Breakthrough in high throughput antifungal drug discovery
A recent study from the University of Manchester has shown that the human pathogenic fungus, Aspergillus fumigatus, can be used to discover antifungal drugs using a high throughput, genetically based profiling technology. This work was funded by the European Commission and Wellcome Trust.
Exposure to airborne spores Aspergillus fumigatus in the environment is constant but the fungus is normally cleared from one’s air passages. However, these spores can cause invasive, chronic and allergic infections of the lungs. One type of infection results in invasive aspergillosis and occurs in patients that are immunocompromised (e.g. those with HIV) and immunosuppressed (e.g. those undergoing immmusuppressive drug treatments), and has a very high mortality rate (>50%).
Aspergillosis diseases are clinically complex to treat and are increasingly associated with antifungal resistance. There is thus a major requirement for new antifungal drugs with novel modes of action to treat human fungal diseases. The interdisciplinary team at the University of Manchester have devised a new method of high throughput antifungal drug discovery against moulds.
Competitive fitness profiling technologies have been used very successfully used with bacterial and yeast systems to identify novel genetic drug targets. However, development of similar systems with the majority of mould species is complicated by the fact that pooled cells of different genetic backgrounds mostly undergo cell fusion resulting in the mixing of genetic material within the cell population. This prevents the detection of different levels of fitness (measured as growth) between distinct mutants in a pool and thus impedes the identification of potentially promising antifungal targets. Research within the Manchester Fungal Infection Group have made the ground-breaking discovery that the extremely important human pathogen, A. fumigatus, does not undergo cell fusion under standard culture conditions, in contrast to the majority of other moulds. This finding paves the way for the exploitation of competitive fitness profiling for drug discovery against A. fumigatus and related moulds. An initial pilot study has shown that this drug discovery technology works extremely well.
Another important discovery made by the Manchester Group was that nutritional and antifungal stress (e.g. starvation or treatment with antifungals) can induce significant cell fusion in A. fumigatus. This raises the intriguing possibility that exposure to antifungals in the natural environment in the wild, and possibly disease settings, may select for mutants with increased fitness and thus resistance against antifungal compounds.
Co-first author, Dr Darren Thomson, is enthralled by this potentially novel resistance mechanism. “It will be exciting to determine whether this fusion trait occurs in nature and in disease situations. This will require multidisciplinary approaches and technologies to visualise and prevent this potential resistance mechanism in vivo.”
MacDonald DM, Thomson DD, Johns A, Contreras AV, Gilsenan JM, Lord KM, Bowyer P, Denning DD, Read ND, Bromley MJ (2019). Inducible cell fusion permits use of competitive fitness profiling in the human pathogenic fungus Aspergillus fumigatus. Antimicrob Agents Chemother 63:e01615-18 https://doi.org/10.1128/AAC.01615-18
Culturing human cells to study fungal lung colonisation
Thursday 20 September 2018
A recent study from the University of Manchester has shown that human cell lines can be used to study lung colonisation by the fungus Aspergillus fumigatus. The work, published in Nature Communications, was partly funded by an NC3Rs Fellowship to Dr Sara Gago.
Exposure to Aspergillus fumigatus in the environment is constant but the fungus is normally cleared from the lungs. Patients with asthma or an immune defect, however, can often have difficulty clearing the pathogen leading to aspergillosis. The team at the University of Manchester have discovered a genetic mutation in humans linked to increased loads of fungal spores in the lungs.
Many laboratories use mouse models to study aspergillosis, which can be associated with welfare concerns as they involve the development of severe lung disease and mild-to-moderate respiratory distress. Human cell-based models can offer a more relevant alternative; for example, the mutation discovered in this study is in a gene for a transcription factor not found in mice. The laboratory developed an in vitro model by using the CRISPR/Cas9 gene editing technology to alter a human cell line to contain the mutation.
Dr Gago comments: “Having developed a way to adapt human cell lines so that they can carry mutations associated with disease, we have avoided using animals entirely.”
