Inorganic phosphate (Pi) is an important macronutrient for all living organisms including yeast, and is essential for ATP, phospholipid and protein synthesis, metabolism and signalling. We are interested in understanding how phosphate homeostasis contributes to fungal virulence and whether enzymes involved are targets for anti-fungal drug design. Key enzymes include inositol polyphosphate (IP) kinases and acid phosphatases. IP kinases covalently attach free Pi to inositol, creating a multitude of IP species with putative roles in transcription regulation, mRNA export, controlling telomere length and protein traffic. Acid phosphatases make Pi available from a variety of organic phosphate sources.
Projects are available to study the role of
Project 1: Role of IP Kinases in fungal pathogenesis Our research group established that fungal phospholipase C1 (Plc1) is the progenitor of IP species in C. neoformans10. Recently, in collaboration with University College London, we identified a number of sequentially-acting IP kinases involved in production of mono (P)- or di(PP)- phosphorylated inositol species2 and obtained NHMRC funding to study the role of these IP kinases (and their IP products) in fungal pathogenesis. We have now created the complete set of cryptococcal IP kinase gene deletion mutants and shown that several have virulence factor defects and reduced ability to cause disease in animal models. We are now in a position to understand how these kinases contribute to pathogenesis, specifically their involvement in signalling, secretion of fungal virulence factors, stimulation of the host immune response and dissemination of infection to the central nervous system.
Project 2: Role of acid phosphatases in fungal pathogenesis We identified phosphate-repressible acid phosphatase (Aph1) in the cryptococcal secretome using proteomics, and demonstrated that it is the major extracellular acid phosphatase in C. neoformans. Sec14-14 was required for optimal export and secretion of Aph1. We labelled Aph1 with a red fluorescent tag and, using fluorescence microscopy, determined that its movement to the cell periphery and vacuoles occurs via endosomes. Aph1 hydrolyzed a range of physiological substrates suggesting that its role is to recycle phosphate from macromolecules in fungal vacuoles and to scavenge phosphate from the extracellular environment. Aph1 also contributes to the cryptococcal virulence profile. The role of other acid phosphatases in phosphate recycling, cellular function and pathogenesis will be investigated.
Techniques include targeted gene disruption using cloning/overlap PCR and biolistic transformation, qPCR, western blotting, transcriptomics, proteomics, virulence profiling in vitro and in vivo using animal models, constructing fluorescent-tagged proteins and tracking their intracellular location using fluorescence microscopy, enzyme assays, phagocytosis and macrophage activation assays using flow cytometry/RT-PCR, drug susceptibility assays.
Outcomes The knowledge gained will further understanding of the role of these enzymes in phosphate homeostasis, signalling and pathogenesis, and may inform new strategies for anti-fungal drug development, enhancing fungal cell recognition by the host and/or manipulating the host immune response to improve infection outcome.
Co-Supervisor Honorary Associate Dr Sophia Lev
The opportunity ID for this research opportunity is 46