Identifying the mechanism of intracellular parasitism by Cryptococcus

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences

Abstract

Cryptococcus is a free-living fungus that is able to cause a fatal infection in humans and other animals. Our current understanding of this disease suggests that fungal spores are inhaled into the lung and then ?hide? from the immune system, sometimes for several years, by living inside a particular type of host cell called a macrophage. Although this cell would kill most other pathogens, Cryptococcus has a remarkable ability to survive and indeed grow inside macrophages. In addition, we have recently discovered that the fungus is also able to ?escape? from inside of this host cell by a novel process called ?reverse phagocytosis?. This process is likely to be advantageous to the pathogen since it does not damage the host cell and is therefore unlikely to trigger a strong inflammatory response.

In order to develop better treatments for this lethal disease, we need to understand how the fungus can manipulate the host cell in this way. Our application is therefore directed at learning more about the molecular process that underlies this behaviour. In particular, we hope to answer two specific questions:
1) can we identify new ways in which human macrophages can be induced to destroy intracellular cryptococci and thereby eradicate latent infections?
2) how great is the risk that particular strains of Cryptococcus will become more dangerous to patients by evolving an improved ability to manipulate host cells in this way?

Technical Summary

Cryptococcosis is a life-threatening disease of the central nervous system caused by the fungus Cryptococcus neoformans. The disease is uniformly fatal without rapid clinical intervention and even with the best available anti-fungal treatment, patient mortality exceeds 10%, rising to 88% in areas of the world with poor healthcare infrastructure. Cryptococcosis is primarily a disease of immunocompromised hosts (especially AIDS patients), although some strains of the closely related species C. gattii have the capacity to infect and kill immunocompetent individuals.

Pathogenic cryptococci show a remarkable ability to survive and proliferate within host macrophages following phagocytosis and are also capable of exiting the host cell via a unique, non-lytic, mechanism. Together these behaviours are thought to underlie the capacity of the pathogen to remain latent within the human host for long periods and, similarly, to cause relapsing episodes of cryptococcosis in patients after initially successful antifungal treatment. Importantly, this aspect of the disease means that infected patients are currently advised to remain on lifelong antifungal medication, despite the considerable cost and toxicity issues associated with this treatment regime. Thus an improved understanding of the cryptococcal-macrophage interaction may lead to significant improvements in our management of latent cryptococcosis.

We therefore propose to build on recent preliminary work from our group and use high-throughput timelapse microscopy to investigate how the normal cell biology of a macrophage is manipulated by Cryptococcus. In particular, we hypothesise that two factors may influence the outcome of this interaction. Firstly, it is known that changes in host inflammatory signalling (for example, in the levels of T-cell derived cytokines) alter cryptococcal disease progression. We therefore propose to investigate whether this may, in part, result from an effect on infected macrophages. Secondly, there is a wealth of data showing that variability in Cryptococcus genotype strongly influences virulence in mammals. We therefore propose to investigate what effect pathogen genotype and phenotype have on intracellular parasitism by this pathogen and thus better understand what risk novel cryptococcal strains may present to human health.

In summary, this proposal is intended to address an important aspect of the host-pathogen interaction in this fatal disease. By understanding the molecular basis of this interaction, we hope to identify possible targets for therapeutic intervention in the future and thereby improve long-term prognosis for infected patients.

Publications

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Farrer RA (2016) Microevolutionary traits and comparative population genomics of the emerging pathogenic fungus Cryptococcus gattii. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

 
Description Lister Fellowship
Amount £200,000 (GBP)
Organisation Lister Institute of Preventive Medicine 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2010 
End 08/2015
 
Description Wellcome Trust Project Grant (Investigating the molecular basis of cryptococcal hypervirulence underlying the Vancouver Outbreak)
Amount £318,000 (GBP)
Funding ID 088148 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2010 
End 12/2012
 
Title Intracellular proliferation assay 
Description Developed assay for quantifying intracellular proliferation of fungal pathogens. Now being widely adopted by other groups internationally. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact Joint publication (PMID: 20421942); ongoing collaboration with Tihana Bicanic, St. Georges, London. 
URL http://europepmc.org/abstract/MED/20421942
 
Description Collaboration with Boekhout Lab, Netherlands 
Organisation Royal Netherlands Academy of Arts and Sciences
Department Fungal Biodiversity Center (CBS)
Country Netherlands 
Sector Learned Society 
PI Contribution Research exchange (Hansong Ma from my group visited and worked with Ferry Hagen in the Boekhout lab), resulting in joint data and a joint publication (PMID: 19651610)
Collaborator Contribution Joint research and publication (PMID: 19651610)
Impact Joint publication (PMID: 19651610) Named collaboration on recently funded grant (Wellcome Trust)
 
Description Collaboration with Heitman laboratory, Duke, USA 
Organisation Duke University
Country United States 
Sector Academic/University 
PI Contribution Research visit by Hansong Ma (PhD student) and subsequently myself to Duke University. Resulted in preliminary data for a grant, recently funded by the Wellcome Trust
Collaborator Contribution Ongoing research collaboration
Impact Resulted in preliminary data for a grant funded by the Wellcome Trust. Joint publication (PMID: 20421942)
Start Year 2008
 
Description Collaboration with Tihana Bicanic 
Organisation St George's University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution Conducting in vitro cell testing of clinical fungal isolates
Collaborator Contribution Provision of clinical samples for analysis
Impact None as yet.
Start Year 2010
 
Description INDEX commercialisation contract 
Organisation Exilica Ltd
Country United Kingdom 
Sector Private 
PI Contribution High-resolution imaging of nanoparticle uptake.
Impact The industrial partner became aware of our MRC-funded work on live cell imaging and approached us for commercial imaging work, which we have now completed.
Start Year 2008
 
Description 'Off-topic' seminar, Culham Centre for Fusion Energy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Seminar to physicists, engineers and mathematicians working at the Culham research facility on fusion energy.

Very positive feedback from the audience.
Year(s) Of Engagement Activity 2010
 
Description School Visit - Bromsgrove 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Ran an interactive session on infectious diseases with 6th formers

Have accepted invitations to return to the same school next year, and an additional invitation to a different school to run the same programme
Year(s) Of Engagement Activity 2008
 
Description School Visit, Sutton Coldfield 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact > 100 pupils attended a "general interest" talk on infectious disease, including our own work.

School requested ongoing outreach connection with our group.
Year(s) Of Engagement Activity 2010
 
Description Science museum day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Ran a 'meet the scientists' day at the Birmingham ThinkTank (science museum) for >200 school-age pupils and their families.

Considerable interest from pupils and parents in having repeat workshops, ideally via their schools.
Year(s) Of Engagement Activity 2011