Date of Award

2026-05-01

Degree Name

Doctor of Philosophy

Department

Biosciences

Advisor(s)

Siddhartha Das

Abstract

Giardia lamblia is a protozoan parasite responsible for the intestinal disease giardiasis worldwide. Giardia is one of the major intestinal parasites in the world, with over 500,000 new cases annually. Symptoms of giardiasis include diarrhea, abdominal pain, and nausea. Children are particularly vulnerable to giardiasis, with cases of malnutrition, stunted growth, and cognitive impairment in countries where the disease is endemic. Giardia consists of two morphologic forms: an infective cyst which transmits the disease and a motile trophozoite which attaches to the epithelium of the small intestine during infection. Metronidazole is a common treatment for giardiasis, but cases of toxicity and resistance have been reported, with treatment failure being on the rise all around the world. Although Giardia attaches to the intestinal epithelium in a host, it does not enter host cells and is non-invasive. As a result, the mechanisms by which it causes infections and symptoms are unclear. Earlier studies from our laboratory have demonstrated that lipid rafts (LRs) and extracellular vesicles (EVs) play important roles during encystation (cyst formation) and host-parasite interactions. These LRs and EVs carry virulence factors that presumably paralyze the host's immune defenses and produce infection in the small intestine. LRs of Giardia are made of cholesterol and sphingolipids that transduce signals downstream of the plasma membrane and play a role in cell signaling and communications. EVs exist as two subtypes: microvesicles (large vesicles; MVs) and exosomes (small vesicles; EXOs). Previous work from our laboratory has demonstrated that oseltamivir (Osm, Tamiflu®), an anti-viral agent, disassembles LRs and the biogenesis of EVs in Giardia. Proteomic analysis revealed that LRs, MVs and EXOs s shared many giardial virulence factors, suggesting that the biogenesis of MVs and EXOs are linked to LR assembly and disassembly by Giardia. The goal of my dissertation is to better understand the mechanisms of disease transmission of Giardia and the roles of the vi parasite's LRs and EVs in this process. To address this, I successfully isolated and characterized EVs (both MVs and EXOs) secreted by Giardia using high-resolution transmission Electron microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). Giardia MVs are larger (~100 200 nm) than EXOs (~80 nm). In experiments with Caco-2 intestinal cells, Giardia, and EVs, we observed that both trophozoites and EVs induce the secretion of chemokines from both the apical and basolateral sites of the Caco-2 cells. Furthermore, it was observed that four oseltamivir (Osm) analogs (synthesized by Dr. Steve Patterson, Dr. Harrison VanKoten, and James Klinkenberg) reduced giardial growth, trophozoite attachment, cyst production, LR assembly, EV biogenesis, and the interactions with Caco-2 cells. Some compounds demonstrated selective activities against MVs or EXOs, suggesting that vesicle subtypes are structurally and functionally different. Finally, I have tested PPMP (1-phenyl-2-palmitoylamino-2-morpholino-1-propanol) and Miglustat (Zavesca®) for their effects on cyst production in culture. The rationale for using these two commercial compounds is that they are inhibitors of glucosylceramide transferase (gGlcT1) (which participates in cyst production by Giardia). My analysis reveals that both compounds inhibit cyst production, suggesting that gGlcT1 could function as a drug target against Giardia. gGlcT1 is a key enzyme in giardial encystation, cyst production, and lipid raft assembly. Next, with these results and new information, I hypothesize that giardial LRs, glucosylceramide transferase (gGlcT1), and EVs play key roles in the production of infection and host-parasite interactions. The Specific Aims of this project are as follows: (1) Determine whether extracellular vesicles (EVs) released by Giardia are involved in host-parasite interactions in a co-culture model; (2) Investigate whether oseltamivir analogs disrupt lipid raft integrity and inhibit extracellular vesicle (EV) release by Giardia during interactions with host intestinal epithelial cells, and (3) vii Investigate the role of Miglustat on infectivity and cyst production by Giardia. Once completed, I propose that the compounds tested (PPMP, Miglustat, and synthetic Osm analogs) will serve as alternative treatments for giardiasis that target pathways unique from what metronidazole targets, potentially allowing these compounds to be useful against metronidazole-refractory giardiasis.

Language

en

Provenance

Received from ProQuest

File Size

134 p.

File Format

application/pdf

Rights Holder

Breanna Chantal Pence

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