Molecular Architecture, Function and Biogenesis of the Ventral Disc in Giardia

Giardiasis is the most common cause of acute protozoan intestinal infection worldwide, and chronic giardiasis is a major contributor to high morbidity in developing countries. Due to the lack of concerted research efforts, giardiasis has been designated a World Health Organization (WHO) neglected disease. <P> Giardia intestinalis is a parasitic protist, and its pathogenicity is dependent upon attachment to the intestinal microvilli via the ventral disc, a novel cytoskeletal structure. Conflicting biophysical support and a conspicuous lack of molecular evidence have hampered the investigation of proposed giardial attachment mechanisms. <P> The primary focus here is to evaluate support for two 'classic' hypotheses that explain giardial attachment: the 'Conformational Change' model and the 'Hydrodynamic' model. The 'Conformational Change' model proposes that conformational changes of the ventral disc cause suction-based attachment. <P> We will investigate putative disc conformational dynamics using high resolution cryoelectron tomography of the ventral disc, live cell imaging, and novel attachment assays (Aim 1). <P> We will also examine the role of disc-associated annexins (1-giardins) in disc conformational dynamics (Aim 2), and identify and characterize novel disc-associated components using a genome-wide, high-throughput random GFP visual screen (Aim 3). The 'Hydrodynamic Model' of giardial attachment posits that the ventral flagella produce a hydrodynamic current enabling suction-based attachment. To test this alternative, we will assay attachment dynamics in various mutants with motility defects (Aim 2). <P> Lastly, to inform our analyses of disc structure and function (Aims 1-3), we will characterize dorsal disc biogenesis and parental disc disassembly during cell division (Aim 4). After mitosis, two dorsal daughter discs are assembled and the parental ventral disc and median body are disassembled. Using live imaging of photoactivatable GFP- tagged strains and analyses of microtubule disassembly mutants, we will test the hypothesis that the median body acts as a reservoir of disc components for dorsal daughter discs. <P> PUBLIC HEALTH RELEVANCE: Giardia intestinalis is one of the ten major parasites in humans, and the current lack of understanding about Giardia has resulted in it being designated a 'neglected disease'. One sixth of the world's population is believed to suffer from giardiasis. Giardia attaches to the host small intestine via an undefined mechanism. The research proposed here directly investigates the attachment mechanism of Giardia, and will offer additional targets for drug discovery. Specifically we investigate the function of the 'ventral disc' a suction cup-like structure.