The team led by Dr Gago and Dr Paul Bowyer showed that the mutation altered the epithelial cells and extracellular matrix the cells excreted, both of which usually provide a protective barrier in the lungs. This led to increased adhesion of the fungal spores to cells, in turn leading to earlier and more successful germination of the spores compared to a human cell line without the mutation. They compared their results to samples from patients with known Aspergillus fungal colonisation and found the mutation present in up to 60% of patients with a high A. fumigatus load, whereas the mutation was not found in patients with a lower fungal load.
The research helps to build confidence in the use of non-animal approaches in studying fungal infectious disease, highlighting how gene editing can be used to study pathogen colonisation in vitro. It has led to a better understanding of why some patients have a higher Aspergillus load than others.
Gago S, Overton NLD, Ben Ghazzi N et al. 2018, Lung colonization by Aspergillus fumigatus is controlled by ZNF77. Nature Communications 9: 3835 (2018) doi:10.1038/s41467-018-06148-7
News | 2017
BSMM award: Dr Margherita Bertuzzi wins 'best oral presentation' award at the BSMM in Birmingham for her work on “Single cell analysis of the Aspergillus fumigatus host-pathogen interaction at the alveolar epithelium”. Sergio Velasquez also gave a talk at the meeting on the paradoxical growth effect caused by Caspofungin in Aspergillus fumigatus.
Presentations: Dr Elaine Bignell give plenery talk at Fungal Genetics meeting in Asilomar, USA. Dr Fabio Gsaller also gave a talk at the meeting.
Podcast interview: David Denning speaks to the CDC on antimicrobial resistance
Acolade: Congratulations to MFIG deputy director Dr Elaine Bignell, who's work on transcriptomics is recognised as exemplar research to represent the field of mycology in PLOS Pathogens: 10 year anniversary collection.
Presentation: Dr Margherita Bertuzzi presented her work “Single cell analysis of the Aspergillus fumigatus host-pathogen interaction at the alveolar epithelium” at the BSMM in Birmingham, UK.
Presentation: Drs Fabio Gsaller and Elaine Bignell gave talks at the Fungal Genetics meeting in Asilomar, USA.
Doctorate award: Congratulations to Akira Alexander from Nick Read's lab on passing his PhD viva. Drs Alex Brand and Mike Bromley were the examiners.
Fellowship Award: Dr Sara Gago is awarded an NC3IR fellowship award to develop new experimental models to understand the genetic basis of ABPA.
Presentation: Dr Margherita Bertuzzi gave a talk and presented a poster on her work "“Outcomes of the Aspergillus fumigatus host-pathogen interaction at the alveolar epithelial interface” at the Gordon research seminar and conference in Galvonston, USA.
Grant Award: Drs Margherita Bertuzzi and Elaine Bignell are awarded an MRC Discovery Award in Single Cell Analysis to study the antifungal potency of the airway epithelium in health and disease.
News | 2016
MFIG celebrates diversity of fungal science and marks 25 years of Fungal Infection Trust (FIT): On the 29th October the Manchester Fungal Infection Group partcipated in the The University of Manchester’s ‘Science Spectacular’ at the Manchester Museum. Adopting the theme ‘Fungi:Friend or Foe’ a 14-strong team of scientists (plus family and friends) used experiments,video games, posters, and films to convey the importance of fungi in all walks of life on the planet. An FIT-sponsored, environmentally-friendly, balloon race designed to exemplify airborne fungal spores as the cause of fungal lung disease attracted >300 entrants and helped to heightened public awareness of fungal lung disease.
MFIG raises > £500 for MacMillan Cancer Support: Manchester Fungal Infection Group hosted its first MacMillan Coffee Morning raising > £500 for the charity. The event was extremely well-attended and required a large area of the CTF ground floor reception in order to accommodate the cakes and visitors. MFIG would like to acknowledge the excellent efforts of Sayema Khan and Zorana Carter for masterminding the event, and of all of the MFIG bakers.
