A lipidomic approach to elucidating the effects of antifungal compounds on signalling in fungi

Abstract

Lipid signalling underpins the majority of intracellular signalling pathways acting in membrane bound compartments to regulate the movement of molecules in and out of cells, the organisation of intracellular structures such as actin and even to organising the recycling of damaged or redundant proteins. The conserved Phosphatidylinositol phosphates (PIPs) are most relevant in these contexts and their tight regulation is essential for normal cell function. Perturbation of PIP signalling is known to be the root cause of very serious disease states such as OCRL, diabetes, obesity and other metabolism related deiseases in humans. The intracellular signalling of PIPS is highly susceptible to extracellular influence and can be hijacked by pathogenic organisms such as Shigella flexneri, Salmonella enterica, Phytophtora infestans to facilitate disease states and some compounds such as Wortmannin and LY294002 have been known to perturb PIP signalling. The crop protection agents Pyrimethanil, Mepanipyrim and Cyprodinil belong to a group of antifungal compounds known as anilinopyrimidines that are used to control plant pathogenic ‘gray mold’ fungus, Botrytis cinerea and Z.tritici in the field context. This work hypothesised that the effects of these compounds will perturb lipid and PIP signalling especially. This was investigated in the laboratory organism S.cerevisiae using methods such as actin phalloidin staining, FM 4-64 staining of vesicles, modified-phosphoinositide ELISA to understand how these antifungals affect the signalling paradigms in fungi. The understandings gained there were ported into the olant-pathogenic fungus Z.tritici and studied further. These studies revealed that the PI(4)P kinase Stt4p is highly relevant to the signalling pathways that these compounds affect. Endocytic and actin modelling systems are perturbed. The presence of Stt4 is protective for the effects of the compounds and while these compounds, especially Cyprodinil upregulate activity of the NADH:Oxidoreductase Complex I, the effect was more profound in the absence of Stt4 in yeasts. These findings may help to direct the course of crop-protective agents in the future.