FUNGAL NEIGHBOURHOOD WATCH: UNDERSTANDING HOW COMBINATORIAL SIGNALS FROM THE HOST NICHE DRIVE PATHOGENESIS IN CANDIDA ALBICANS

The fungal kingdom is estimated to contain 1.5 million species with only 70,000 species currently described. Many of these species are pathogenic to plants and animals. Most fungi that infect humans are classed as opportunistic pathogens because they seldom cause disease in healthy individuals, but "target" immune suppressed patients. The increased use of immune suppression therapies and an increasing aging population, together with the development of antifungal resistant isolates and inadequate diagnostic techniques has led to a dramatic rise in fungal infections over the years. Therefore, there is urgent need for the development of new fungal diagnostics and anti-fungal therapies.

Fungi are capable of colonising and subsequently infecting many different human body sites including the skin, nails, lungs, brain, organs and oral, genital and gastrointestinal tracts. Each of these sites has its own environmental characteristics and local defence mechanisms to which the fungus must be able to adapt to, and resist, in order to maintain its position in the niche. Currently, we only have basic knowledge of what these environments are, how they are sensed, and how the fungus uses these environmental signals to drive disease progression. However, what we do know, from in vitro studies dissecting the response of single signals, is that many host-derived signals including serum, pH and temperature drive fungal virulence, while microbe-derived signals dampen these characteristics. In the niche, the fungal pathogen will be simultaneously exposed to both host and microbe-derived signals. Therefore, understanding how the dual response to such opposing traits is orchestrated is an essential goal for the host-pathogen interaction field.

In addition to environments affecting fungal pathogenesis, different environments are likely to impact on how our immune system sees the pathogen. Fungi are surrounded by a multi-layered cell wall, which serves as a protective shield against the environment and host immune defences. The cell wall is a highly dynamic structure and its composition is dependent on the local environment. The outer layer of the cell wall is formed from proteins that are highly decorated with sugars. These sugars are recognised by specific receptors on the surface of white blood cells, called phagocytes, which form part of the host's immune system. After recognition, the fungus is engulfed and killed by the phagocyte. Therefore, changes in the fungal cell wall, due to environmental signals, will affect the ability of the phagocyte to recognise and subsequently kill the pathogen.

The majority of our understanding of environmental sensing, comes from in vitro studies using isolated signals at high concentrations, with no attempt to address how the fungus perceives the multifactorial signals it will encounter in the host niche. I aim to address this fundamental gap in our knowledge of host-pathogen interactions by using one of the major human fungal pathogens, Candida albicans, as a model for fungal infection. My application will use fundamental pathogen cell biology in order to understand the behaviour of C. albicans in diverse host niches. Specifically, I will address how this successful opportunistic fungal pathogen perceives multiple environmental signals encountered during infection, how these combinatorial environments affect the fungus, and how in turn the fungus uses these environments to hide and escape from our immune system.

Understanding the effects of the interactions between C. albicans and the host environment opens up the potential to improve diagnostic tools and discover targets for novel anti-fungal therapies.

Candida albicans is the most common fungus isolated from patients. This fungus infects the oral and vaginal mucosa of healthy individuals causing local inflammation and increased morbidity. In immune compromised hosts, it infects major organs, resulting in life threatening infections. Our understanding of the response the fungus has to the niche environment is based largely on the analysis of single environmental stimuli, not the multifactorial signals that occur in the niche. I will addresses this gap in our knowledge by studying combinations of stimuli that more accurately represent in vivo environments. In phase 1, I will employ large scale screening to deduce how combinatorial environmental stimuli (CO2, pH, quorum sensing molecules etc.) impact on the growth, biofilm formation, and morphology of the fungus. I will identify core environment-regulated genes by RNA-Seq and compare in vitro transcript profiles to those from in vivo samples. I will determine the role of environment-regulated genes in fungal virulence using the concurrent oral-vaginal mucosal and disseminated candidiasis infection models. In phase 2, I will determine how combinatorial signaling affects the fungal cell wall. I will use state of the art proteomics to identify proteins in the cell wall, NMR to determine the structure of the cell wall glycans, and TEM to visualise the cell wall architecture. In phase 3, I will characterise how these cell wall perturbations affect the host's immune response, focusing on epithelial cells, and neutrophil and macrophage phagocytosis. I will quantify epithelial damage by histology, cytokine secretion by ELISA and phagosome maturation by live cell imaging. In phase 4, I will develop fungal reporter strains that respond to changes in specific environmental conditions. These strains will then be use as an indicator of the environmental parameters experienced in the host niche. This proposal has the potential to identify novel diagnostics and therapeutic targets.

Candida albicans causes 400,000 bloodstream infections per year worldwide, in patients with weakened immune function, which are associated with high mortality rates (up to 40%). In addition to this, C. albicans causes mucosal infections of the oral and vaginal tracts in immune competent individuals. It is estimated that around 75 million women suffer from at least one episode of vaginitis per year, while oral candidiasis is common in infants, HIV infected patients and in 60% of denture wearers. Therefore, outputs from this proposal will be of direct relevance to pharmaceutical companies such as NovaBiotics, which have active research programs in antifungal drug development. For example, the characterisation of fungal cell surface proteins, which are directly involved in mucosal colonisation and infection, could be potential therapeutic targets for the treatment of mucosal infections. These potential targets may also be required for systemic disease, further increasing the importance of these research outputs to the industry. Alternatively, these cell surface markers could be exploited as novel diagnostic tools to increase sensitivity and specificity of the current mannan/glucan/fungal DNA diagnostics. Discussions with the pharmaceutical industry about the commercialisation of research findings will be communicated as outlined in the pathway to impact statement. Drug development is a time consuming process (5-15 years), so industrial outputs will be a long-term goal of the proposal.

During treatment of infections, antimicrobial resistance is used to assess the sensitivity of the microbe to the available treatment options. This is normally performed as a standard assay, in standard media that does not represent the local conditions of the infection, and these conditions may affect drug efficacy. Therefore, the identification of medium that closely resembles specific host niches will permit refinement of the minimal inhibitory concentration (MIC) assay. Therefore, outputs from this proposal will be of direct relevance to the medical sector, including the NHS because niche specific drug selection could be made based on the new MICs. If for example, the acidic pH of the vagina increases fungal resistance to some antifungals, e.g. the azoles, then this type of therapeutic could be avoided for the treatment of vaginal candidiasis. This could lead to more proficient treatment of the infection reducing the cost of treatment.

A clearer understanding of how the invading fungal pathogen interacts with the microbiome will benefit the medical sector in several ways. For example, pro-longed exposure to antibiotics is known to increase the risk of candidiasis, due to removal of bacteria from mucosal niches. Presumably this is due to the microflora competing with C. albicans for nutrients, and deploying quorum sensing molecules to restrict fungal growth. Therefore, if we know which bacteria are required to prevent fungal growth we may be able to combine antibiotic treatment with probiotics to reduce the risk of the patient developing a secondary fungal infection. On the other hand, this increased knowledge could also be exploited for selection of treatment.
Characterisation of host niches and a clearer understanding of how and which environmental conditions drive fungal pathogenesis could provide beneficial information to individuals at risk from developing candidiasis. For example, if we show that fluctuations in pH towards a more acidic environment promote virulence in C. albicans, we could inform the general public, especially females prone to recurrent vaginitis, through front-line primary care workers to avoid using soaps and ointments that naturally acidify the niche environment. This type of advice comes at minimal expense and is a short to medium term goal of the proposal.