De-cloaking the cell wall: investigating the molecular mechanism(s) of cell wall remodelling during adaptation to environmental pH in Candida albicans

The development of new antifungals is slower than antibacterials as fungi are eukaryotes and are therefore very similar to humans. However, the sugar structures that make up the cell wall are specific to fungi and are made by fungal specific enzymes. Therefore, the pathways that synthesise the cell wall are excellent targets for the development of new antifungal agents. However, at the moment we have little information on how the production of the cell wall is regulated, and how the cell wall changes upon contact with the human host. A deeper understanding of these processes will enable us to develop better antifungals and diagnostics in the future.

All fungi are surrounded by a thick exterior cell wall, comprised of sugars. The fungal cell wall is essential for maintaining the shape of the fungus, and for fungal survival, by providing a protective barrier from the external environment. The cell wall is also the key fungal component that is recognised as foreign material by our immune system, initiating clearance from infected sites. Therefore, this sugary coat plays many important roles during fungal infection.

The sugars that comprise the fungal cell wall are chitin, glucan and proteins decorated with sugars called mannoproteins. These sugars are assembled in a particular fashion so that the chitin and glucan are closest to the fungal cell, and provide the rigidity of the fungal cell, with the mannoproteins forming the outermost hair-like layer, that enables the fungus to stick to surfaces and other cells.

Candida albicans is the fungal pathogen most often associated with genital thrush. Almost all women will experience at least one episode of thrush in their lifetime; while up to 15% will experience multiple infections. Although not life-threatening these infections are painful and have a significant affect on the well-being of women. In this infection, the fungus has to survive in the acidic environment of the female reproductive tract. We have shown that low environmental pH affects the structural organisation of the fungal cell wall. This change in the fungal cell wall results in over stimulation of the immune system, resulting in the symptoms associated with thrush.

We now want to know how fungi mediate these changes in their cell wall in response to the low pH of the female reproductive tract, and whether these modifications are required for survival of the fungus in this environment. Addressing these questions will enable us to develop better anti-fungal drugs in the future and provide a rationale as to why some women experience more painful or recurrent infections, and this information will inform measures to improve women's health and well-being.

The fungal cell wall plays critical roles in maintaining cell shape, viability and in host-pathogen interactions. However, we have very little knowledge of how cell wall biosynthesis is regulated in fungi, and more importantly almost nothing is know about how the host environment influences cell wall biogenesis. We have recently shown that the cell wall of Candida albicans, the major causative agent of genital thrush, undergoes significant cell wall remodelling in environments of low pH, similar to those encountered in the vaginal mucosa. These cell wall modifications result in a strong pro-inflammatory innate immune response and enhanced neutrophil recruitment, due to the increased exposure of glucan at the cell wall periphery. This non-protective hyper-activation of the immune system is a hallmark of vaginal thrush. We aim to decipher the role of the host environment in regulating the host-pathogen interaction. We hypothesise that pH-dependent glucan unmasking is mediated by active depletion of the outer mannan shield. Here, we will test this hypothesis by focusing our attention on two interlinked mechanisms, the direct sensing of external pH by the fungus, affecting the expression of cell wall remodelling enzymes, in combination with external pH reducing cytoplasmic pH disrupting vesicle trafficking, and therefore preventing synthesis of the outer mannan shield. Using reverse genetic approaches we will identify the signalling pathway(s) that regulate glucan exposure. Using state-of-the-art super resolution microscopy, we will characterise the role of the secretory pathway in delivering cell wall modifying enzymes to sites of carbohydrate exposure and determine the impact of cytoplasmic pH on vesicle trafficking. As the cell wall is dynamic, we will investigate how the cell wall is remodelled over time, the regulatory processes that govern this dynamic remodelling and determine whether these cell wall modifications are essential for survival at low pH.

The continued use of antimicrobials and immunosuppressive therapies is paving the way for fungal infections. Compared to bacteria we have significantly less classes of therapeutic compounds to treat fungal infections. Like antibacterial agents, there are only a few new antifungal agents in the pipeline, and this is greatly hindered by the similarity between fungal and humans, making fungal specific targets rare. The major target for antifungal therapy is the fungal cell wall, due to the enzymes in the cell wall biosynthesis pathway being fungal specific. However, we have very limited knowledge on how the cell wall is synthesised and how this is perturbed during colonisation of the host. In this proposal we will address this gap in our fundamental knowledge by elucidating how the host environment affects the structure and regulation of the fungal cell wall.

Human Health: Although basic in its approach this research programme investigates the molecular mechanisms behind the unmasking of beta-glucan, a strong pro-inflammatory carbohydrate, during adaptation to the low pH environment found at the vaginal mucosa. On average 75% of the female population will develop at least one case of symptomatic Candida colonisation in their lifetime, with 10-15% experiencing recurrent infection (4 or more episodes in a 12 month period). Deciphering this molecular mechanism will identify essential proteins to which small molecule inhibitors will be designed to, and used in conjunction with standard treatments for genital thrush (fluconazole) to reduce glucan exposure and symptoms associated with thrush. These small molecule inhibitors will also be beneficial to women who suffer with recurrent infections. Although the underlying reasons why some women suffer from repeated infections are still unknown, reducing the exposure of this pro-inflammatory carbohydrate could reduce the occurrence of symptomatic colonisation, increasing well-being. Women that suffer with recurrent symptomatic Candida colonisation are often on long-term azole treatment to prevent recurrence. Reducing the possibility of symptomatic colonisation, by preventing the immune system from eliciting the strong pro-inflammatory innate immune response associated with thrush, would in turn reduce the azole usage in these women reducing the possibility for the development of azole resistant isolates.

Industry: The identification of small molecule inhibitors of fungal specific targets will be of interest to pharmaceutical companies. Understanding how the cell wall is modified during infection will also enable the development of better diagnostics that could be used to discriminate between asymptomatic and symptomatic colonisation.

Students/outreach: This project provides essential training for students in microbiology, molecular biology and cell imaging. Mycology is often under represented in undergraduate degrees and often research projects in my group are the only exposure students get to mycology. Therefore, it is imperative to maintain fungal-based research and to develop student's enthusiasm for this subject. Techniques from this project will also be used in the annual Institute for Microbiology and Infection Summer School I organise, which provides 26 year 12 students with the opportunity to develop hands on laboratory in microbiology, and in our UK fungus day outreach activities further increasing the awareness of fungal research within the UK.