New arrival: Welcome to Jennifer Scott who will be working in Dr Jorge Amich's lab.
Doctrate award: Congratulations to Smija Kurian from Nick Read's lab on passing her PhD viva "Live-cell imaging of the early stages of colony development in Fusarium oxysporum in vitro and ex vivo during infection of a human corneal model".
Publication: Congratulations to Nick Read on his publication: Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell–cell recognition and fusion, Weichart et al., 2016, PNAS
Publication: Congratulations to Nicola Overton, Paul Bowyer and David Denning on thier publication:Genetic susceptibility to allergic bronchopulmonary aspergillosus in asthma: a genetic association study, Oveton et al., 2016, Allergy, Asthman and Clinnical Immunology
MFIG poster award: Dr Fabio Gsaller presented a talk and won the poster prize at the 50th German Mycological Society meeting in Essen, Germany. Fabio also presented his work at the 5th CESC /2nd Rising Stars meeting on Mycology in Szeged, Hungary.
Publication: Congratulations to Patricia Hernandez-Ortiz and Nick Read on their publication: Antifungal mechanisms of a plant defensin MtDef4 are not conserved between the ascomycete fungi Neurospora crassa and Fusarium graminearum, El-Mounadi et al., 2016, Molecular Microbiology
Publication: Congratulations to Alberto Munoz and Nick Read on their publication: Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus, Zhang et al., 2016, PLoS Genetics
Publication:Congratulations to David Cabellero-Lima, David Denning and Nick Read on their publication: Electrophilic, Activation-Free Fluorogenic Reagent for Labeling Bioactive Amines, Sintes et al., 2016, Bioconjugate Chemistry
Publication: Congratulations to Dr Fabio Gsaller, Dr Takanori Furukawa, Paul Car, Bharat Rash, Dr Paul Bowyer and Dr Mike Bromley on their publication:Sterol Biosynthesis and Azole Tolerance Is Governed by the Opposing Actions of SrbA and the CCAAT Binding Complex, Gsaller et al., 2016, PLoS Pathogens
Publication:Congratulations to Jorge Amich and Elaine Bignell on their review: Amino acid biosynthetic routes as drug targets for pulmonary fungal pathogens: what is known and why do we need to know more? Amich & Bignell, 2016, Current Opinion in Microbiology
Publication: Congratulations to Can Zhao and Professor Nick Read on thier recent paper which generates new diagnostics to visualise fungal infections; Nature Communications: Spacer-free BODIPY fluorogens in antimicrobial peptides for direct imaging of fungal infection in human tissue
MFIG take several awards at the 7th Advances Against Aspergillosus meeting: Congratulations to Dr Nicola Overton, Dr Jorge Amich and Dr Fabio Gsaller (2) who won 4 poster prizes representing MFIG at the meeting in Manchester. Dr Margherita Bertuzzi, Dr Elaine Bignell and Dr Mike Bromley also gave talks. Meeting co-ordinator David Denning also made an appearance on BBC breakfast to highlight the dangers of fungal spores in our households and their impact on health.
New arrival: A big MFIG welcome to Dr Sylvain Brun from Paris who will be spending 6 months in Professor Nick Read's lab learning advanced live-cell imaging techniques to study mating and chemotropic mechanisms in Neurospora crassa.
Visitors: MFIG are delighted to host visitor Michelle Momany and her graduate student Earl as they learn infection techniques and image analyses.
News | 2015
MRC Funding for network biology of the pathogenic mould Aspergillus fumigatus: Researchers from the Manchester Fungal Infection Group (MFIG), and FLS have secured a major new grant to define the basis of pathogenicity in the environmental mould Aspergillus fumigatus.
Airborne spores of the common mould, Aspergillus fumigatus, are agents of widespread, often fatal, lung diseases but a lack of vital molecular insight precludes a mechanistic understanding of pathogenicity. Amongst a genomic repertoire of >400 transcription factors, less than 10 have been characterised for roles in pathogenicity. This knowledge gap poses a critical hindrance to the rationalised discovery of new therapies, a need which is heightening in light of increasing resistance to azoles, the only available oral anti-mould agents.
