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FP7

FUNGIBRAIN Report Summary

Project ID: 607963
Funded under: FP7-PEOPLE
Country: United Kingdom

Periodic Report Summary 1 - FUNGIBRAIN (Sensing and integration of signals governing cell polarity and tropism in fungi)

An emerging concept is that of a “fungal brain”, which integrates exogenous and endogenous signals to determine both the morphogenesis and the direction of growth of fungal cells, at the level of the individual cells and the fungal colony. In spite of the universal importance of these processes, surprisingly little is yet known about their mechanistic basis.

The main project objectives of the FungiBrain project are to:
(1) Establish a detailed understanding of conserved fungal signalling networks that regulate polarized and directed fungal cell growth in a wide range of fungal models and pathogens.
(2) Identify novel conserved targets for antifungal drug discovery in these signalling networks
(3) Develop novel high throughput, live-cell fungal tropism screens of mutant and chemical libraries
(4) Develop an outstanding interdisciplinary Training Programme on analysing, manipulating and inhibiting the polarized growth and tropisms of fungi

These objectives are being addressed in seven Work Packages (WP). Work performed since the beginning of the project and the main results achieved so far are as follows.

WP1. SIGNALS GENERATING TROPISMS. (1) The chemical signal responsible for negative cell tropisms that result in germ tubes and mature vegetative growing away from each other in the human pathogen Aspergillus fumigatus has been investigated. This signal is probably a volatile but not a secondary metabolite, C02 or NO. Also germ tubes have been found to have a propensity to invade their agar substratum which may also be influenced by gradients of the same volatile signal (ESR1). (2) Synthetic sex pheromones induce positive hyphal tropisms in the plant pathogen Fusarium oxysporum (ER2).
WP2. SIGNAL RECEPTION AND TRANSDUCTION. (1) The role of sex pheromone-induced hyphal tropisms in F. oxysporum has been analysed. Evidence from deletion mutants has shown that the cognate pheromone-receptor pairs are required for these tropisms. (2) The artificial recruitment of the active rho-GTPase Cdc42 to the plasma membrane using a light activated system in the human pathogen Candida albicans has been found to perturb polarised hyphal growth (ESR4). (3) Methods for the routine imaging of intracellular calcium dynamics with the genetically encoded probe GCaMP6 have been developed in Neurospora, Aspergillus and Candida (ESR1, ESR3 and ER1 – also see WP3 below). The polarized formation of conidial anastomosis tubes (CATs) from germ tubes of N. crassa has been found to be dependent on the frequency of intracellular calcium spiking, the calcium channel proteins CCH-1 and MID-1, and on calcineurin and calmodulin (ER1). (4) cAMP pathway signalling has been shown to play a role in promoting polarized penetration peg formation in Saccharomycopsis during predation of C. albicans and Ashbya gossypii cells (ESR9). (5) The influence of the subcellular localisation of the MAP kinase MAK-2 during chemotropic growth of CATs in N. crassa has been analysed using live-cell imaging and chemical genetics. Its localization has been found to strongly influence its level of activity probably by assembling and/or isolating regulatory factors and targets (ESR8).
WP3. SIGNAL INTEGRATION (THE ‘FUNGAL BRAIN’). (1) The Cdk5-Pcl12 kinases act and Crk1 are necessary for infection by the plant pathogen Ustilago maydis and have been found to act in a synergistic way to promote polar growth and cell cycle arrest (ESR11). (2) Calcium spiking in hyphal tips of N. crassa and A. fumigatus does not seem to be involved in regulating hyphal tip growth and localized changes in cytosolic free calcium have not been correlated with negative tropisms (CAT chemoattraction in N. crassa) or positive tropisms (germ tube avoidance in A. fumigatus or N. crassa) (ESR1, ER1). (3) The role of a trimeric methytransferase signal transduction cascade during the reception of environmental signals that influence polarized cell growth and differentiation in A. nidulans and A. fumigatus have been analysed and evidence has been obtained that their modes-of-action is different in these two species (ESR7). (4) By using live-cell imaging, genetics and pharmacological approaches, evidence has been obtained for cooperation between the actin and microtubule cytoskeletons during the polarized growth of both the yeast cells and hyphae of the dimorphic fungus Schizosaccharomyces japonicus (ESR6).
WP4. OUTCOMES AND CONSEQUENCES OF TROPIC GROWTH. (1) A high resolution method for imaging and measuring the dynamic localized changes in cell wall thickness in S. pombe has been developed (ESR5). (2) The quantitative image analysis of germ tube avoidance in A. fumigatus expressing cytoplasmically targeted GFP (ESR1) is forming the basis of an automated, unbiased high throughput, quantitative assay and screen of a chemical library supplied by Partner 11 to be used in the second half of the project.
WP5. PATHOGENICITY RESULTING FROM TROPISMS. (1) ESR1, ESR2, ESR4, ESR10, ESR11, ER2 and ER3 have been analysing the role of different tropisms and tropism control proteins involved in infection by the human pathogens A. fumigatus and C. albicans and the plant pathogens F. oxysporum and P. infestans (see WP1-4 above). (2) The focal adhesion protein homologues Cst20 (a PAK kinase) and Pxl1 (Paxillin) in C. albicans have been identified and shown to be involved in hyphal steering, substrate penetration and interactions with macrophages (ESR3). (3) Intracellular pH measurements in F. oxysporum using the genetically encoded pH reporter, pHluorin, combined with mutant analyses and measurements of MAP kinase phosphorylation, have revealed that intracellular pH dynamics play important roles in the transmission of environmental cues to the cell signalling machinery (particularly MAP kinase signalling) during pathogenesis (ESR2).
WP6. TARGETTING TROPISMS WITH NOVEL ANTIFUNGALS. (1) Live-cell imaging and quantitative analyses have been used to analyse the influence on hyphal growth and tropic responses of a novel antifungal compound (F901318), discovered by F2G Ltd, that targets the enzyme DHODH involved in pyrimidine synthesis in fungal mitochondria. The drug target has been fluorescently labelled, a fluorescent analogue of the drug has been made for live-cell imaging, and quantitative live-cell imaging of F901318-treated hyphae has revealed the kinetics of the fungistatic and fungicidal effects of the drug (ESR10). (2) Rho-GTPases encoded by the oomycete plant pathogen P. infestans have been analysed a novel Rho-GTPase (PiRAC) of the Rac subfamily has been discovered and has potential as an oomycete-specific antifungal drug target (ER3).
WP7. COMPARISION OF CELL POLARITY REGULATION AND TROPIC GROWTH IN DIFFERENT FUNGI. It is still too early in this project to identify important novel common features as well as important novel differences with the different tropic systems in the different fungi. These aspects will become clearer in the second half of the project.

