Biological and biophysical characterisation of novel virulence factors from pathogenic fungi of humans

Pathogenic fungi kill an estimated 1.2 million people every year. The global incidence of life-threatening fungal infections is rising, and the threat to public health from drug resistant isolates and emerging pathogens such as Candida auris is serious and growing. However, despite the global impact of fungal diseases on human health, little is known about the virulence factors that drive pathogenesis. Recently, we discovered that the fungal pathogen Candida albicans secretes a peptide virulence factor called candidalysin, the first cytolytic toxin identified in a fungal pathogen of humans. Our discovery of candidalysin has created an exciting new field of research investigating the toxin biology of pathogenic fungi that cause disease in humans.

Using a bespoke computational algorithm to screen 350 fungal genomes, we have identified 219 new putative toxins in 37 different species of medically relevant fungal pathogens, some of which cause oral infections (Candida albicans, C. dubliniensis, C. glabrata, C. orthopsilosis, C. parapsilosis, C. tropicalis, C. lusitaniae and Paracoccidioides brasiliensis) skin infections (Coccidioides posadasii, Malassezia globosa, Mucor circinelloides, Rhizopus delemar, R. microsporus, Sporothrix brasiliensis, S. schenckii, Trichophyton interdigitale and T. rubrum), and lung infections (Aspergillus fumigatus, Cryptococcus neoformans, C. gattii, Histoplasma capsulatum and Pneumocystis jirovecii).

We hypothesize that some of these molecules are likely to be novel virulence factors that promote fungal pathogenicity and disease progression. Toxins associated with fungi that pose the greatest threat to human health will be prioritised for analysis.

This research project will use biological, cellular, immunological and biophysical techniques to characterise the role of selected toxins in fungal pathogenicity.

The goals of this PhD project are to:
1. Characterise fungal toxins associated with oral, skin and lung infections for their ability to cause cellular damage and induction of host immune responses.
2. Assess the role of toxins during fungal infection of human cells using in vitro and in vivo model systems.
3. Ascertain the mechanism of toxin action on human cells and artificial plasma membranes.

Initial characterisation of toxins will be performed on oral epithelial cells. Toxins will be evaluated for their ability to cause plasma membrane damage, induction of cellular stresses (calcium influx, mitochondrial dysfunction, depletion of intracellular ATP, generation of reactive oxygen species), and activation of host immune responses and intracellular signalling pathways (EGFR/p38/c-Fos).

To characterise the interaction of toxins with synthetic lipid membranes, permeabilisation assays will be performed on fluorophore-containing small unilamellar vesicle (SUVs) comprised of different lipids. In parallel, electrical conductance spectroscopy will be performed on synthetic lipid bilayers to determine the duration of membrane destabilisation, diameter of toxin pores and the role of specific lipids in toxin-induced membrane destabilisation. Genetic techniques will be used to knock out toxin-encoding genes in selected fungal pathogens and compare the ability of wild type and mutant fungi to cause disease using established in vitro and in vivo model systems (costs for in vivo work met through alternative sources).

This PhD project will significantly advance our understanding of fungal disease at mucosal surfaces and create future avenues of therapeutic intervention.