Disc and Actin-Associated Protein 1 Influence Attachment in the Intestinal Parasite Giardia lamblia

Abstract

The deep-branching eukaryote Giardia lamblia is an extracellular parasite that attaches to the host intestine via a microtubule-based structure called the ventral disc. Control of attachment is mediated in part by the movement of two regions of the ventral disc that either permit or exclude the passage of fluid under the disc. Several known disc-associated proteins (DAPs) contribute to disc structure and function, but no force-generating protein has been identified among them. We recently identified several Giardia actin (GlActin) interacting proteins at the ventral disc, which could potentially employ actin polymerization for force generation and disc conformational changes. One of these proteins, Disc and Actin Associated Protein 1 (DAAP1), is highly enriched at the two regions of the disc previously shown to be important for fluid flow during attachment. In this study, we investigate the role of both GlActin and DAAP1 in ventral disc morphology and function. We confirmed interaction between GlActin and DAAP1 through coimmunoprecipitation, and used immunofluorescence to localize both proteins throughout the cell cycle and during trophozoite attachment. Similar to other DAPs, the association of DAAP1 with the disc is stable, except during cell division when the disc disassembles. Depletion of GlActin by translation-blocking antisense morpholinos resulted in both impaired attachment and defects in the ventral disc, indicating that GlActin contributes to disc-mediated attachment.Depletion of DAAP1 through CRISPR interference resulted in intact discs but impaired attachment, gating, and flow under the disc. As attachment is essential for infection, elucidation of these and other molecular mediators is a promising area for development of new therapeutics against a ubiquitous parasite.Author SummaryGiardia lamblia is a single-celled organism and one of the most common gastrointestinal parasites worldwide. In developing countries, recurrent Giardia infections are common, due to lack of access to clean water. Giardia infections can lead to diarrhea, vomiting, dehydration, disruption of the intestinal microbiome, and chronic infections can lead to colitis and irritable bowel syndrome. Because existing drug treatments have side effects and Giardia’s resistance to drugs is increasing, new treatment strategies are needed. The parasite’s attachment to the host’s intestine is mediated by a Giardia-specific structure that resembles a suction cup and is called the ventral adhesive disc. We previously identified DAAP1, a protein which interacts with Giardia actin and localizes to the ventral disc. Here, we explore the relationship between these two proteins and investigate their role in disc-based attachment. Most disc proteins, including DAAP1, are unrelated to any human proteins, making them appealing drug targets to inhibit parasite attachment and infection.

The Giardia ventrolateral flange is a lamellar membrane protrusion that supports attachment

“…Therapeutic strategies targeting attachment would therefore effectively clear infection. The results presented here in combination with our recent study of Gl Actin’s role in ventral disc function indicate that Giardia’s highly divergent actin homolog has multiple roles in supporting attachment [ 5 , 6 ]. Inhibiting Gl Actin would simultaneously disrupt ventral disc and ventrolateral flange function, making it a promising therapeutic target.…”

“…We observed fewer Gl Rac- and Gl Actin-knockdown cells attached to the culture tubes compared to the control ( S7 Fig ). The reduced attachment of Gl Rac- and Gl Actin-depleted cells likely resulted from gross disorganization of the cytoskeleton [ 14 ] which can disruption both the flange and ventral disc [ 6 ]. Due to multiple roles for each of these proteins in cellular organization specific conclusions about the role of the ventrolateral flange in attachment cannot be made with this assay.…”

“…The prominent microtubule based ventral adhesive disc plays a central role in supporting parasitic attachment [ 4 ]. Recently we used proteomics to identify actin binding proteins in Giardia and found several that localized to portions of the ventral disc which indicates that the actin cytoskeleton also plays a role in parasitic attachment [ 5 , 6 ].…”