Structure-function analysis of a pH-responsive molecular switch required for fungal pathogenicity

Lead Research Organisation: University of Manchester
Department Name: School of Biological Sciences

Abstract

Most fungi are extremely useful, for example, the production of antibiotics, bread, wine, and beer all involve fungi. However, like bacteria and viruses, fungi can cause life-threatening diseases. Unlike bacteria and viruses, fungal cells share many similarities with those of humans. For this reason it has been very difficult to identify drugs which will kill fungi, without causing harm to the human patient.

Infectious diseases caused by fungi are a worldwide problem and recent estimates suggest that fungal infections cause at least as many deaths as malaria and tuberculosis. There are currently only three classes of drugs with which to treat fungal infections, and there are limitations associated with all of them. This research project will characterize a group of fungal proteins, already proven to be required for survival in the mammalian host, with a view to finding drugs which inhibit them. We will focus upon a specific class of signaling protein which is outstandingly amenable to drug discovery, known as G protein coupled receptors (GPCRs). Fungal cells use GPCR-like proteins to gather information about their surroundings, it is therefore likely that chemistries to inhibit these proteins already exist but have been overlooked in conventional drug screening approaches.

Roughly half of currently licensed drugs target GPCRs. GPCR-like proteins are located at the surface of the cell, and have a characteristic structure composed of seven membrane-spanning segments. GPCRs are sensory molecules which detect signals outside of the cell, and convey this information via intracellular signaling molecules, to elicit a cellular response. The success of GPCRs as drug targets is due to their location at the surface of the cell, the existence of important pockets or folds in the proteins which can be blocked by drugs, and the availability of multiple technologies and tools to monitor GPCR-mediated signaling.

Most fungi use a GPCR-like protein (called PalH or Rim21) to sense the pH of their surroundings. In pathogenic fungi the loss of PalH, or the transcription factor (PacC) it communicates with, prevents fungal survival in the mammalian host. Recently, in the major mould pathogen of humans, Aspergillus fumigatus, we found that loss of pH signalling impacts multiple processes associated with fungal infection including cell wall biosynthesis and penetration of the lung lining. Additionally we found that mutants defective in pH signaling became highly susceptible to killing by echinocandin antifungal drugs, which are usually unable to kill Aspergillus species. When we tested this observation in a mammalian infection model, we found that pH non-signaling mutants were cleared much more efficiently in echinocandin-treated mice. Thus alone, and in combination with existing antifungal drugs, inhibition of fungal pH signaling could be a valuable treatment option.

During this research we will find out how PalH sensors detect extracellular pH by systematically mutating the sensor. Mutations which prevent pH signaling will inform us upon which segments of the protein are required for signaling. Additionally we will try to make enough of the PalH protein in the laboratory to allow formation of crystals and analysis of the protein structure. In practice the resolution of membrane protein structure has been very challenging but recent methodological advances have improved success rates and access to a 3D model of protein structure will unlock many possibilities for computational analysis of the protein and possible inhibitors. Finally, informed by the structural and mutational analyses, we will develop assays using fluorescent proteins, which will be useful for high throughput screening of PalH inhibitors. The tools and insight generated by this research will open new avenues for antifungal drug discovery. In fungal genomes many hundreds of GPCR-like proteins are encoded but to date the number characterized is less than 1 per cent.

Technical Summary

Invasive fungal infections (IFIs) pose a severe and fatal threat to patients undergoing organ or stem cell transplantation, anti-cancer therapy, or suffering from AIDs. On a global scale IFIs cause equivalent mortality to TB and malaria, but our ability to cure IFIs remains critically restricted by the limited repertoire and suboptimal efficacy of licensed antifungal drugs.

Fungi use a GPCR-like protein (called PalH or Rim21), absent in humans, to sense external pH. In pathogenic fungi the loss of PalH, or the transcription factor (PacC) it communicates with, prevents fungal survival in the mammalian host. Recently, a major mould pathogen of humans, Aspergillus fumigatus, we found that loss of pH signalling impacts multiple processes associated with fungal infection including penetration of the lung lining.

The simplest model for PalH-mediated pH sensing would be one in which the PalH pH sensor exists in multiple conformational states, each having a different affinity for, and/or ability to activate, the PalF arrrestin partner. Plausible pH-dependent triggers of altered conformational equilibrium might include binding of a ligand, altered protonation state of amino acid side chains, modulation of interactions between PalH amino terminus and extracellular loops (ECLs), PalH oligomerisation, altered lipid-protein interactions, or alteration of physical characteristics of the cell such as cell volume, or cell wall composition. To distinguish between these multiple possibilities, we will probe the relative contributions of particular amino acid side chains, and carboxy- and amino-terminal domains to functionality of the PalH receptor. We will then redirect mutant constructs into recombinant protein expression vectors, yeast membrane two hybrid (YTMH) vectors, and A. fumigatus FRET and bimolecular fluorescence complementation strains to interrogate the effects of mutations on protein structure, arrestin engagement and receptor oligomerisation.