To permanently eradicate this shortfall an interdisciplinary team led by Dr Elaine Bignell, Dr Mike Bromley and Dr Paul Bowyer (MFIG) and Prof Magnus Rattray (FLS), and which will include two postdoctoral scientists, an infection technician, and a PhD student, will take a state-of-the-art approach using a toolkit of systems-level methods. By combining a first-in-field library of bar-coded null mutants in 401 of the predicted 436 transcription factors encoded by the A. fumigatus genome, and a method via parallel fitness analyses for ethical testing of the mutants in the mammalian host, the team will, with minimal animal usage, identify the full cohort of transcription factors driving pathogenicity, and the network of gene products they govern.
The results of this study will revolutionise our view of mould pathogenesis in the mammalian lung, and fortify productivity within the community of scientists, clinicians and industries engaged in combating mould-related disease.
MFIG secures 5 year MRC Career Development Award : Dr Jorge Amich (University of Wurzburg) will join MFIG for a period of 5 years commencing January 2016 to start up his own group that will conduct a study entitled: Re-engineering of fungal sulphur metabolism to limit mould viability and virulence. Dr Amich is a talented fungal geneticist and infection biologist who has worked extensively on the fungal host-pathogen interaction.
‘From Bug to Drug’: A new iCASE partnership with Blueberry Therapeutics for antifungal peptide aptamers: Dr Elaine Bignell (MFIG) has secured a BBSRC ICASE studentship (commencing October 2015) to initiate a new discovery programme for antifungal peptide aptamers. During this 4 year BBSRC ICASE project, genetically engineered microbes will be used to synthesise designer drugs active against fungal diseases of man and poultry. By coupling a bespoke molecular interaction assay with a patented nanoparticle drug delivery technology, developed at Blueberry Therapeutics. The aim of the study is to deliver potent inhibitors of fungal growth directly into the intracellular environment of the pathogen cell. This combination of technologies will establish a ‘Bug to Drug’ discovery pipeline capable of producing novel fungicides.
Peptide aptamers are designer proteins composed of short amino acid chains, encased within a protein scaffold. A unique property of PAs is their ability to fold into highly diverse 3D structures which are capable of binding very specifically to other proteins. The construction of libraries of randomised aptamer sequences creates a source of molecules having a plethora of unique conformations, and able to recognise individual combinations of charge, shape and hydrophobicity. In an era of antibiotic resistance access to such a diverse array of molecular structures provides an invaluable source of new drugs. The challenge with PAs has been efficient delivery into fungal cells (PAs are, in general, unable to penetrate fungi), but by using nanoparticle-based drug delivery, developed at Blueberry Therapeutics, we can overcome this limitation. PAs have several advantages over chemically-based medicines. As they bind very specifically to other proteins, PAs are likely to cause fewer side-effects than standard pharmaceuticals. The discovery pipeline requires a much shorter cycle time to obtain a potent active molecule and PAs exhibit modes of action which are not easily accessible via pharmaceutical screening, such as interfering with protein folding, or protein-protein interactions. The metabolism of peptide aptamers yields naturally occurring amino acids, thereby limiting toxic side effects.
News | 2014
Paper: Congratulations to Margherita Bertuzzi on her publication in PLoS Pathogens: The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial invasion during pulmonary aspergillosis (October 2014)
Paper: Congratulations to Fabio Gsaller on his publication in The EMBO Journal: The Janus transcription factor HapX controls fungal adaptation to both iron starvation and iron excess (Aug. 2014)
Paper: New review article by MFIG's Nicola Smith and David Denning published in Immunology: Clinical implications of interferon gamma genetic and epigenetic variants (July 2014)
Press Release: New data from MFIG has indicated that crop spraying on British farms could be aiding a life-threatening fungus suffered by tens of thousand of people in the UK each year. MHS news hub - Fungicides for crops: worrying link to fungal drug resistance in UK warn scientists
New member: MFIG welcomes a new Research technician, Neuza Duarte to the Bignell group, who will work on structural studies of fungal pH sensors.
