Final Report Summary - FUNGIBRAIN (Sensing and integration of signals governing cell polarity and tropism in fungi)
FINAL PUBLISHABLE SUMMARY
An emerging concept is that of a “fungal brain”, which integrates exogenous and endogenous signals to determine the morphogenesis and the direction of growth of fungal hyphae, at the level of single cells and the fungal colony. However, very little is known about the mechanistic basis of these processes. The MAIN OBJECTIVES of the FUNGIBRAIN project (http://research.bmh.manchester.ac.uk/FungiBrain/) were 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 fungal polarized growth and tropisms. These objectives were addressed in 7 Work Packages (WPs). Significant results achieved so far by the FUNGIBRAIN fellows are listed below by work package:
WP1. SIGNALS GENERATING TROPISMS. (1) Chemical responsible for negative cell tropisms is likely a volatile but not a secondary metabolite, C02 or NO. Germ tubes have a propensity to invade the agar substratum, which is influenced by gradients of the same volatile signal. (2) Conidial anastomosis tubes (CATs) of N. crassa and B. cinerea responded to each positively but do not fuse, indicating that the chemotropic signals of each species must possess key similarities for mutual chemoattraction. (3) The structure-activity relationship of α mating pheromone from F. oxysporum was determined. (4) Polar cell growth in S. pombe is accompanied by oscillations in cell wall thickness that negatively influences cell growth, and is dependent on mechano-sensing activities of the cell wall integrity pathway which adjusts cell wall synthase activity. (5) Fruit juice extracts are found to act as natural inducers of hyphal formation in the dimorphic fungus S. japonicus.
WP2. SIGNAL RECEPTION AND TRANSDUCTION. (1) Only one Rho-GTPase of the Rac subfamily (PiRac1) is present in P. infestans and is important for pathogenicity. PiRAC complemented a C. albicans rac1Δ strain for invasive growth. (2) Blue light-induced recruitment of active Cdc42 was used to artificially alter polarization of hyphal growth in C. albicans. (3) Mutant analyses and live-cell imaging identified and characterized several proteins involved in hyphal steering in response to microtopographical signals in C. albicans. (4) Extracellular pH governs infectious growth in F. oxysporum by reprogramming phosphorylation levels in MAP kinases. Evidence was obtained for intracellular pH acting as a second messenger regulating MAP kinase activity. (5) Polarized penetration peg formation in Saccharomycopsis during predation of C. albicans and A. gossypii was developed as an experimental system for analysing polarized and tropic growth during yeast pathogenicity. cAMP signalling plays a role in promoting peg formation.
WP3. SIGNAL INTEGRATION (THE ‘FUNGAL BRAIN’). (1) Cdk5/Pcl12 and Crk1 were found to control cell cycle arrest and found necessary for plant infection by Ustilago maydis via synergistically promoting polar growth. (2) Oscillatory membrane recruitment of the MAP kinase MAK-2 was found to be essential for the coordination of cell behaviour during N. crassa CAT chemotropism. The subcellular localisation of MAK-2 during CAT chemotropism strongly influences its level of localized activity probably by assembling and/or isolating regulatory factors and targets. (3) Intracellular oscillations of cytosolic free calcium occur during CAT homing and fusion, but do not correlate with the oscillatory recruitment of MAK-2 or the protein SO to CAT tips or regulate their mutual homing attraction. CAT induction and calcium oscillations were dependent on the extracellular calcium concentration, the CCH-1/MID-1 calcium channel complex, calmodulin and calcineurin. (4) The filamentous form of the dimorphic fungus S. japonicus lack a Spitzenkörper (apical vesicle cluster), typical of growing hyphae of filamentous fungi. Actin-based transport is the dominant form of polarized cargo delivery, although partial co-localisation in both yeast and hyphal forms suggests cooperation in delivery of cargo to tips. RNAseq of the transition between yeast and filamentous growth indicated that S. japonicus filaments are substantially distinct from classical fungal hyphae. (5) Genome sequencing of five Saccharomycopsis species revealed that they belong to the CTG yeast clade. RNAseq showed that a number of highly up-regulated genes are likely involved in degradation of cell walls of yeast prey.
WP4. OUTCOMES AND CONSEQUENCES OF TROPIC GROWTH. Three novel medium-high throughput, live-cell, chemical and mutant screens/assays for analysing the different outcomes and consequences of polarised and tropic growth were developed. These provided automated, unbiased image capture and quantitative analysis from multiple regions of interest at multiple time points of multiple living fungal samples grown in 96-well plates using a Leica SP8 confocal microscope; a widefield Nikon TiI inverted fluorescence microscope; and a Leica DV8 fluorescence stereo microscope. Subsequent, automated image analysis was performed.
WP5. PATHOGENICITY RESULTING FROM TROPISMS. The diverse roles of different tropisms and tropism regulatory proteins operating during infection was analysed in the human pathogens A. fumigatus and C. albicans, the plant pathogens F. oxysporum, U. maydis and P. infestans, and the predacious yeast Saccharomycopsis.
