The Physiological Role of Intracellular Zinc Trafficking in Fungi

All life, from humans, to the microbes that infect us, requires micronutrients for growth and development. Zinc is an essential micronutrient because it is a cofactor for approximately 10% of eukaryotic cell proteins, but it can also be very toxic at high concentrations. The human immune system has harnessed both the essentiality and toxicity of this trace metal to combat microbial infections: a process known as “nutritional immunity”. In turn, pathogens have developed counter-mechanisms in order to maintain zinc homeostasis in the nutritionally-challenging environment of the infected host [1,2]. We have found that the human fungal pathogen, Candida albicans, can assimilate significant intracellular stores of zinc in compartments that are trafficked dynamically within the fungal cell.

The aim of this project is to understand how fungi control the intracellular trafficking of zinc, a fundamental yet understudied biological process which regulates this essential, yet poisonous, metal cofactor within the cell.

This project will employ a diverse repertoire of interdisciplinary technologies in order to build a detailed model of zinc homeostasis, and how changes in zinc status impact cell physiology. We know that C. albicans can accumulate and mobilise zinc, but we do not yet understand the underlying mechanisms. We will therefore use live-cell imaging and zinc-specific fluorescent probes to monitor the dynamics of intracellular trafficking within filamentous C. albicans cells. This novel bioimaging technology will be combined with organelle-specific probes and chemical inhibitors of defined cellular components. These will uncover the processes driving zinc mobilisation and the nature of zinc-containing compartments, which have been referred to as ‘zincosomes’. We will then explore zinc mobility in C. albicans mutants that lack intracellular zinc transporters and proteins involved in vacuolar biogenesis. Localisation studies will be performed under zinc starvation, zinc overload and oxidative stress, during which zinc serves a crucial role in detoxifying reactive oxygen species.

To complement subcellular localisation studies, inductively-coupled plasma mass-spectrometry will be used to provide an absolute quantification of metal levels in fungal cells. These interdisciplinary approaches will illustrate the molecular and cellular mechanisms which drive zinc-trafficking in C. albicans and how this dynamic process contributes to environmental adaptation.

To assess whether zinc mobilisation is a conserved process in fungi, the bioimaging tools developed in C. albicans will be applied to Aspergillus nidulans, a relatively distantly-related filamentous fungus. Indeed, it is anticipated that the fundamental mechanisms discovered in this project will be broadly applicable to many other species, including industrially-important species and both fungal plant pathogens and symbionts.

Importantly, C. albicans is a major pathogen of humans. Therefore, in the final element of this project, we will elucidate the role of zinc trafficking in pathogen-host interactions. Macrophages kill their microbial meals via oxidative mechanisms, but can also be manipulated, via cytokine stimulation, to either accumulate or exclude zinc [3]. This permits investigation of both facets of zinc nutritional immunity, at a single cell level. Fungal intracellular zinc trafficking will be assessed within stimulated macrophages using fluorescent probes. Fungal mutants that exhibit defective zinc mobilisation (above) will also be tested for their ability to survive confrontations with these immune cells. Using these models, we will uncover the role of fungal zinc trafficking in resisting attack by the immune system.