Dissection of A. fumigatus alkaline adaptation and virulence (with a view to inhibiting fungal growth in vivo)

Lead Research Organisation: Imperial College London
Department Name: Unlisted

Abstract

Aspergillus is a common fungus in the environment, dwelling in pot plants and air conditioning ducts. Aspergillus produces huge numbers of airborne spores which disperse on air currents. The spores are small enough to reach the extremities of the human lung when inhaled. This is not usually dangerous as white blood cells are able to kill them efficiently. However, patients having white blood cell deficiency or abnormality, cannot kill spores which germinate and grow inside the body causing a disease called invasive aspergilloisis. From the lung aspergillus can penetrate the blood vessels and spread to other organs.

Invasive aspergillosis affects up to one quarter of bone marrow transplant recipients and leukaemia patients. Around 60,000 people per year become infected worldwide, as many as 9 out of 10 infected patients die. Diagnosis is difficult and comes after significant fungal growth so aggressive treatment is required to stem infections. Only a handful of antifungal drugs are available. They act on the fungal cell wall or cell membrane and are designed to kill the fungus without harming the patient. Unfortunately they have varying effectiveness, particularly against aspergillus. They are toxic to the liver and often cross-react with other medicines. Furthermore, some aspergillus species are developing resistance to these compounds in the same way that bacteria become resistant to antibiotics. There is, therefore, a desperate need for new therapies against aspergillus infection.

My work has identified aspergillus genes which allow growth inside a mouse lung by sensing the pH (concentration of hydrogen ions) and adapting accordingly. Two of the genes, called palH and palI, mediate pH-sensing at the fungal cell membrane. If palH and palI function could be impaired experimentally, fungal growth may be preventable. This project will examine how these genes mediate pH-sensing by identifying the proteins they interact with and provide a means for future screening for chemicals which impair their function by blocking the protein-protein interactions. A third gene controlling the fungal pH response in mice is pacC, which acts after pH?sensing to regulate an appropriate fungal response. Identification of functions under pacC control or which act upstream of pacC will provide further opportunities for interfering with this essential fungal adaptation mechanism.

Technical Summary

Aspergillus fumigatus is the commonest cause of death from fungal disease worldwide, causing life-threatening infection in chronically immunosuppressed patients. No specific A. fumigatus virulence factors are identified but the repertoire of genes having demonstrated roles in vivo is growing. The majority are involved in nutrient biosynthesis or uptake. I have recently described two exceptions: sidA, catalysing siderophore biosynthesis and pacC, mediating alkaline adaptation, which together form a new class of A. fumigatus virulence determinants controlling programmed adaptive responses to the host environment. Both employ fungal-specific proteins and therefore have potential as targets for therapeutic intervention.

Alkaline adaptation is critical for fungal virulence and is mediated, in Aspergillus nidulans, by the transcription factor PacC. PacC, its appropriate processing, and six upstream pH-sensing Pal proteins are indispensable for murine pathogenicity. PacC/Rim101 fungal proteins control cell wall biosynthesis, morphogenesis, sporulation and exported enzyme and metabolite production. Many PacC targets function at, or beyond, the cell boundary.

HYPOTHESES:

Factors under PacC regulation mediate virulence as PacC is indispensable for murine pathogenicity and its constitutive activation enhances virulence.

Inhibition of PacC activation in vivo may prevent infection.

This research explores the novel concept that a non-essential fungal adaptation mechanism (which is essential for virulence) can provide valid opportunities for, and novel means of, preventing fungal growth in vivo.

Using in vivo Aspergillus fumigatus transcriptional profiling, phenotypic screening in Saccharomyces cerevisiae, and a yeast-based A. fumigatus proteomic split-ubiquitin screen, novel virulence-essential A. fumigatus functions acting up- and downstream of the transcription factor PacC, which is a pivotal regulator of Aspergillus virulence will be identified. Moreover, a new proteomic tool for identifying A. fumigatus membrane-bound protein-protein interactions will be established using the virulence-essential pH-sensing A. fumigatus PalH and PalI proteins as prototypes. This will provide a screening platform for future identification of inhibitors of characterised A. fumigatus membrane protein-protein interactions and, ultimately, a means to screen for potential inhibitors of A. fumigatus growth in vivo.

The project has three key goals.

A) Identification of functions under PacC transcriptional control in vivo.

B) Identification of novel A. fumigatus PacC regulators.

C) Establish a split-ubiquitin screening system2 for identification of A. fumigatus membrane protein-protein interactions using the pH-sensing membrane proteins PalH and PalI (having known roles in virulence) as prototypes to identify A. fumigatus PalH and PalI protein partners.

