophozoites and discuss the possible mechanism involved in its antigiardial effect.
Research on Giardia lamblia has accumulated large information about its molecular cell biology and infection biology. However, giardiasis is still one of the commonest parasitic diarrheal diseases affecting humans. Additionally, an alarming increase in cases refractory to conventional treatment has been reported in low prevalence settings. Consequently, efforts directed toward supporting the efficient use of alternative drugs, and the study of their molecular targets appears promising. Repurposing of proton pump inhibitors is effective in vitro against the parasite and the toxic activity is associated with the inhibition of the G. lamblia triosephosphate isomerase ( Gl TIM) via the formation of covalent adducts with cysteine residue at position 222. Herein, we evaluate the effectiveness of omeprazole in vitro and in situ on Gl TIM mutants lacking the most superficial cysteines. We studied the influence on the glycolysis of Giardia trophozoites treated with omeprazole and characterized, for the first time, the morphological effect caused by this drug on the parasite. Our results support the effectiveness of omeprazole against Gl TIM despite of the possibility to mutate the druggable amino acid targets as an adaptive response. Also, we further characterized the effect of omeprazole on trophozoites and discuss the possible mechanism involved in its antigiardial effect.
CRISPR/Cas9 system is an extremely powerful technique that is extensively used for different genome modifications in various organisms including parasitic protists. Giardia intestinalis, a protozoan parasite infecting large number of people around the world each year, has been eluding the use of CRISPR/Cas9 technique so far which may be caused by its rather complicated genome containing four copies of each gene in its two nuclei. Apart from only single exception (Ebneter et al., 2016), without the use of CRISPR/Cas9 technology in its full potential, researchers in the field have not been able to establish knock-out cell lines to study the functional aspect of Giardia genes. In this work, we show the ability of in-vitro developed CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. Moreover, we used self-propagating CRISPR/Cas9 system to establish full knock out cell lines for mem, cwp1 and mlf1 genes. We also show that the system function even for essential genes, as we knocked-down tom40, lowering the amount of Tom40 protein by more than 90%. Further, we tested the length of homologous arms needed for successful integration of homology recombination cassette used for genome editing. Taken together, our work introduces CRISPR/Cas9 to Giardia for routine use in the lab, further extending the catalogue of molecular tolls available for genetic manipulation of the protist and allowing researchers to study the function of Giardia genes properly for the first time.
After ingestion of dormant cysts, the widespread protozoan parasite Giardia lamblia colonizes the host gastrointestinal tract via direct and reversible attachment using a novel microtubule organelle, the ventral disc. Extracellular attachment to the host allows the parasite to resist peristaltic flow, facilitates colonization and is proposed to cause damage to the microvilli of host enterocytes as well as disrupt host barrier integrity. The 9 um in diameter ventral disc is defined by a highly complex architecture of unique protein complexes scaffolded onto a spiral microtubule (MT) array of one hundred parallel, uniformly spaced MT polymers that bend approximately one and a quarter turns to form a domed structure. To investigate the role of disc-mediated attachment in causing epithelial cell damage, we used a new approach to rapidly create a stable quadruple knockout of Giardia of an essential ventral disc protein, MBP, using a new method of CRISPR-mediated gene disruption with multiple positive selectable markers. MBP quadruple KO mutant discs lack the characteristic domed architecture and possess a flattened crescent or horseshoe-shaped conformation that lacks the overlapping region, with severe defects in the microribbon-crossbridge (MR-CB) complex structure. MBP KO mutants are also unable to resist fluid flow required for attachment to inert surfaces. Importantly, MBP KO mutants have 100% penetrance off positive selection, which is essential for quantification of in vivo impacts of disc and attachment mutants with host cells. Using a new gastrointestinal organoid model of pathogenesis, we found that MBP KO infections had a significantly reduced ability to cause the barrier breakdown characteristic of wild-type infections. Overall, this work provides direct evidence of the role of MBP in creating the domed disc, as well as the first direct evidence that parasite attachment is necessary for host pathology, specifically epithelial barrier breakdown.
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