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Study of the mechanisms impairing cell wall maintenance by abnormal sterols in fungi

Grant number: 16/07896-8
Support type:Scholarships in Brazil - Master
Effective date (Start): June 01, 2017
Effective date (End): May 31, 2019
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Agustín Hernández López
Grantee:Leydi Roxana Gutiérrez Armijos
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated research grant:14/10443-0 - Mechanisms and consequences of intracellular traffic impairment by 8- and 14-dehydrosterols in fungal parasite models, AP.JP

Abstract

Parasitic fungi are responsible for ca 70% of plant diseases. Amine fungicides are a class of compounds essential for crop protection since they can control the proliferation of parasites affecting high value crops, for example yellow sigatoka, a parasite responsible for the loss of ca 50% of the banana crop yield in Brazil. The targets of this class of compounds are the enzymes sterol-”8,”7-isomerase and sterol-”14-redutase. In yeast, these enzymes are encoded by the genes named ERG2 and ERG24, respectively. Inhibition of these enzymes induces the accumulation of abnormal sterols. Nevertheless, despite many years since the initial release of these fungicides, the cellular mechanisms these abnormal sterols affect cell viability and control fungal proliferation are still unknown. In all organisms bearing cell wall, it is well known there exists a correlation between defects in sterol homeostasis and cell wall defects. However, the mechanisms behind this fenomenon are still obscure. The cell wall represents the fungal protection layer against biotic and abiotic stresses. In addition, in parasitic fungi, host invasion requires remodelling of this organelle and, also, it bears the majority of the targets initially recognised by the host. Therefore, the importance of knowing the mechanisms that involve cell wall and sterols is paramount. Among the majoritary and most important components of the fungal cell wall responding to damage are the glucans. In yeast, sinthesis of ²-1,3-glucan is carried out by synthases Fks1-3p, although only Fks1p is the only important under normal vegetative conditions. Glucan synthases (GS) are plasm membrane embeded proteins and they need to reach their destination through the exocitic pathway. In previous studies, our group showed that some plasma membrane proteins like Pma1p are re-directed towards the vacuole when the cell accumulates abnormal 8-dehydroesterols. In yeast, cell wall maintenance is chiefly regulated through the TORC2/CWI (Cell Wall Integrity) pathway. Cell wall defects are detected by a series of plasma membrane sensors, the most important being Wsc1p. Under stressful conditions, these sensors are relocated to the vacuole; therefore, it is reasonable to think that a misslocalization of the these sensors could lead to cell wall defects. Our group has shown that, in abnormal sterol-accumulating cells, endocytosis is inhibited due to loss of the capacity of lumenal acidification through the V-ATPase. In addition of the plasma membrane sensors, TORC2 complex is a major regulator of the response to cell wall damage. This complex is located at the plasma membrane and, among other functions, controls clathrin-independent endocytosis and cell wall maintenance. In this context, it is reasonable to think that plasma membrane lipid composition affects its localisation or function and, hence, the Fks1p-dependent regulation of cell wall maintenance. In addition, other regulators, like Rho1p, may be involved in regulating GS catalytic activity. In this sense, the hypothesis behind this project is that accumulation of abnormal sterols affects cell wall maintenance through one or several of the following mechanisms: directly through lipid-mediated inhibition of GS catalytic activity or indirectly through inhibition of the endo/exocytic traffic. Among the methods to be used in this study they can be mentioned reverse genetics techniques, site-directed mutagenesis, fluorescence microscopy and enzyme kinetics.