Planned Impact

The global market for antifungal therapeutics was estimated to be in the region of $9.4 billion US in 2010 (GBI research) and is anticipated to exceed $12 billion by 2016 with expenditure in the EU accounting for around a third of this (BCC research). The highest grossing agents in 2010 were the systemic drugs, voriconazole ($825 million), caspofungin ($611 million) and itraconazole ($341 million) (Evaluate Pharma). The significance of Aspergillus and candida sp. to health has lead one pharmaceutical company (Merck) to express a public interest in acquiring an Aspergillus/Candida (or mould specific) anti-infective agent.

The health burden of diseases caused by fungi is considerable. Where severe invasive infections are concerned, deep-organ fungal infection occurs as a negative corollary to otherwise successful, but nonetheless expensive, treatments for serious illness. According to a working group of the Health Protection Agency Advisory Committee for Fungal Infection and Superficial Parasites, in 2002, almost 10,000 patients in England were estimated to have suffered a deep-organ fungal infection; almost half of them died from their fungal disease. New classes of antifungal drugs are urgently required and the antifungal drug development pipeline is currently sparse.

The annual healthcare costs go way beyond just the cost of therapeutics. The cost of invasive infections caused by Aspergillus sp. in the US alone was estimated at $633 million in 1996 and includes the cost of failed chemotherapy, bone marrow or organ transplantation procedures (Dasbach EJ, Davies GM, Teutsch SM. Burden of aspergillosis-related hospitalizations in the United States. Clin Infect Dis 2000;31:1524-8.).

The pipeline for new antifungal compounds is sparse. Very few antifungal compounds are currently in development. Three new triazoles (albaconazole, isavuconazole and ravuconazole) are in clinical trials however, as with the current triazoles they all target the lanosterol 14-demethylase and are likely to be dogged by similar resistance issues. The glucan synthase inhibitor in development by Merck has been stalled in Phase I since 2009, as have the novel compounds corifungin (Acea biotech), T-2307 (Toyama) and the HDAc inhibitor of Methylgene. A number of other compounds show pre-clinical promise, particularly E1210 an inhibitor of GPI-anchor biosynthesis (EISAI) however this too has stalled. All of this indicates potential problems in development.

Molecular switches: Currently synthetic molecular switches are of interest in the field of nanotechnology for application in molecular computers. The pH-responsive molecular switch under study in this proposal could be of future value for the development of novel medical devices, drug delivery strategies, and medical monitoring devices. We will work to train academic and commercial partners in the application and use of these tools, protecting where necessary any intellectual property.

Live cell imaging assays for fungal signal transduction: Researchers and commercial enterprises seeking live cell assays for inhibition of intracellular signalling pathways will benefit from this research. The FRET and Bimolecular Fluorescence reporter strains are a novel development for Apsergillus species as the platform we have developed allows a single-step transformation to introduce interacting partners of interest. These tools have commercial worth as they can facilitate high throughput screening for chemicals which inhibit fungal signalling pathways. We will work to train academic and commercial partners in the application and use of these tools, protecting where necessary any intellectual property.

Publications

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Bertuzzi M (2018) Anti-Aspergillus Activities of the Respiratory Epithelium in Health and Disease. in Journal of fungi (Basel, Switzerland)

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Bignell E (2016) Secondary metabolite arsenal of an opportunistic pathogenic fungus. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

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Farhadi Cheshmeh Morvari S (2023) Conserved and Divergent Features of pH Sensing in Major Fungal Pathogens in Current Clinical Microbiology Reports

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Gsaller F (2018) Mechanistic Basis of pH-Dependent 5-Flucytosine Resistance in Aspergillus fumigatus. in Antimicrobial agents and chemotherapy

 
Description Irish Fungal Society invited lecture: Modus operandi of an accidental fungal pathogen of humans
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
Impact 100 attendees
 
Description Chemigenetic analysis and efficacy of novel antifungal drugs that target fungal pH signalling
Amount £532,867 (GBP)
Funding ID BB/V017004/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2021 
End 06/2024
 
Description Epithelial cytotoxins research grant
Amount £30,000 (GBP)
Organisation Chelsea and Westminster Hospital 
Sector Hospitals
Country United Kingdom
Start 06/2017 
End 12/2018
 
Description MRC Confidence in Concept Award
Amount £40,220 (GBP)
Organisation Medical Research Council (MRC) 
Department MRC Confidence in Concept Scheme
Sector Charity/Non Profit
Country United Kingdom
Start 02/2015 
End 12/2015
 
