Award details

The Cryptococcus neoformans Redoxome: The role of Rac GTPases in ROS Signal Transduction and Titanisation

Reference	BB/M014525/1
Principal Investigator / Supervisor	Dr Elizabeth Ballou
Co-Investigators / Co-Supervisors
Institution	University of Aberdeen
Department	School of Medical Sciences
Funding type	Research
Value (£)	293,179
Status	Completed
Type	Fellowships
Start date	27/03/2015
End date	03/04/2017
Duration	24 months

Abstract

This Fellowship addresses the role of Reactive Oxygen Species (ROS) as signalling molecules that regulate genome plasticity in the fungal pathogen Cryptococcus neoformans and tests the hypothesis that C. neoformans has co-opted conserved Rac-GTPase-mediated ROS signalling to regulate a niche specific stress response. Rac is a conserved component of the NADPH Oxidase (Nox) complex responsible for the oxidative burst in phagocytes. Rac-Nox-dependent ROS generation is conserved across eukaryotes, but no CnNox homologue is yet known. C. neoformans causes 600,000 deaths annually. In the lung, small haploid yeast can convert to large, polyploid Titan cells that resist phagocytosis and can bud off haploid cells, acting as reservoirs of infection. The yeast-to-Titan switch is likely a stress response, however the underlying mechanisms remain unclear. I have shown that CnRac regulates ploidy: Rac over-expression suppresses polyploid mutant phenotypes, and rac mutants are anueploid. Additionally, Rac is required for ROS localisation during hyphal polarisation. These data suggest a novel role for Rac and ROS in genome plasticity and polarisation. Targets of ROS (the Redoxome) are known to regulate stress responses in other species. I hypothesise that a CnRac-Nox pathway has been adapted for the Titan cell stress response. Here, I will: 1. Define conserved and fungal-specific elements of the C. neoformans Rac-Nox complex and build a model of Rac-Nox regulation of the yeast-to-Titan switch. 2. Identify redox targets of Rac-Nox ROS signal transduction through whole Redoxome mapping during yeast and Titan-inducing conditions. 3. Identify chemical inhibitors of Rac-Nox function and test for inhibition of the yeast-to-Titan switch. This work will contribute to fundamental understanding of genome plasticity, the Redoxome, and cellular proliferation. The low homology between human and CnNox makes these promising drug targets and may lead to novel antifungal therapies.

Summary

When cells grow and divide, they must simultaneously coordinate a number of complex events. The mother cell must direct all of its growth to the budding daughter cell and then must duplicate and correctly share out its DNA, giving one copy to the daughter and keeping one copy for itself. Without tight coordination of these events, daughter and mother cells die. As a consequence, cells have evolved detailed mechanisms to tightly coordinate growth, DNA duplication and DNA distribution. Some cells have found another way around this problem, however: They are able to survive even when they have the wrong amount of DNA. This survival is called genome plasticity because in these cells, the number of copies of DNA, also known as the genome, is malleable. Cancer is one key example of cells displaying genome plasticity. In these cells, having many copies of the genome gives the cell more tools to grow and to evade drug treatment. Another example of cells with genome plasticity is the fungal pathogen Cryptococcus neoformans. Cryptococcus affects nearly 1 million people each year worldwide, and kills nearly two thirds of those infected within three months of infection. Cryptococcus grows in the lungs. In healthy people, the immune system is able to combat this growth, but in people with underlying diseases, including HIV and auto-immune diseases, the immune system is weakened. Among people on long-term immune suppressors, such as steroids to combat organ transplant rejection, 1 in 20 develop cryptococcosis. In these individuals, Cryptococcus escapes the lung and goes to the brain, where it causes meningitis and death if left untreated. Normal Cryptococcus cells have a single copy of their DNA, but Cryptococcus also makes a unique structure called a Titan cell that contains many DNA copies. What is particularly striking is that Titan cells are still able to keep this DNA organised and give only one copy to their daughters during budding. As the name suggests, Cryptococcus Titan cells are much larger than ordinary cells -10 times bigger. This is similar to a cherry ballooning to the size of a football. These huge cells are too big for our immune cells to destroy, and they also produce new small cells that can escape into the blood and cause disease. No one knows how Cryptococcus Titan cells are formed. One clue is that Cryptococcus uses molecules called ROS as messengers in the cell. In other organisms, ROS send signals to help coordinate growth. I have shown that Cryptococcus mutants that are defective in ROS are also defective in growth. Additionally, the same factors that control ROS also control genome plasticity. Together, this suggests that ROS, growth and genome plasticity may be related. For example, ROS may act as messengers during budding that tell the mother cell when it is time to distribute DNA. Titan cells may form when this signal is altered. The research in this Fellowship will investigate how Cryptococcus accomplishes this task. Because Cryptococcus uses Titan cells to resist drug treatment, understanding how Titan cells work and how to prevent their formation will help us develop better drugs.