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)

 
Description BBSRC Responsive Mode Grant
Amount £426,226 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2008 
End 09/2011
 
Description BBSRC Responsive Mode Grant
Amount £489,937 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2009 
End 01/2012
 
Description BIRAX Israeli-British Partnership
Amount £15,000 (GBP)
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2010 
End 05/2011
 
Description Epithelial cytotoxins research grant
Amount £30,000 (GBP)
Organisation Chelsea and Westminster Hospital 
Sector Hospitals
Country United Kingdom
Start 07/2017 
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 Wellcome Trust Research Grant
Amount £245,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2008 
End 05/2011
 
Description Wellcome Trust Research Grant
Amount £450,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2011 
End 04/2013
 
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 10/2014 
End 09/2017
 
Title A split ubiquitin protein interaction screening tool 
Description A system of vectors and a library to promote the discovery of, and characteriation of, membrane protein-protein interactions in A. fumigatus 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact A successful funding application to the BBSRC 
 
Title In vivo transcription profile for Aspergillus fumigatus 
Description The first description of global gene expression in the host-adapting fungus 
Type Of Material Biological samples 
Year Produced 2008 
Provided To Others? Yes  
Impact Pubmed ID 18787699 and a successful funding application to the 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 Dr Gustavo Goldman 
Organisation Universidade de São Paulo
Country Brazil 
Sector Academic/University 
PI Contribution We have performed virulence anlayses of mutaully interesting A. fumigatus mutants
Collaborator Contribution This collaboration has provided network anlayses of time-series microarray data and will continue to underpin our methodology in future experimentation.
Impact Pubmed 18538268 and 18298443
Start Year 2007
 
Description Dr Hubertus Haas 
Organisation Medical University of Innsbruck
Country Austria 
Sector Academic/University 
PI Contribution We have performed virulence anlayses of A. fumigatus strains having mutual relevance and authored joint publications
Collaborator Contribution The haas laboratory has constructed mutant strains on our behalf and performed analyses of siderophore biosynthesis, oxidative stress and growth rate determinations. All of which have provided substantive insight on A. fumigatus pathogenesis
Impact Pubmed 17845073
 
Description Dr Matthew Fisher 
Organisation Imperial College London
Department Department of Epidemiology and Biostatistics
Country United Kingdom 
Sector Academic/University 
PI Contribution Advise and training on development of fungal virulence models
Collaborator Contribution Comparative genomic analyses on the evolution of fungal virulence
Impact A successful collaborative funding application to the Wellcome Trust
Start Year 2008
 
Description Dr Nir Osherov 
Organisation Tel Aviv University
Country Israel 
Sector Academic/University 
PI Contribution Advise on microarray data mangement and interrogation
Collaborator Contribution Shared expertise on ex vivo infection assays and A. fumigatus gene deletion strategies
Impact This collaboration has resulted in successful application to the British Council for collaborative funding.
Start Year 2008
 
Description Dr Sven Krappmann 
Organisation University of Wurzburg
Country Germany 
Sector Academic/University 
PI Contribution We have provided training in murine modelling of aspergillosis and tested virulence of mutants [constructed in the partner laboratory] having relevance to both partners' research.
Collaborator Contribution This collaboration has provided heightened expertise in construction of recyclable genetic markers in Aspergillus fumigatus which has increased the sophistication of gene deletion methodology. This collaborator also checks modes of integration in many of our transformants and has collaborated on the construction of bioluminescent Aspergillus fumigatus strains
Impact PubMed ID 18249572 and 19564390
 
Description ESF Fuminomics Consortium 
Organisation Pasteur Institute, Paris
Country France 
Sector Charity/Non Profit 
PI Contribution I am a steering committee member and my team regularly present at the scientific meetings
Collaborator Contribution This consortium provides a platform for us to present our research directly to the European A. fumigatus community. It enables access to new methodologies and funds our attendance at regular consortium research meetings. It also promotes and strengthens 'omics' tools development in the EC.
Impact Training, tools development and a planned bid for Eurpean funding.
Start Year 2008
 
Description JCVI Partnering 
Organisation J Craig Venter Institute
Country United States 
Sector Charity/Non Profit 
PI Contribution We provide infected samples for analysis and co-author all manuscripts resulting from this collaboration
Collaborator Contribution The Nierman laboratory performs microarray experimentation on a regular basis for my group and has begun to develop next generation sequencing protocols using material provided from my laboratory.
Impact Pubmed ID 18787699
Start Year 2006
 
