The fungal "septasome": dividing and conquering cells of fungal pathogens

Fungi are opportunistic pathogens capable of causing life-threatening infections in immuno-compromised patients and chronic infections in a very large number of immuno-competent people. This makes them a major problem in the intensive care unit (ICU), where fungal infections often undo the good done in the treatment of cancer, transplant recipients and other critically ill patients. The incidence of fungal infections in the ICU rivals that of the bacterial pathogens such as MRSA, often with higher mortality rates. It is difficult to develop drugs that target fungi without also harming the patient because human biochemistry is in many ways similar to that of fungi. Candida species cause the most fungal infections in the ICU and have a 30% mortality rate, cost of over £16 million per year and result in around 700 deaths per year.

As fungal cells grow and divide, an ordered series of events occur, ending in the formation of cross-walls, called septa, which act as stabilising barriers between new and old cells. Septa are made of chitin, a carbohydrate that is an essential component of the cell wall of all fungi, but is not found in humans. Surprisingly, little is known about the mechanism of septum formation in fungi, and this proposal focuses on this process. Understanding this process is important because septum formation and chitin synthesis are both essential and fungal-specific and therefore make excellent targets for future generations of antifungal drugs.

The objective of this project is to understand how chitin is made in the septa of the most common serious fungal pathogen of humans, Candida albicans. This fungus can grow in both unicellular (yeast) and filamentous (hyphal) forms which makes it an ideal model organism for all fungi which grow in one or the other of these two growth forms. C. albicans has four enzymes that make chitin and my previous work unexpectedly revealed that all four enzymes are present at the site of septum formation. The cellular components that assemble at septation sites and associate with the enzymes that make chitin will be identified, as well as how this process is regulated. This will be done by carefully observing the position of fluorescently labelled versions of proteins inside live cells under normal growth conditions, as well as in cells lacking specific proteins or where some cellular processes have been disrupted using chemical inhibitors and antibiotics. The specific proteins that the enzymes that make chitin interact with at the site of septum formation will also be identified.

This work will provide insights into the mechanism and regulation of chitin synthesis in the septa of all fungi, and will serve as useful models for other unicellular and filamentous fungi which have more expanded and complicated families of enzymes that make chitin. Once we understand how these enzymes make chitin in septa and how this process is controlled, we will be able to investigate ways to prevent this essential process. In the future, we might be able to save lives by identifying drugs that block essential interactions between the enzymes that make chitin and other components of the cell division machinery, or that prevent these enzymes from reaching sites of septum formation in the first place.

Chitin synthesis and septum formation are both essential processes for the viability of fungi. The main objective of this proposal is to examine the molecular mechanisms of chitin synthesis during septation in the dimorphic fungal pathogen of humans Candida albicans, and how this process is regulated. This will inform our understanding of septation in all fungi and create an attractive platform for the identification of novel targets for antifungal therapies. This will be achieved by:

(i) Determining how and when the four chitin synthase enzymes are recruited to septation sites in yeast cells. Live cell fluorescence microscopy will be used to observe the position and dynamic recruitment of fluorescently-tagged chitin synthase proteins in relation to known components of the cell division machinery in wild-type cells. Mutant strains and chemical inhibitors will be used to determine the effects of interfering with protein localisation to the mother-bud neck.

(ii) Identifying the proteins that the chitin synthases interact with at septation sites, i.e. defining the fungal "septasome". The membrane yeast two-hybrid system will be used to detect direct protein-protein interactions between the chitin synthases themselves and with proteins co-localised at septation sites, and to identify the specific domains of the proteins required for these interactions. In parallel, immuno-precipitation based screens and affinity chromatography will be used to identify novel binding partners of the chitin synthases by pull-down.

(iii) Assessing how chitin synthase assembly mechanisms differ in yeast and hyphal cells. Chitin synthase recruitment and assembly at hyphal septa will be defined and will serve as a useful model for filamentous fungi. The differences and, more importantly, points of convergence with septum formation in yeast cells will be identified which might be explored as novel targets for antifungal therapies with future funding.

From the one gene-one enzyme studies of George Beadle and Edward Tatum working with Neurospora, to the cell cycle regulation experiments of Paul Nurse and Lee Hartwell in budding yeast, fungi have a distinguished history as paradigm shifting model systems in cell and molecular biology. My research vision is to understand the essential process of septum formation in a fungus that enables front-line molecular genetics to be applied, and in which the product of our understanding can be used to generate new drugs that are urgently needed to tackle the diseases in humans attributed to fungal pathogens. Examining the molecular mechanisms and regulation of chitin synthesis during septation in Candida albicans will inform our understanding of septation in all fungi and create an attractive platform for the exploration of novel targets for antifungal therapies. This work is therefore is firmly placed within the remit of the IIB under the heading of "basic research into mycology" as well as the research priority area of "healthcare acquired infections".

Who will benefit from this research?
The main and immediate benefit of this work will be the contribution to knowledge and understanding of basic aspects of fungal cell biology. The academic impact of this work is described in the Academic Beneficiaries section. In the longer-term, beneficiaries include:

(i) Industry and SMEs. Companies with interests in developing anti-microbial therapeutics are focussed on biochemical drug targets in essential microbial processes that are not represented in mammals and which could deliver specific, non-toxic, broad-spectrum antimicrobial agents. Aberdeen is Scotland's commercial drug discovery capital and has excellent relations with both big pharma and SMEs.

(ii) General public. Life-threatening, systemic fungal infections occur in severely immuno-compromised individuals, the number of which is increasing as an unfortunate consequence of advancements in modern medical practices. These "at-risk" patient groups include those receiving cancer treatments, organ transplant recipients, those who have had abdominal surgery and seriously ill patients being treated in the intensive care unit.

How will they benefit from this research?
(i) Industry and SMEs: Both chitin synthesis and septum formation are fundamental, essential, fungal-specific processes, and therefore make attractive targets for antifungal therapeutics. An objective of this work is to identify the proteins that interact with the chitin synthase enzymes at septation sites. Chemicals libraries will be screened to identify compounds that block essential interactions between the chitin synthases and the cell division machinery which could be developed as novel antifungal drugs. This work will also identify common control points for the recruitment and/or assembly of the chitin synthase enzymes to the site of septum formation. These may also be drugable targets for novel antifungal therapies. The Kosterlitz Centre for Therapeutics at the University of Aberdeen will provide the facilities, expertise and important links to industry including Pfizer, NovaBiotics and the Scottish Biologics Screening Facility, for the development of future projects that will exploit the findings of this work.

(ii) General public: The development of better treatments for opportunistic fungal infections will ultimately save lives.