<i>Giardia's</i> domed ventral disc architecture is essential for attachment and contributes to epithelial barrier disruption

<i>Giardia lamblia</i> is a widespread anaerobic protistan parasite causing significant diarrheal disease worldwide. <i>Giardia</i> trophozoites attach extracellularly to the host gastrointestinal epithelium using a unique microtubule (MT) organelle, the ventral disk. The complex, dome-shaped disk is composed of microribbon-cross-bridge (MR-CB) protein complexes scaffolded onto a spiral MT array. Attachment is dynamic and reversible, facilitating parasite contact and colonization of the gastrointestinal epithelium. To investigate possible contributions of disk-mediated attachment to host pathobiology, we generated a stable quadruple allelic knockout (KO) of an abundant disk-associated protein, MBP, using a new method of CRISPR-mediated gene disruption. MBPKO mutants had flattened crescent- or horseshoe-shaped discs, severe MR-CB defects, and complete phenotypic penetrance off selection. MBP mutants also had aberrant surface contacts and were unable to resist shear forces under fluid flow. Using a human gastrointestinal organoid model, we discovered that MBPKO mutants had a significantly reduced ability to cause the host epithelial barrier breakdown characteristic of wild-type infections. In contrast, the addition of spent medium or lysed parasites had no impact on epithelial barrier breakdown. Overall, this pioneering work provides direct evidence that MBP is required for the domed-disk architecture and that disk-mediated attachment contributes to host pathobiology, specifically epithelial barrier breakdown.