Impact Summary

This proposal focuses on the pressing global health issue of Cryptococcus infection, which carries significant morbidity, mortality and financial burdens. The need for novel drug therapies across infectious fungal species will be a growing problem in the future as expanding vulnerable patient populations are placed on long-term prophylactic treatment. Understanding how fungal genetic plasticity facilitates persistence within the host will be important to efforts to preserve available therapies and prevent the emergence of resistant strains and will answer basic biological questions about fungal adaption to host microenvironments. In order to maximise the impact of this research, I will pursue the following specific objectives: 1) Contribute to the economic competitiveness of the UK through researcher career development. 2) Strengthen pan-UK researcher ties and establish new links with researchers abroad. 3) Promote greater public understanding of science in general and fungal pathogenesis in particular through public-engagement activities. 4) Investigate the potential commercial exploitation of research outcomes. 1) Investment in researcher training has a significant impact on the economic competitiveness of the UK through the development of a pool of highly skilled individuals. Through this Fellowship, I will gain training and expertise in state-of-the-art laboratory techniques and will also expand and strengthen transferable skills including effective communication and presentation, personal effectiveness, and networking. The University of Aberdeen has demonstrated its commitment through a strong Research Staff Development programme (http://www.abdn.ac.uk/develop/), and I will continue to utilise this network to build the transferable skills needed to fully develop my career. 2) The successful completion of this project will require open communication and research exchange with the May Lab at the University of Birmingham and the Kozubowski Lab at Clemson University, USA. My planned research visits and web-based meetings with these two groups will strengthen connections between the AFG and the wider research community within the UK and abroad. 3) I am committed to the dissemination of scientific knowledge to the public. My efforts to expand public awareness of the impact of fungal pathogens on all aspects of our economy and society -from food to energy to health- are ongoing. I maintain a blog (www.erballou.com) that reports scientific findings in fungal research for a lay audience and I will use this platform to disseminate the findings of this work. In addition, I will continue to participate in public outreach events, including those I organise myself and those organised by others. I will undertake further training from the University of Aberdeen Public Engagement with Research Unit (PERU, www.abdn.ac.uk/science), and I will seek out new platforms for public engagement. Research generated through this Fellowship will be presented at national and international scientific meetings and will be published in a timely manner in journals with OpenAccess policies and will be publicised on the University website. Publications will be accompanied by lay summaries explaining the major findings, placing these findings in context in society, and demonstrating the role of the scientific method of hypothesis generation and testing. 4) A major aim of this proposal is to screen mutants for sensitivity to a library of FDA approved drugs, giving this project strong translational potential to identify drugs that are likely to be fungistatic in vivo and have low cross-reactivity with human proteins. In order to pursue commercial opportunities arising from this research, I will work together with Prof. May and the University of Aberdeen Kosterlitz Centre for Therapeutics (abdn.ac.uk/kosterlitz) whose central mission is to link academia, industry and the business sector to drive the translation of basic research.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Microbiology, Pharmaceuticals
Research Priority	X – Research Priority information not available
Research Initiative	Fellowship - Future Leader Fellowship (FLF) [2014-2015]
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/M014525/2 The Cryptococcus neoformans Redoxome: The role of Rac GTPases in ROS Signal Transduction and Titanisation

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