Description MRC Discovery Award (Internal funding stream)
Amount £90,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 11/2016 
End 03/2018
 
Description MRC Project grant
Amount £720,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 03/2016 
End 02/2019
 
Description Real time responses to antifungal drugs: A microfluidics-assisted confocal approach
Amount £140,000 (GBP)
Organisation Gilead Sciences, Inc. 
Sector Private
Country United States
Start 06/2021 
End 09/2022
 
Description Wellcome Trust strategic award for medical Mycology & Immunology
Amount £112,000 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Strategic Award
Sector Charity/Non Profit
Country United Kingdom
Start 09/2014 
End 09/2017
 
Title Bespoke peptide aptamer library 
Description This tool will allow academics to screen for small molecule inhibitors. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Novel aptamers blocking pH signal 
 
Title Genetic screening methodology for antifungal drug discovery 
Description A genetic screen was developed to identify compounds directly inhibiting fungal pH signalling. A methodology for high throughput screening was developed. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? No  
Impact A new funding application was submitted to BBSRC 
 
Description CryoEM collaboration 
Organisation University of Manchester
Department Division of Infection, Immunity & Respiratory Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We have identified a critical pH sensor having a 7TMD which is indicated as an important new drug target. we are working to provide recombinant protein for structural studies.
Collaborator Contribution we are benefiting from our partners' expertise in generating sufficient protein for preliminary protein studies.
Impact In progress
Start Year 2017
 
Description Identification and definition of SARs driving activity of compounds which inhibit pH signalling in the human fungal pathogen A. fumigatus. 
Organisation MedChemica Ltd
Country United Kingdom 
Sector Private 
PI Contribution We developed a genetic screen for inhibitors of pH signalling
Collaborator Contribution In silico predictions of active motifs. Identification and sourcing of chemical analogs for pinpointing structure-activity relationships.
Impact A new funding application has been written and submitted to BBSRC
Start Year 2019
 
Description MCCIR 
Organisation University of Manchester
Department Manchester Collaborative Centre for Inflammation Research (MCCIR)
Country United Kingdom 
Sector Academic/University 
PI Contribution Training in the manipulation of fungal pathogens
Collaborator Contribution Training in FACS analyses of the host-pathogen interaction
Impact Trained personnel in my research group, access to state-of-the-art FACS analyses
Start Year 2014
 
Description MIB 
Organisation University of Manchester
Department Manchester Institute of Biotechnology MIB
Country United Kingdom 
Sector Academic/University 
PI Contribution We have developed assays amenable to fragment-based screening for novel anifungal drugs
Collaborator Contribution Gene synthesis
Impact Successful synthesis of expression clones for a fungal pH sensor
Start Year 2014
 
Description MRC Laboratory Molecular Biology 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Constructs for expression of recombinant integral membrane protein in HEK cells (for reconstitution of multimolecular machine).
Collaborator Contribution Specialist expertise in heterologous protein expression (integral membrane proteins); vectors, cell lines.
Impact Multidisciplinary: Medical Mycology-Protein structure
Start Year 2022
 
Description MRC Laboratory Molecular Biology 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Constructs for expression of recombinant integral membrane protein in HEK cells (for reconstitution of multimolecular machine).
Collaborator Contribution Specialist expertise in heterologous protein expression (integral membrane proteins); vectors, cell lines.
Impact Multidisciplinary: Medical Mycology-Protein structure
Start Year 2022
 
Description MRC Laboratory Molecular Biology 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Constructs for expression of recombinant integral membrane protein in HEK cells (for reconstitution of multimolecular machine).
Collaborator Contribution Specialist expertise in heterologous protein expression (integral membrane proteins); vectors, cell lines.
Impact Multidisciplinary: Medical Mycology-Protein structure
Start Year 2022
 
Description Pfizer Global R&D 
Organisation Pfizer Global R & D
Country United States 
Sector Private 
PI Contribution Training in peptide aptamer biology
Collaborator Contribution Training (via an 8 week placement) in antibody technologies, protein expression, hybridoma manipulations and antibody isolation.
Impact The production of an anti-PalH antibody
Start Year 2014
 
Description Chair of Fungal Biology Research Committee of the British Mycological Society 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Fungal Biology Research Committee Mission Statement
Raise the profile of Fungal Biology Research in the UK and Internationally.
Promote networking across the fungal biology community.
Maintain strong links and communications with other mycological Societies and the International Mycological Association (IMA)
Strengthen links with UK and international funding bodies to ensure fungal research has a voice where funding and strategy decisions are being made.
Organise and support conferences, meetings and workshops for the international mycological community.
Engage and inspire future generations of fungal biologists.
Year(s) Of Engagement Activity 2016,2017,2018
URL https://www.britmycolsoc.org.uk/science/
 