Student Scholarships: Bethany McCann awarded a Summer Scholarship by the British Mycological society to study pH receptor polymorphism in clinical and environmental A. fumigatus isolates, with Elaine Bignell.
MFIG in the Scientific Community: MFIG's Elaine Bignell has accepted a prestigious position on the editorial board of the journal Microbiology, part of the Society for General Microbiology (SEM) publication portfolio. Elaine will hold the post of Editor for five years from August 1st 2014.
TV interviews: David Denning and Jo Armstead appeared on BBC breakfast to discuss their recent findings on A. fumigatus infections in cystic fibrosis patients (8.15am , 29.06.14)
Press Release: New data from the National Aspergillosis Centre indicating that nearly half of adults with Cystic Fibrosis are infected by fungi. Data published in the journal PLOS ONE June 10th 2014
New members: MFIG welcomes a new Research technician, Neuza Duarte to the Bignell group, who will work on structural studies of fungal pH sensors. (June 2014)
Conferences: Elaine Bignell presented latest research findings at the 2014 Gordon Research Conference on Fungal cell and Molecular Biology, Holderness, New Hampshire, USA. (June 2014)
Invited talks: Elaine Bignell presented an overview of A. fumigatus pathogenicity at Pfizer Global R&D (Vaccine Development), Pearl River, New Jersey, USA. (June 2014)
Student Scholarships: Bethany McCann awarded a Summer Scholarship by the British Mycological society to study pH receptor polymorphism in clinical and environmental A. fumigatus isolates, with Elaine Bignell. (June 2014)
Paper: Congratulations to Alberto Muñoz et al in the Read group for the publication this month in Molecular Microbiology: Specific domains of plant defensins differentially disrupt colony initiation, cell fusion and calcium homeostasis in Neurospora crassa, Muñoz et al, 2014, Mol Microbiol.
Paper: Congratulations to Paul Bowyer and Nicola Smith on their recent publication in Clinical Microbiology and Infection: Reduced expression of TLR3, TLR10 and TREM1 by human macrophages in CCPA, and novel associations of VEGFA, DENND1B and PLAT, Smith et al, 2014, Clin Microbiol Infec.
Departures: We say a sad farewell to Rosie Hutchinson and Erik Zajta this month. It was great to meet you both and we wish you the best of luck in your future endevours! (May 2014)
Secondment: Christopher Grice awarded a travel bursary by the British society for Medical Mycology to pursue antibody production project at Pfizer Global R&D (Vaccine development), Pearl River, New Jersey, USA.
New member: Sayema Khan has join MFIG as a group technician this month, welcome Sayema.
New member: Welcome to Inigo Perez who joins MFIG to work with Mike Bromley and his team as a technician.
New member: Welcome to Dr Patricia Hernandez Ortiz who joined the Read Group for 2 years from May 1st, 2014 to work as post-doctoral research associate as part of the FungiBrain consortium.
Paper: Congratulations to the Read group for their recent publication in the Journal of Cell Science: Cdc42 and RAC-1 regulate opposite chemotropisms in Neurospora Crassa. Lichius et al, 2014, J Cell Sci.
New member: Welcome to Pavlos Geranios a new PhD. student in MFIG. Pavlos will be working as part of the FungiBrain consortium and obtaining a PhD. at the University of Manchester supervised by Professor Nick Read and Dr. Mike Bromley. (April 2014)
Student Scholarships: Erik Zajta awarded a 3 month travel bursary by the Fungal Infection Trust to support a Campus Hungary scholarship visit to the Bignell group to study the role of secreted Aspergillus fumigatus proteins in epithelial damage. (March 2014)
News | 2013
Press Releases: University of Manchester press release on the formation of MFIG (Aug 2013)
MFIG social events
Next Beer and Pizza evening: TBA