Elbeyioglu, Firat (2014) Lipid rafts and cell signalling in 'Schistosoma mansoni'. (MSc(R) thesis), Kingston University, .

Abstract

Schistosoma mansoni is a blood-dwelling parasite with the ability to cause schistosomiasis, a neglected tropical disease that is second in prevalence to malaria. This study focussed on the potential presence of lipid rafts in S. mansoni schistosomules (somules) and adult worms. Lipid rafts are characterised by a high content of cholesterol and sphingolipids and have been identified in many organisms with a critical role in cell signalling. Using a lipid raft staining kit, lipid rafts were found to be present at the surface of one-day old somules and adult worms when imaged using confocal laser scanning microscopy. Western blotting with antibodies against a number of proteins known to be present in lipid rafts revealed that the GTPase switch protein Ras (21 kDa), the Gqa subunit (45 kDa) and, most importantly, flotillin (48k Da) were present and detectable in S. mansoni worms and somules. Exposure of somules to 15 ng/ml human EGF resulted in the increased phosphorylation (activation) of extracellular signal-regulated kinase (ERK) after five minutes of EGF incubation. A similar result was observed for protein kinase C (PKC). A detergent-resistant membrane separation was next performed on adult worms which identified the presence of flotillin in the Triton X-100 insoluble fraction only, indicating the association of rafts with flotillin in schistosomes; this result was further confirmed when beta-tubulin, a cytosolic marker, was identified only in the cytosolic fraction. Immunohistochemistry identified the expression of flotillin, Ras and Gq in the tegument of the somules as well as the sub-tegumental musculature and other structures to varying degrees. Moreover phosphorylated ERK was also localised to these regions. Cholesterol depletion assays on EGF 15 ng/ml-treated somules using 1 mM methyl-beta-cyclodextrin identified loss of ERK signalling after 10 or 20 minute methyl-beta-cyclodextrin treatment. Collectively, the results of this study indicate the presence of lipid rafts at the surface of S. mansoni and highlight the potential importance that these domains play in this parasite.

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