WP6. TARGETTING TROPISMS WITH NOVEL ANTIFUNGALS. (1) The DHODH drug target of the novel antifungal F901318 was fluorescently labelled and localized to mitochondria. Automated, quantitative live-cell imaging of labelled-F901318 treated hyphae revealed the kinetics of the fungistatic and fungicidal effects of this drug. (2) A novel Rho-GTPase (PiRAC) of the Rac subfamily has been discovered in P. infestans and has potential as an oomycete-specific antifungal drug target.
WP7. COMPARISION OF CELL POLARITY REGULATION AND TROPIC GROWTH IN
DIFFERENT FUNGI. Key common features of cell polarity and tropic growth in fungi were identified in the FUNGIBRAIN project by a comparative, interdisciplinary, multi-tropism, multi-organism approach.
EXPECTED FINAL RESULTS AND THEIR POTENTIAL IMPACT AND USE
(1) Training of 11 ESRs and 3 ERs for improved employability. They have been provided with an exceptional skill set and team work experience in analysing, manipulating and inhibiting fungal polarized growth and tropisms of fungi which will be invaluable in their future academic/industrial careers. (2) Socio-economic impact. Fungi have a devastating impact on agriculture and human health. Crops, worth billions of € per year, are destroyed by fungal diseases. At least as many people die each year from fungal infections as die from tuberculosis, malaria or breast cancer. Resistance against the limited number of commercial available antifungals is rising. There is a critical need for the development of new antifungals. Global markets in agricultural fungicides and medical antifungals are increasing steadily and in 5 years will be >€30 billion p.a. The FUNGIBRAIN project has characterized and identified key components of signalling networks involved in fungal cell tropisms that have provided candidates for novel antifungal drugs and fungicides. It has also provided novel automated live-cell screens and assays for the identification and analysis of new antifungal compounds. (3) Scientific impact. Research conducted in FUNGIBRAIN has provided 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 is being mainly communicated via publications and in conference presentations. (4) Collaboration and networking between Partners. This network partnership led to a considerable transfer of knowledge, experience and expertise between partners as well as providing current and future collaborative and joint funding opportunities. (5) Collaboration between academic and private sectors. FUNGIBRAIN has greatly facilitated valuable exchanges of ideas, methodologies and personnel between these diverse groups. (6) Outreach and communication with the general public about the importance of fungal biology, and of fungal diseases and their control has played a significant role during this project.
An emerging concept is that of a “fungal brain”, which integrates exogenous and endogenous signals to determine the morphogenesis and the direction of growth of fungal hyphae, at the level of single cells and the fungal colony. However, very little is known about the mechanistic basis of these processes. The MAIN OBJECTIVES of the FUNGIBRAIN project (http://research.bmh.manchester.ac.uk/FungiBrain/) were 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 fungal polarized growth and tropisms. These objectives were addressed in 7 Work Packages (WPs). Significant results achieved so far by the FUNGIBRAIN fellows are listed below by work package:
WP1. SIGNALS GENERATING TROPISMS. (1) Chemical responsible for negative cell tropisms is likely a volatile but not a secondary metabolite, C02 or NO. Germ tubes have a propensity to invade the agar substratum, which is influenced by gradients of the same volatile signal. (2) Conidial anastomosis tubes (CATs) of N. crassa and B. cinerea responded to each positively but do not fuse, indicating that the chemotropic signals of each species must possess key similarities for mutual chemoattraction. (3) The structure-activity relationship of α mating pheromone from F. oxysporum was determined. (4) Polar cell growth in S. pombe is accompanied by oscillations in cell wall thickness that negatively influences cell growth, and is dependent on mechano-sensing activities of the cell wall integrity pathway which adjusts cell wall synthase activity. (5) Fruit juice extracts are found to act as natural inducers of hyphal formation in the dimorphic fungus S. japonicus.
WP2. SIGNAL RECEPTION AND TRANSDUCTION. (1) Only one Rho-GTPase of the Rac subfamily (PiRac1) is present in P. infestans and is important for pathogenicity. PiRAC complemented a C. albicans rac1Δ strain for invasive growth. (2) Blue light-induced recruitment of active Cdc42 was used to artificially alter polarization of hyphal growth in C. albicans. (3) Mutant analyses and live-cell imaging identified and characterized several proteins involved in hyphal steering in response to microtopographical signals in C. albicans. (4) Extracellular pH governs infectious growth in F. oxysporum by reprogramming phosphorylation levels in MAP kinases. Evidence was obtained for intracellular pH acting as a second messenger regulating MAP kinase activity. (5) Polarized penetration peg formation in Saccharomycopsis during predation of C. albicans and A. gossypii was developed as an experimental system for analysing polarized and tropic growth during yeast pathogenicity. cAMP signalling plays a role in promoting peg formation.
