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Identification of growth inhibiting compounds in a Giardia lamblia high-throughput screen.

ABSTRACT: Giardia lamblia is one of the most common eukaryotic pathogens and is classified by the CDC as a category B agent of bioterrorism. In a departure from more traditional research focused on specific pathways or molecules, we have developed a high-throughput assay for screening libraries of small compounds for inhibitors and enhancers of trophozoite multiplication. Following a 24-h period of culture in 384-well plates in the presence of compounds, trophozoites were fixed, stained and enumerated. Quadruplicate screening of 1520 compounds from two libraries of known bioactives detected numerous inhibitory compounds. Based on a stringent cut-off of 5 standard deviations from the plate mean, 50 compounds (3.3%) were inhibitory. The activity of 3 compounds was confirmed in conventional culture. Although not meeting the threshold, one compound (indirubin) was identified as an agonist of trophozoite proliferation. Demonstrating the potential of high-throughput screening for rapidly finding new compounds which perturb G. lamblia multiplication, most of the hits identified by high-throughput screening do not appear to have been tested previously for their ability to affect G. lamblia trophozoites. High-throughput screening of bioactive compounds will open new avenues to a system-wide analysis of pathways affecting G. lamblia proliferation, and eventually to other phases of the life cycle.

SUBMITTER: Bonilla-Santiago R

PROVIDER: S-EPMC2597095 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Identification of growth inhibiting compounds in a Giardia lamblia high-throughput screen.

Bonilla-Santiago Rubén R Wu Zhijin Z Zhang Linghui L Widmer Giovanni G

Molecular and biochemical parasitology 20080829 2

Giardia lamblia is one of the most common eukaryotic pathogens and is classified by the CDC as a category B agent of bioterrorism. In a departure from more traditional research focused on specific pathways or molecules, we have developed a high-throughput assay for screening libraries of small compounds for inhibitors and enhancers of trophozoite multiplication. Following a 24-h period of culture in 384-well plates in the presence of compounds, trophozoites were fixed, stained and enumerated. Qu ...[more]

PMID: 18796315

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Project description:The deep-branching protozoan parasite Giardia lamblia is the causative agent of the intestinal disease giardiasis. Consistent with its proposed evolutionary position, many pathways are minimalistic or divergent, including its actin cytoskeleton. Giardia is the only eukaryote known to lack all canonical actin-binding proteins. Previously, our lab identified a number of noncanonical Giardia lamblia actin (GlActin) interactors; however, these proteins appeared to interact only with monomeric or globular actin (G-actin) rather than with filamentous actin (F-actin). To identify F-actin interactors, we used a chemical cross-linker to preserve native interactions followed by an anti-GlActin antibody, protein A affinity chromatography, and liquid chromatography coupled to mass spectrometry. We found 46 putative actin interactors enriched under the conditions favoring F-actin. Data are available via ProteomeXchange with identifier PXD026067. None of the proteins identified contain known actin-interacting motifs, and many lacked conserved domains. Each potential interactor was then tagged with the fluorescent protein mNeonGreen and visualized in live cells. We categorized the proteins based on their primary localization; localizations included ventral disc, marginal plate, nuclei, flagella, plasma membrane, and internal membranes. One protein from each of the six categories was colocalized with GlActin using immunofluorescence microscopy. We also co-immunoprecipitated one protein from each category and confirmed three of the six potential interactions. Most of the localization patterns are consistent with previously demonstrated GlActin functions, but the ventral disc represents a new category of actin interactor localization. These results suggest a role for GlActin in ventral disc function, which has previously been controversial. IMPORTANCE Giardia lamblia is an intestinal parasite that colonizes the small intestine and causes diarrhea, which can lead to dehydration and malnutrition. Giardia actin (GlActin) has a conserved role in Giardia cells, despite being a highly divergent protein with none of the conserved regulators found in model organisms. Here, we identify and localize 46 interactors of polymerized actin. These putative interactors localize to a number of places in the cell, underlining GlActin's importance in multiple cellular processes. Surprisingly, eight of these proteins localize to the ventral disc, Giardia's host attachment organelle. Since host attachment is required for infection, proteins involved in this process are an appealing target for new drugs. While treatments for Giardia exist, drug resistance is becoming more common, resulting in a need for new treatments. Giardia and human systems are highly dissimilar, thus drugs specifically tailored to Giardia proteins would be less likely to have side effects.

Dataset Information

Identification of growth inhibiting compounds in a Giardia lamblia high-throughput screen.

Publications

Identification of growth inhibiting compounds in a Giardia lamblia high-throughput screen.

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