The Organization and Clustering of Giardial Lipid Raft Domains After Treatment with Oseltamivir by Direct Stochastic Optical Resolution Microscopy

Abstract

Lipid rafts contain combinations of glycosphigolipids, cholesterol, and protein receptors organized into lipid microdomains that form ordered structures. They play a role in signal transduction in order for cells to respond efficiently to changes in their environment and have the ability to undergo self-assembly and disassembly. Proteomic analysis reveals the presence of several proteins with biological functions in giardial lipid rafts (gLRs), including variable surface proteins, kinases, and glycosylphosphatidylinositol anchored proteins, giving rise to the idea that these lipid rafts could be a potential drug target. Previous data has shown that oseltamivir (Tamiflu), a neuraminidase inhibitor and FDA-approved drug, disrupts the growth and differentiation of Giardia by inhibiting formation of gLRs. Recalcitrant giardiasis is frequently contributed to drug resistance with treatment failures to the commonly prescribed drugs, metronidazole or albendazole, providing a need for new drug targets. We have determined the nanoscale clustering characteristics of GM1 ganglioside, a functionally important glycolipid in lipid rafts, using dSTORM, before and after treatment with oseltamivir. GM1 was labeled with CTxB-AF647 and Giardia were fixed in 4% agarose to prevent motion during acquisition for optimal resolution for dSTORM. In untreated Giardia, we observe clusters of GM1 with a mean radius of 19.3nm, with single probe density at 0.75E-3nm−2 and multi-probe density at 3.5E-5nm−2. After treatment with oseltamivir, we see a significant decrease in the size of GM1 clusters, with a mean radius of 15.1nm. Single probe density significantly increased to 1.5E-3nm−2, and multi-probe density significantly decreased to 1.25E-5nm−2. These results validate that at the nanoscale, oseltamivir disrupts gLR assembly and could be used as an anti-giardial drug, targeting specific raft clusters.