Phosphoinositide 3-kinase signalling pathways are critical in the regulation of several cellular mechanisms studied in mammalian and yeast cells. Recent studies have identified and characterised two putative phosphoinositide 3-kinase (PI3K) genes in Giardia intestinalis called GiPI3K1 and GiPI3K2, which have homology to Class I and Class III PI3K isoforms, respectively. G. intestinalis is a parasitic protozoan causing diarrhoea and malabsorption throughout the world. It has been hypothesised that Giardia processes such as cell growth, differentiation and vesicle trafficking, all of which are important for parasite proliferation and disease transmission, may be mediated by the PI3K signalling mechanism. This research aims to test this hypothesis using bioinformatics and a range of experimental approaches. We used BLAST and Clustal W analysis to further characterise PI3K genes and identify and characterise additional putative ancillary components of lipid signalling cascades. A limited number of such components was identified in the Giardia genome by homology searching, but two genes with high homology to Saccharomyces cerevisae glycogen synthases kinase-3 (GSK-3) were found with 40% identity to Giardia GSK sequence alignment. To investigate the putative functionality of these lipid signalling kinases in Giardia, we exposed parasite trophozoites to a range of well characterised specific inhibitors of GSK and PI3K and measured their effect on parasite growth. Also, we initiated encystation of trophozoites to cysts cells and exposed to inhibitors to observe their effect. As an alternative to cell counting, we developed a quantitative spectrophotometric method that correlates cell number to methylene blue staining. The PI3K inhibitor LY294002 showed the strong inhibition of trophozoite growth. However PI-103, a recently described specific inhibitor of PI3K, showed no evidence of reducing trophozoites’ proliferation. Neither did wortmanin, a potent and irreversible inhibitor of PI3K. The structural features of Giardia PI3K that may explain these observations will be discussed. Exposure to inhibitory concentrations of CT99021, a specific inhibitor of GSK-3, had no detectable effect on Giardia growth. This was not unexpected as, in mammalian cells and other eukaryotes, protein kinase B-mediated phosphorylation of GSK-3 prevents cell cycle arrest as a result of GSK3-mediated phosphorylation of specific cyclins. This result, however, rules out the possibility that the growth inhibition of Giardia trophozoites observed with LY294002 could be the result of non-specific GSK-3 inhibition.
|Award date||1 Feb 2015|
|Publication status||Unpublished - 2015|