Role of Aspergillus fumigatus gene clusters in mammalian niche-adaptation

Lead Research Organisation: Imperial College London
Department Name: Dept of Medicine

Abstract

A small number of fungi are able to cause infection in humans. Among them, the soil-dwelling fungus Aspergillus fumigatus is a serious threat to human health. Aspergillus fumigatus spores are extremely small and remain airborne for long periods of time. If inhaled by humans with defective or deficient immunity they can germinate and grow within the lung, causing invasive infection which is often fatal. It is currently unknown why Aspergillus fumigatus is able to cause human disease whereas closely related fungal species are not. The Aspergillus fumigatus genome has been sequenced and comparisons to genomes of non-pathogenic relatives identify the greatest differences, at the level of DNA sequence, at the ends of chromosomes, which are known as telomeres. We previously used a technique called microarray analysis to record gene activity, simultaneously from every Aspergillus fumigatus gene, in an attempt to identify genes involved in establishing mammalian infection. There were two surprising features of the resulting dataset. Firstly, the vast majority of active genes were located at the telomeres of genes. Secondly we identified many clusters of neighbouring genes which seem to be co-regulated. Recently, gene clusters have been identified in other disease-causing fungi, such as those infecting plants, and have been implicated in infection. We therefore wish to investigate the function of the Aspergillus gene clusters to determine whether, and how, they are involved in establshing disease. Aspergillus fumigatus may have developed the ability to colonise the human lung as a result of evolving to withstand stresses in the soil environment. We therefore plan to investigate the process of adaptation to the mammalian lung environment to identify factors important for establishing infection. The process of adapting to the stresses of varying habitats is known as niche-adaptation.

Technical Summary

In our recent gene expression screens for niche-adapting gene functions in the major mould pathogen of humans, Aspergillus fumigatus, we discovered multiple clusters of physically-linked co-ordinately regulated genes preferentially expressed during germinative growth in the murine lung relative to laboratory culture. The likely physiological significance of this discovery is heightened by the presence of known virulence loci among those we identified, including that encoding biosynthesis of the immunotoxin gliotoxin, and a siderophore biosynthetic gene cluster containing genes essential for virulence. Similar experimentation in the biotrophic fungal phytopathogen, Ustilago maydis, recently identified a similar phenomenon, linking gene function to virulence for a number of loci. We hypothesise that co-regulation of genes located within the A. fumigatus 'infection clusters' indicates concerted function during initiation of mammalian infection and we now wish to determine the role of such 'infection' clusters (ICs) in A. fumigatus niche-adaptation, host damage and virulence. In order to do this we plan to expand our microarray anlayses, assessing time series gene expression profiles during initiation of infection in two murine models. We will also optimise a recombineering protocol suitable for high throughput aspergillus fumigatus gene deletion cassette construction . This will enable us to delete genes within, and surrounding, important ICs. Using this approach we will make full deletions of up to 96 ICs. IC mutants will be characterised for defects in growth, development, proteinase secretion, cytotoxicty, virulence and adhesion. We will further interrogate the transcriptional regulation of five important ICs using RT-PCR analysis. Epigenetic regulation will also be assessed using A. fumigatus mutants defective in histone deacetylase activity and methyltransferase activity.

Publications

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Yasmin S (2012) Mevalonate governs interdependency of ergosterol and siderophore biosyntheses in the fungal pathogen Aspergillus fumigatus. in Proceedings of the National Academy of Sciences of the United States of America

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Cairns T (2010) The host-infecting fungal transcriptome. in FEMS microbiology letters

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

 
Description Genome-wide temporal profiling of Aspergillus fumigatus gene expression was performed during early infection in two murine models of infection, and in growth-matched laboratory cultured germlings. The analysis identified genes both up- and down-regulated in response to the host niche, relative to laboratory culture. The data have been complied into a compendium of A. fumigatus gene expression and, upon publication, will represent the only extant source of data derived from host-infecting A. fumigatus.



The compendium will be accessible via the Bignell laboratory home-page and will permit the research community to interrogate, simultaneously, data derived from more than 200 microarrays to study the expression profile of their favourite gene(s). The compendium is expected to go live from the end of May 2012, and will be accompanied by a publication (to be submitted to BMC Genomics) which documents the relevant experimentation and key findings.



A methodology for deletion of A. fumigatus gene clusters, utilising bacterial artificial chromosome recombineering was developed. The entire pseurotin biosynthetic gene cluster was replaced with a single dominant selectable marker conferring resistance to hygromycin. Mutants deficient in pseurotin biosynthesis were found to have reduced virulence in murine models of pulmonary aspergillosis, thereby identifying a second A. fumigatus secondary metabolite which impacts host defence against Aspergillus infection.
Exploitation Route The study highlights potential biomarkers of Aspergillus infection and might inform the design of new diagnostic strategies This research highlights gene functions which are employed by the fungus during infection and will inform the development of novel antifungal therapies
Sectors Healthcare

 
Description A study to identify novel regulators of calcineurin in aspergillus fumigatus
Amount £220,000 (GBP)
Funding ID WT093596MA 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2011 
End 05/2014
 
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 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 Recombineering 
Description A means to manipulate large gene clusters in A. fumigatus 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact A new paper so further activities will be reported next year 
 
Title A Compendium of Aspergillus fumigatus transcriptome data 
Description A compendium of quantile-normalised transcript abundance data curated from 200 sample sources, and representing a wide variety of in vivo and in vitro conditions. A pre-release version of the site is currently undergoing testing by members of research community. Imperial College website (Bignell Homepage) 
Type Of Material Database/Collection of data 
Year Produced 2012 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Description Hubertus Haas Innsbruck Medical University 
Organisation Medical University of Innsbruck
Country Austria 
Sector Academic/University 
PI Contribution An on-going collaboration with Innsbruck Medical University examined the role of a siderophore biosynthetic gene cluster in Aspergillus fumigatus virulence. The study resulted in a joint first authorship PNAS manuscript (2012) for the postdoc employed on the BBSRC grant.
Start Year 2007
 
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 Professor Nick Read - calcium signalling 
Organisation University of Edinburgh
Department Institute of Cell Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Our transcriptome analyses identified several major stresses exacted by the mammalian host upon infecting fungi. One of these was extracellular calcium stress, requiring upregulation of transporters which remove cytosolic calcium.
Start Year 2009
 
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 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 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 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
 
Description Woman's Hour Radio 4 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A broadcast on BBC Radio 4 discussed the problem of black yeasts in domestic appliances. This also provided a platform to discuss the problem of fungal pathogens in general.

Many enquiries from the general public highlighted the importance of this issue to the audience.
Year(s) Of Engagement Activity 2011