The Assembly of GM1 Glycolipid- and Cholesterol-Enriched Raft-Like Membrane Microdomains Is Important for Giardial Encystation

Abstract

Although encystation (or cyst formation) is an important step of the life cycle of Giardia, the cellular events that trigger encystation are poorly understood. Because membrane microdomains are involved in inducing growth and differentiation in many eukaryotes, we wondered if these raft-like domains are assembled by this parasite and participate in the encystation process. Since the GM1 ganglioside is a major constituent of mammalian lipid rafts (LRs) and known to react with cholera toxin B (CTXB), we used Alexa Fluor-conjugated CTXB and GM1 antibodies to detect giardial LRs. Raft-like structures in trophozoites are located in the plasma membranes and on the periphery of ventral discs. In cysts, however, they are localized in the membranes beneath the cyst wall. Nystatin and filipin III, two cholesterol-binding agents, and oseltamivir (Tamiflu), a viral neuraminidase inhibitor, disassembled the microdomains, as evidenced by reduced staining of trophozoites with CTXB and GM1 antibodies. GM1-and cholesterol-enriched LRs were isolated from Giardia by density gradient centrifugation and found to be sensitive to nystatin and oseltamivir. The involvement of LRs in encystation could be supported by the observation that raft inhibitors interrupted the biogenesis of encystation-specific vesicles and cyst production. Furthermore, culturing of trophozoites in dialyzed medium containing fetal bovine serum (which is low in cholesterol) reduced raft assembly and encystation, which could be rescued by adding cholesterol from the outside. Our results suggest that Giardia is able to form GM1-and cholesterol-enriched lipid rafts and these raft domains are important for encystation.

Arachidonic Acid Induces the Migration of MDA-MB-231 Cells by Activating Raft-associated Leukotriene B4 Receptors

“…Student’s t tests were performed for the results shown in Fig. S1, and a P value of < 0.05 was considered statistically significant [26]. The results obtained from HPLC data (Fig.…”

Microvesicles released from Giardia intestinalis disturb host-pathogen response in vitro

“…Moreover nystatin and filipin III, two well known LR disrupting agents inhibited the CTXB binding indicating that lipid rafts contain cholesterol and the removal destabilized the microdomains. De Chatterjee et al (2015) discussed the possible connection between lipids rafts and sphingolipids metabolism in Giardia regulating the encystation process.…”

A Targeted Mass Spectrometric Analysis Reveals the Presence of a Reduced but Dynamic Sphingolipid Metabolic Pathway in an Ancient Protozoan, Giardia lamblia

“…Cells were allowed to encyst for 72 h, and cysts were isolated by centrifugation (2,500 × g for 10 min at 4°C), washed three times in cold, distilled water, and kept in water for 1-h at 4°C. Cells were immunostained with antibodies against trophozoite and cyst proteins as previously described (De Chatterjee et al, 2015).…”

“…To observe the expression of trophozoites-specific proteins, parasites were fixed in 4% paraformaldehyde for 15 min and immunostained with FITC-conjugated trophozoite antibody (Troph-O-Glo, anti-rat polyclonal antibody, Waterborne Inc., New Orleans, LA) as described by us earlier (De Chatterjee et al, 2015). For cyst-wall proteins, we used cyst antibody (1:100, monoclonal: Santa Cruz Biotechnology, Inc., Santa Cruz, CA).…”

“…For intensity measurements, cells were selected randomly from 10 fields of 3–5 separate experiments. Approximately 50 cells were considered for each condition and were analyzed by Zeiss 2009 ZEN confocal software (De Chatterjee et al, 2015).…”