Description Professor Herbert N Arst Jnr 
Organisation Imperial College London
Department MRC Centre for Molecular Bacteriology and Infection
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide advice on analysis of membrane protein-protein interactions using the slpit-ubiquitin analysis platform
Collaborator Contribution The Herb Arst group supports development of molecular tools and provides mechanistic insight into A. fumigatus environemtal adpatation via classical genetic analyses in A. fumigatus
Impact A successful collaborative funding application to the BBSRC Pubmed ID 19754306
 
Description Professor Nick Read, Edinburgh 
Organisation University of Edinburgh
Department Institute of Molecular Plant Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Joint grant application to the wellcome trust Infection and Immunity Panel - Funded 2010
Collaborator Contribution A new collaboration to study cell biology of invasive A. fumigatus growth, measurement of intracellular calcium transients and natural product screening. Use of the MRC-funded split-ubiquitin platform to identify protein-protein interactions required for calcium homeostasis in A. fumigatus
Impact Joint grant application to the wellcome trust Infection and Immunity Panel - Funded 2010
Start Year 2009
 
Description dsRNA viruses 
Organisation Imperial College London
Department Faculty of Natural Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Isolation of virus-infected A. fumigatus isolates Development of a transfection protocol for virus infection Analysis of virulence of infected isolates
Collaborator Contribution We have recently identified, chracterised and sequenced the first chrysoviruses in A. fumigatus
Impact £20 000 funding from the Chelsea and Westmister Charitable trust Publication in 2010 (see list)
Start Year 2007
 
Description Advances Against Aspergillosis 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact 5th Advances Against Aspergillosis Conference
Istanbul, Turkey, 26-28 January 2012
Aspergillus fumigatus survival in the lung environment
Invited speaker


A review article was commissioned. Extensive dicussions and networking reaulted, including crucial new contacts in antifungal drug discovery.
Year(s) Of Engagement Activity 2012
 
Description BSMM host 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Health professionals
Results and Impact The Society is comprised of healthcare workers, routine microbiologists, medics originating from various clinical backgrounds and pharmaceutical representatives. I hosted the 2009 Annual Meeting of the Society at Imperial College in March 2009, an event which attracted >100 attendees.

Two invitations to present work at ISHAM Tokyo May 2009 and the University of Canterbury in September 2009
Year(s) Of Engagement Activity 2009
 
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 Elected to committe for education and outreach 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The British Mycological Society supports science and education on fungi in the UK. The Education and Outreach Committee monitors content of scool curricula, petitions education ministers to include fungi on the school curricula, orgainises events and meetings to promote fungal biology and research, end ecology in the UK.

The education minister Michael Gove was petitioned on the ommission of fungi fom UK school curricula.
Year(s) Of Engagement Activity 2012,2013
URL http://www.britmycolsoc.org.uk/
 
Description Gordon Research Conference on Fungal Cell and Molecular Biology 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact An opportunity to review systems biology and its impact upon the field of fungal infection biology. Many new high profile contacts made.

Gordon Conference Cellular & Molecular Fungal Biology
Holderness School, New Hampshire, USA. June 17-22, 2012
Discussion Leader for Systems Biology
Year(s) Of Engagement Activity 2012
 
Description Hosting a Nuffield Foundation student 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact A sixth form student worked in the laboratory for 2 months on a split-ubiquitin assay developed using MRC funding

Placement requested for further students next year
Year(s) Of Engagement Activity 2011
 
Description Human Fungal Pathogens Lecture Course 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact 150 attendees enjoyed talks from International experts on molecular and cellular biology of human fungal pathogens.

A number of prizes were awarded to outstanding young scientists.
Year(s) Of Engagement Activity 2009,2011,2013
URL http://www.pasteur.fr/infosci/conf/hfp2013/
 
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 Pozaconazole update meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Health professionals
Results and Impact I presented a talk to clinicians and pharmaceutical professionals about the genome of Aspergillus fumigatus and how its dynamic regulation is likely to affect antigen presentation during early disease, thereby impacting design of novel diagnostics

Prompted multiple enquiries from Microbiology lab workers on likely dominant antigens for diagnostics development.
Year(s) Of Engagement Activity 2009
 
Description Royal Society Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Health professionals
Results and Impact A 30 minute lecture delivered at the Royal Society of Medicine entitled 'Diagnosis of invasive aspergillosis - what the fungi can tell us.....' this took place on Thursday 29th May, 2008 and was delivered to a lay audience of varying medical backgrounds, intending to raise the profile of Aspergillus-related disorders and to publicise the importance of this research with respect to understanding disease and identifying important new diagnostic markers.

An invitation to present our work at the AsTeC - Aspergillus Technology Consortium in Bethesda, Maryland, November 2008.
Year(s) Of Engagement Activity 2008