Description Fungal Education and Outreach Committee 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The committee organises UK Fungus Day - a Nationwide initiative to increase awareness and understanding of fungi

More than 50 events took place on this day, schools, forays, lectures, exhibitions
Year(s) Of Engagement Activity 2014
URL http://www.ukfungusday.co.uk/
 
Description Invited lecture: British Mycological Society Annual Scientific Conference 2023 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Modus operandi of an accidental fungal pathogen of humans - delivered to 100+ delegates
Year(s) Of Engagement Activity 2023
 
Description January 2022 MRC Seminar Series: Tackling fungi that cause human lung disease (Elaine Bignell) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Tackling fungi that cause human lung disease - A talk delivered to the Directorate, Board & Programme Managers and Institute staff of the Medical Research Council
Year(s) Of Engagement Activity 2022
URL https://www.youtube.com/watch?v=_WKtneF1wrk&list=PLSus4fp7v7sQuXdUwREyHbUMR8cqNZKYj&index=15
 
Description Killer Fungus Event at Manchester Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact A 40-strong team of clinicians and scientists from the Universities of Aberdeen and Manchester, University Hospital South Manchester, National Aspergillosis Centre and Mycology Reference Laboratory Manchester, delivered the 'Killer Fungus' event at the Manchester Museum of Science and Industry in October. Visited by more than 1300 members of the public in a single day, the exhibit utilised the entirety of the enormous MOSI Power Hall to deliver two parallel events depicting the breadth, wonder and deadly nature of fungal contributions to life, and death, on Earth. New to the Killer Fungus exhibit for 2018, an app-based role playing game 'Outbreak' pitched 38 teams of impromptu scientists against a deadly killer fungus to resolve a mystery illness sweeping Manchester.
Arriving to breaking news from the BBC of a mystery illness causing fatal meningitis amongst the Greater Manchester population, teams were immersed into a battle, alongside real-life clinicians and scientists, to collect field samples, witness the effects of the disease on human patients, and examine clinical data and pathogen genetic code and to work against the clock to crack the source of the Outbreak.
At a complementary Platform for Investigation exhibit, visitors learned about fungi and the diseases they cause by playing computer games, viewed microscopic examples of fungal pathogens colonising agar plates and lung tissue, and made Play Dough models of innate immune cells attacking fungi.
Year(s) Of Engagement Activity 2017
URL http://www.killerfungus.org/
 
Description MRC Seminar Series: Tackling fungi that cause lung disease 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Third sector organisations
Results and Impact Elaine Bignell delivered a talk for the MRC Seminar Series attended by all MRC Centre members and MRC personnel. The talk focused upon the fundamental basis of environmental sensing in fungi and how such research can become translated for drug discovery. The talk resulted in a new collaboration with Chris Tate (of the MRC Laboratory of Molecular Biology) and joint authorship of an opinion-piece publication on novel routes to antifungal drug discovery.
Year(s) Of Engagement Activity 2022
URL https://www.youtube.com/watch?v=_WKtneF1wrk&list=PLSus4fp7v7sQuXdUwREyHbUMR8cqNZKYj&index=15
 
Description MRC Seminar Series: Tackling fungi that cause lung disease 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Third sector organisations
Results and Impact Elaine Bignell delivered a talk for the MRC Seminar Series attended by all MRC Centre members and MRC personnel. The talk focused upon the fundamental basis of environmental sensing in fungi and how such research can become translated for drug discovery. The talk resulted in a new collaboration with Chris Tate (of the MRC Laboratory of Molecular Biology) and joint authorship of an opinion-piece publication on novel routes to antifungal drug discovery.
Year(s) Of Engagement Activity 2022
URL https://www.youtube.com/watch?v=_WKtneF1wrk&list=PLSus4fp7v7sQuXdUwREyHbUMR8cqNZKYj&index=15
 
Description Mycotalks: S1 E12 Antivirulence strategies for tackling Aspergilloses 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact A talk delivered to Medical Mycologists of all career stages that describes molecular genetic approaches to novel antifungal drug discovery
Year(s) Of Engagement Activity 2022
URL https://www.youtube.com/watch?v=iGB64q16cgc
 
Description Studies of Aspergillosis at the MRC Centre for Medical Mycology: World Aspergillosis Day 2022 (Elaine Bignell) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact An overview (for patients suffering from Aspergilloses) of ongoing research aimed at tackling the problem
Year(s) Of Engagement Activity 2022
URL https://www.aspergillosisday.org/WAD2022/national-aspergillosis-centre-seminar-series/