WP3. SIGNAL INTEGRATION (THE ‘FUNGAL BRAIN’). (1) Cdk5/Pcl12 and Crk1 were found to control cell cycle arrest and found necessary for plant infection by Ustilago maydis via synergistically promoting polar growth. (2) Oscillatory membrane recruitment of the MAP kinase MAK-2 was found to be essential for the coordination of cell behaviour during N. crassa CAT chemotropism. The subcellular localisation of MAK-2 during CAT chemotropism strongly influences its level of localized activity probably by assembling and/or isolating regulatory factors and targets. (3) Intracellular oscillations of cytosolic free calcium occur during CAT homing and fusion, but do not correlate with the oscillatory recruitment of MAK-2 or the protein SO to CAT tips or regulate their mutual homing attraction. CAT induction and calcium oscillations were dependent on the extracellular calcium concentration, the CCH-1/MID-1 calcium channel complex, calmodulin and calcineurin. (4) The filamentous form of the dimorphic fungus S. japonicus lack a Spitzenkörper (apical vesicle cluster), typical of growing hyphae of filamentous fungi. Actin-based transport is the dominant form of polarized cargo delivery, although partial co-localisation in both yeast and hyphal forms suggests cooperation in delivery of cargo to tips. RNAseq of the transition between yeast and filamentous growth indicated that S. japonicus filaments are substantially distinct from classical fungal hyphae. (5) Genome sequencing of five Saccharomycopsis species revealed that they belong to the CTG yeast clade. RNAseq showed that a number of highly up-regulated genes are likely involved in degradation of cell walls of yeast prey.
WP4. OUTCOMES AND CONSEQUENCES OF TROPIC GROWTH. Three novel medium-high throughput, live-cell, chemical and mutant screens/assays for analysing the different outcomes and consequences of polarised and tropic growth were developed. These provided automated, unbiased image capture and quantitative analysis from multiple regions of interest at multiple time points of multiple living fungal samples grown in 96-well plates using a Leica SP8 confocal microscope; a widefield Nikon TiI inverted fluorescence microscope; and a Leica DV8 fluorescence stereo microscope. Subsequent, automated image analysis was performed.
WP5. PATHOGENICITY RESULTING FROM TROPISMS. The diverse roles of different tropisms and tropism regulatory proteins operating during infection was analysed in the human pathogens A. fumigatus and C. albicans, the plant pathogens F. oxysporum, U. maydis and P. infestans, and the predacious yeast Saccharomycopsis.
WP6. TARGETTING TROPISMS WITH NOVEL ANTIFUNGALS. (1) The DHODH drug target of the novel antifungal F901318 was fluorescently labelled and localized to mitochondria. Automated, quantitative live-cell imaging of labelled-F901318 treated hyphae revealed the kinetics of the fungistatic and fungicidal effects of this drug. (2) A novel Rho-GTPase (PiRAC) of the Rac subfamily has been discovered in P. infestans and has potential as an oomycete-specific antifungal drug target.
WP7. COMPARISION OF CELL POLARITY REGULATION AND TROPIC GROWTH IN
DIFFERENT FUNGI. Key common features of cell polarity and tropic growth in fungi were identified in the FUNGIBRAIN project by a comparative, interdisciplinary, multi-tropism, multi-organism approach.
EXPECTED FINAL RESULTS AND THEIR POTENTIAL IMPACT AND USE
(1) Training of 11 ESRs and 3 ERs for improved employability. They have been provided with an exceptional skill set and team work experience in analysing, manipulating and inhibiting fungal polarized growth and tropisms of fungi which will be invaluable in their future academic/industrial careers. (2) Socio-economic impact. Fungi have a devastating impact on agriculture and human health. Crops, worth billions of € per year, are destroyed by fungal diseases. At least as many people die each year from fungal infections as die from tuberculosis, malaria or breast cancer. Resistance against the limited number of commercial available antifungals is rising. There is a critical need for the development of new antifungals. Global markets in agricultural fungicides and medical antifungals are increasing steadily and in 5 years will be >€30 billion p.a. The FUNGIBRAIN project has characterized and identified key components of signalling networks involved in fungal cell tropisms that have provided candidates for novel antifungal drugs and fungicides. It has also provided novel automated live-cell screens and assays for the identification and analysis of new antifungal compounds. (3) Scientific impact. Research conducted in FUNGIBRAIN has provided 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 is being mainly communicated via publications and in conference presentations. (4) Collaboration and networking between Partners. This network partnership led to a considerable transfer of knowledge, experience and expertise between partners as well as providing current and future collaborative and joint funding opportunities. (5) Collaboration between academic and private sectors. FUNGIBRAIN has greatly facilitated valuable exchanges of ideas, methodologies and personnel between these diverse groups. (6) Outreach and communication with the general public about the importance of fungal biology, and of fungal diseases and their control has played a significant role during this project.