THE ROLE OF EXTRACELLULAR VESICLES IN REGULATING DIVISION OF LABOUR IN FUNGI

Many animal species use teamwork in order to maximize fitness through Division of Labour: for instance, via the allocation of guard duties and food gathering duties to different individuals in social insects. However, a recent and surprising discovery has been that single-celled microbes are also capable of Division of Labour. Moreover, such behaviours often underlie important processes such as antimicrobial resistance in bacteria.

We have recently discovered a novel Division of Labour behaviour in the fatal fungal pathogen Cryptococcus gattii. Upon entry into the host, individual fungal cells communicate in order to adopt two different fates: some individuals stop growing and become quiescent "guard cells", which then protect the other "proliferative" individuals from being killed by the host. This enables the fungus to grow rapidly and overwhelm the host; something that neither fungal cell type can achieve alone.

The mechanism by which this Division of Labour occurs at the molecular level is completely unknown. However, we have recent data demonstrating that the cell-to-cell communication it entails is carried out by extracellular vesicles, which act as long-distance "carrier pigeons" to enable fungal cells to coordinate their behaviour over large distances.

The aim of this proposal is therefore to dissect, for the first time, the nature of this novel virulence mechanism by:

1. discovering how extracellular vesicles coordinate fungal behaviour

2. characterizing the contents of these vesicles

3. identifying how these vesicles are transported between fungi and their surrounding host cells

4. revealing how host cells respond to this fungal manipulation.

In doing so, we aim to reveal a completely new type of pathogenesis mechanism in an important human and animal disease. In addition, since i) Division of Labour mechanisms operate in a diverse range of microbes, and ii) extracellular vesicles are known to be shed by a large number of bacterial and fungal species, it is possible that the processes we will unveil within this project may provide a broad paradigm for understanding Division of Labour across the full breadth of microbiology.

A major revelation in microbiology within the last decade has been the discovery of microbial "teamwork" and, in particular, Division of Labour in microbial communities. Such behaviour underpins phenotypes ranging from colony morphology to antimicrobial resistance and indicates that Division of Labour may be a widespread occurrence in single-celled organisms. To date, however, the underlying mechanism of most Division of Labour processes remains unknown.

We recently uncovered a Division of Labour process that drives hypervirulence in the fatal fungal pathogen Cryptococcus gattii. During this process, individual fungal cells adopt either a "guard" or "proliferative" fate, and a mixture of both phenotypes is essential for effective pathogenesis in the host.

Such teamwork requires communication between individual microbial cells to ensure coordinated heterogeneity within the population. We have now discovered that, in C. gattii , this communication is carried out through the exchange of fungal extracellular vesicles. Specifically, addition of purified vesicles to otherwise non-pathogenic strains of C. gattii triggers Division of Labour and rapid proliferation within the host.

The aim of this proposal is thus to reveal the molecular mechanism of this Division of Labour by:
1. Determining how fungal-derived extracellular vesicles trigger Division of Labour in recipient cells
2. Determining the composition of these vesicles and the components that are critical for driving Division of Labour
3. Determining the cellular pathways that mediate trafficking of extracellular vesicles between donor and recipient cells
4. Determining how host cells respond to vesicle-mediated communication and thereby facilitate fungal virulence

In so doing, we aim to provide a) a full molecular characterization of a hitherto unknown mechanism of fungal virulence and b) a broader paradigm for investigating Division of Labour in otherwise unrelated microbial pathogens.

At heart, this is a basic science proposal that aims to address fundamental questions about teamwork in single celled organisms, the evolution of novel virulence mechanisms and cell-to-cell communication in fungi. Consequently, the major impact within the three years of this proposal will be on academic colleagues. However, we are aware that our proposed investigations hold long term impact for a number of industrial/commercial disciplines. Specifically:

1) Our work focuses on a major human and animal pathogen. Dissecting the molecular basis of virulence in this organism clearly offers the potential to develop novel therapeutic interventions and/or prevention strategies. Whilst this is a medium to long term objective of our work, we note that strategies to intervene in Division of Labour virulence processes may offer considerable benefits over traditional antimicrobials, since such 'antivirulence' interventions do not kill the microbe, thereby reducing the selective pressure to evolve resistance.

2) Microbial teamwork phenomena occur in a number of industrial microbiology processes. These can be either beneficial (e.g. with multiple steps of a biosynthetic pathway being "split" across different microbes) or detrimental (e.g. the formation of aggregating cell types that clump during brewing processes). Identifying the molecular pathway(s) that drive Division of Labour in a well-studied pathogen such as Cryptococcus gattii may therefore highlight potential avenues of intervention into these phenomena in less well-characterised microbial species that hold industrial importance.

3) Lastly, a major part of our proposal is the detailed characterization of extracellular vesicles. These unusual, highly stable lipid compartments hold considerable promise as biologically active delivery systems, and are under active investigation by many groups as potential drug delivery or transfection vehicles. To date, cryptococcal vesicles have not been investigated in this way, but the fact that they are highly resistant to host-imposed damage means that they may offer substantial advantages to such approaches. Consequently, the biochemical characterization we will undertake may be of considerable value to colleagues in these other, more applied, fields.