Biophysical and genetic approaches to uncover ventral disc contractile attachment mechanisms in Giardia

Giardia lamblia is a widespread zoonotic intestinal parasite that causes acute and chronic diarrheal disease, affecting over 280 million people annually. Hosts ingest cysts, which excyst into trophozoites that extracellularly attach to the small intestine using the ventral disc, a complex and unique microtubule (MT) organelle. Attachment allows Giardia to resist host peristalsis and colonize the epithelium and may also directly damage and shorten the microvilli of epithelial cells, disrupt the intestinal epithelial barrier, or trigger apoptosis and malabsorption. The ventral disc is eight microns in diameter and is composed of upwards of 100 disc-associated proteins (DAPs) that create unique substructural disc components whose movements may generate forces required for attachment. How these disc substructures function to generate the varied biophysical forces underlying disc-mediated attachment is not well understood. Using live imaging, we recently provided direct evidence of disc contraction during attachment, supporting early observations of contractile attachment mechanisms causing damage to the epithelium. Here we leverage new CRISPR-based genetic tools to create quadruple allelic knockouts in this double diploid parasite. We then employ live imaging approaches with fluorescently tagged marker strains and state-of-the-art biophysical assays using rigid and deformable surfaces to evaluate the contribution of three key disc structural elements (the MR-CB complex, the lateral crest, and the overlap zone) to the modes and forces of disc contraction governing attachment. Both new and existing DAP mutants that comprise these substructural elements will be used to test three different hypothetical force-generating mechanisms for disc-mediated contraction and attachment. More than one DAP associated with a given substructure is tested, as null mutants of different DAPs may have different disc functional phenotypes. New DAPs contributing to disc contractility will also be identified and tested using two unbiased CRISPRi-based attachment and contractility screens. Unravelling the molecular intricacies of how disc substructures generate forces of attachment will enhance our understanding of this key virulence component of giardiasis and offer potential therapeutic targets to combat Giardia infection and its associated pathology.