EXPECTED FINAL RESULTS AND THEIR POTENTIAL IMPACT AND USE
(1) Training of ESRs and ERs. They will be provided with an exceptional skill set and team work experience in analysing, manipulating and inhibiting the polarized growth and tropisms of fungi which will be invaluable for their future academic or industrial careers. This will include enhanced academic-industrial training collaborations.
(2) Socio-economic impact. Fungi have a devastating impact on agriculture and human health. Crop production, worth billions of € per year is destroyed by fungal diseases. At least as many people die each year from fungal infections as die from tuberculosis or malaria. Resistance against antifungals is rising. Global markets in agricultural fungicides and medical antifungals are increasing steadily and in 5 years these will top €30 billion per year. There is a critical need for the development of new antifungals The FungiBrain project should identify key components of signalling networks involved in regulating polarized and directed cell growth. The project will also produce novel live-cell screens assays for antifungal drugs.
(3) Scientific impact. Research conducted in the FungiBrain will provide a major improvement in our understanding of the sensing and integration of signals governing cell polarity and tropisms in a wide range of fungi, including plant and human pathogens. This will mainly be communicated via publications and in conference presentations.
(4) Collaboration and networking between Partners. This will be greatly facilitated during the project and will lead to considerable transfer of knowledge, experience and expertise between Partners as well as provide future collaborative and joint funding opportunities.
(5) Collaboration with the private sector allowing exchange of ideas, methodologies and personnel.
(6) Outreach and communication with the general public about the importance of fungal biology and of fungal diseases and their control.

The FungiBrain logo and our WORPACKAGES diagram can be found in Appendix A1.
The FungiBrain website: http://www.inflammation-repair.manchester.ac.uk/FungiBrain/
Contact details of the Project Coordinator: Prof Nick D. Read (nick.read@manchester.ac.uk)

Contact

Claire Faichnie, (EU Funding and Development Manager)
Tel.: +441612757114
E-mail

Subjects

Life Sciences
Record Number: 182536 / Last updated on: 2016-05-19
Information source: SESAM