Epigenetics of stress-induced genome instability in the human fungal pathogen Candida albicans

Candida albicans is an opportunistic fungus (a form of yeast) that normally lives on the human body without causing any harm. However, C. albicans can cause devastating diseases especially in immunocompromised patients who have undergone organ transplants, chemotherapy, or HIV treatment. During infections, C. albicans colonises several parts of our body and therefore needs to thrive in the many different environments found in the body. For example, pathogenic C. albicans survives high temperatures (i.e. fever) and drug resistant C. albicans strains thrive in the presence of anti-fungal drugs. The main goal of this proposal is to answer the key question: How can C. albicans adapt to such different environments?
In all living organisms, the genome contains the information (genes) needed to build the organism and allow it to grow and develop. In most organisms, genome organisation must be maintained to ensure the right balance of genes (and therefore of the instructions for making our body function correctly).
This is different in C. albicans: this fungus, like many other human fungal pathogens, has a flexible genome. This means that its genome structure changes and that C.albicans can live without the right proportion of its genes or even lose a part of a chromosome. Importantly, C. albicans' genome flexibility is enhanced when C. albicans encounters hostile environments (such as for example drug treatments) this allows selection of new genome organisations with a combination of genes that allow survival in these hostile environments.
My laboratory has discovered that genome flexibility originates by breaking the DNA at specific DNA sites: the TRE hotspots. Identical TRE sites are found at each of C. albicans chromosomes and therefore, once broken, TRE sites can interact and fuse with each other. As a result, the C. albicans genome can rearrange itself, causing loss of some sequences and duplication of others. It can also bring two separate sections of the genome together. Work from my laboratory has demonstrated that the DNA packaging (chromatin) of TRE sites can direct or stop breaks at TRE hotspots.
It is unknown what makes TRE sites special. This is impossible to predict because TRE hotspots are not found in other organisms and therefore it is not possible to guess their function. In this proposal, we will use our expertise in chromatin and C. albicans biology to answer 3 key questions:
1. Why are TRE sites of DNA breaks?
We will use genetics and genomics to determine why TRE hotspots are sites of DNA breakage.
2. How does the DNA packaging control TRE breaks?
We hypothesise that the DNA packaging of TRE hotspots changes in different environments. This allows TRE hotspots to be exposed in hostile environments and to be protected in unstressed cells.
3. What are the consequences of TRE breaks?
We will use CRISPR/Cas9 genome editing to generate a library of C. albicans strains carrying all the possible combination of TRE genomic shuffling. We will test which of these genomic organisation are beneficial for C. albicans virulence (ie how efficiently it causes infection).

Understanding how TRE hotspots work will help the development of strategies to block genome flexibility stopping infections and drug resistance. Importantly, TRE DNA sequences are not found in humans and therefore blocking DNA shuffling at these sites will not have any deleterious effect in our body.

Fungal pathogens are a threat to human health because they can thrive in different host environments, and there is a rising occurrence of anti-fungal drug resistance. Candida albicans, the most common human fungal pathogen, colonises almost every organ in the human body where it can cause life-threating infections. C. albicans clinical isolates are often characterised by chromosome rearrangements, and genome instability increases in host-relevant stress conditions. These findings suggest that stress-induced genome instability plays pivotal roles in C. albicans adaptation to the host environment.
Drivers, regulatory mechanisms and consequences of stress-induced genome instability are still unknown.
We have recently identify the first C. albicans instability hotspot: the subtelomeric TRE (TLO Recombination Element) hotspot. This discovery offers for the first time the opportunity to understand how genome plasticity leads to adaptation in C. albicans.
We hypothesise that genome instability is unlocked in hostile host environments via alteration of chromatin states, leading to gene expression rewiring and development of virulent traits.
In this proposal, we will use our long-term experience in chromatin biology, genome plasticity and human fungal pathogen biology to test this hypothesis. Our approach will be to combine genetics, epigenomics and CRISPR-Cas9 editing approaches to discover drivers, mechanisms and physiological outcomes of stress-induced genome plasticity at TRE hotpots.

The aims of this proposal are:
1. To understand why TREs are instability hotspots,
2. To understand how chromatin states affect TRE genome plasticity
3. To identify the causality of genome-instability at TREs.

Who will benefit from this research?
This basic research tackles questions which can find applications beneficial to areas of health and wealth.
Academia will be the most direct sector to benefit by the research since the proposed work relates to scientific and knowledge advancement. The research disciplines which will most benefit are the ones related with microbiology, infectious diseases and epigenetics. In the longer term, this research has the potential to impact in areas of healthcare and pharmaceutical industries. Beneficiaries beyond academia are the biopharmaceutical sector with interest in anti-fungal drug discovery and the wider general public.
How will they benefit from this research?
The Bioscience research community: Chromatin biology in the fungal pathogen Candida albicans is a surprisingly poorly explored area. Our research will generate data and knowledge on this area of research that will benefit the whole C. albicans community and those interested in fungal biology. This project will also deliver increased capacity and capability in this area of biology through the provision of training and further development of key methodologies. Scientists interested in epigenetics and global regulation of gene expression will benefit from our discoveries of mechanisms underlying modulation and propagation of chromatin domain under different environmental conditions.
The biopharmaceutical sector with interest in anti-fungal drug discovery: C. albicans is the most prevalent human fungal pathogen. One of the major obstacles in defeating this pathogen is the high occurrence of anti-fungal drug resistance, creating an urgent need for innovative anti-fungal drugs. Our research will unveil epigenetic mechanisms regulating C. albicans genome stability and pathogenicity and will have the potential to identify new anti-fungal targets. This research in this proposal could potentially be exploited for commercial applications.
The wider general public: C. albicans infection is initial cause of important life-threating diseases with mortality rates up to 50%. These diseases have huge cost implications (~£12 billion/ annum) and our research, in the longer term, will benefit the National Health Service, the patient and ultimately the UK economy by the development of new drugs.
In summary, our research will provide substantial and widespread benefit to our society with respect to scientific output, the economy, quality of life and health.