Identification of Actin Filament Interactors in Giardia lamblia

Abstract 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 non-canonical Giardia lamblia actin ( Gl Actin) interactors; however, these proteins appeared to interact only with monomeric or globular actin (G-actin), rather than filamentous actin (F-actin). To identify interactors, we used a chemical crosslinker to preserve native interactions, followed by an anti- Gl Actin antibody, Protein A affinity chromatography, and liquid chromatography coupled to mass spectrometry. We found 46 putative actin interactors enriched in 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 category was co-localized with Gl Actin using immunofluorescence microscopy. We also co-immunoprecipitated one protein from each category and confirmed three interactions. Most of the localization patterns are consistent with previously demonstrated Gl Actin functions, but the ventral disc represents a new category of actin interactor localization. These results suggest a role for Gl Actin in ventral disc function, which has previously been controversial. Importance The single-celled eukaryote Giardia lamblia is an intestinal parasite that colonizes the small intestine and causes diarrhea and vomiting, which can lead to dehydration and malnutrition. Giardia actin ( Gl Actin) 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 Gl Actin’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 unlikely to have side effects.