DEALING WITH THERAPY-RESISTANT CRYPTOCOCCOSIS BY TARGETING INTRACELLULAR PATHOGENS
Lead Research Organisation:
University of Birmingham
Department Name: Sch of Biosciences
Abstract
Cryptococcosis is a life-threatening fungal disease that kills more than 600 000 patients per annum worldwide. The majority of these individuals are newly diagnosed HIV-positive patients in the developing world, for whom the prognosis is relatively good, PROVIDED that they survive the cryptococcal infection. In addition, there is a globally increasing incidence of cryptococcal infection in otherwise healthy people, which appears to be due to the occurrence of localized, hypervirulent populations of the fungus. Thus understanding more about the biology of this pathogen, and developing ways to improve treatment of it, is critical.
One major problem is that the 'best practice' antifungal treatment available still fails in more than 10% of patients. One reason for this is that, during an infection, some cryptococcal cells 'hide' within white blood cells of the patient. These intracellular fungi are not exposed to high enough levels of antifungal drugs and can thus act as 'reservoirs' of disease.
To try and address this problem, our proposal aims to identify drugs that can be used to stimulate the expulsion of intracellular cryptococci from white blood cells, thus exposing them to lethal levels of antifungal drugs in the circulation. Our group has recently discovered just such an expulsion process, which we call vomocytosis, and shown that it can be stimulated by applying different agents, such as the anti-malarial drug chloroquine.
In order to move this finding into the clinic, we need to follow two lines of investigation. Firstly, we need to understand the molecular events that happen within infected white blood cells and lead to fungal expulsion. Such information is essential if we are to devise methods of manipulating that process without causing unwanted 'collateral damage' (for instance, dramatically impairing the body's ability to tackle other pathogens that may be infecting at the same time as Cryptococcus). Secondly, we plan to undertake a high-throughput screen to identify new, more effective, compounds that stimulate cryptococcal expulsion. Importantly, we will take a 'new tricks for old drugs' approach, focusing our search on the list of (>1000) compounds that have already been approved by the Food and Drug Administration. These drugs therefore meet minimum safety standards and have already been used to treat other disease conditions. Therefore compounds that we identify can be moved into clinical practice more rapidly and more cheaply than totally new molecules.
At the end of the project, we will thus have provided both important new information about a fatal human disease and revealed a set of compounds that might, in the near future, be utilized to help treat that disease.
One major problem is that the 'best practice' antifungal treatment available still fails in more than 10% of patients. One reason for this is that, during an infection, some cryptococcal cells 'hide' within white blood cells of the patient. These intracellular fungi are not exposed to high enough levels of antifungal drugs and can thus act as 'reservoirs' of disease.
To try and address this problem, our proposal aims to identify drugs that can be used to stimulate the expulsion of intracellular cryptococci from white blood cells, thus exposing them to lethal levels of antifungal drugs in the circulation. Our group has recently discovered just such an expulsion process, which we call vomocytosis, and shown that it can be stimulated by applying different agents, such as the anti-malarial drug chloroquine.
In order to move this finding into the clinic, we need to follow two lines of investigation. Firstly, we need to understand the molecular events that happen within infected white blood cells and lead to fungal expulsion. Such information is essential if we are to devise methods of manipulating that process without causing unwanted 'collateral damage' (for instance, dramatically impairing the body's ability to tackle other pathogens that may be infecting at the same time as Cryptococcus). Secondly, we plan to undertake a high-throughput screen to identify new, more effective, compounds that stimulate cryptococcal expulsion. Importantly, we will take a 'new tricks for old drugs' approach, focusing our search on the list of (>1000) compounds that have already been approved by the Food and Drug Administration. These drugs therefore meet minimum safety standards and have already been used to treat other disease conditions. Therefore compounds that we identify can be moved into clinical practice more rapidly and more cheaply than totally new molecules.
At the end of the project, we will thus have provided both important new information about a fatal human disease and revealed a set of compounds that might, in the near future, be utilized to help treat that disease.
Technical Summary
The etiological agents of cryptococcosis, Cryptococcus neoformans and C. gattii , are both facultative intracellular pathogens that can survive and proliferate within macrophages. Intracellular yeast represent a reservoir of disease that is difficult to eradicate with current antifungal regimes. We have recently identified a novel expulsion process, termed vomocytosis, by which intracellular cryptococci exit macrophages. In addition, we have shown that vomocytosis can be enhanced by the application of specific cytokine subsets, gaseous stimuli or chemical agents.
Here we propose a programme of work to both investigate the intracellular behaviour of cryptococci and identify agents that manipulate this behaviour. Specifically, we will:
a) identify the molecular steps that lead to cryptococcal expulsion
b) determine how differing execution of these steps leads to inter-strain variation in these infectious species
c) identify compounds that induce vomocytosis, and
d) reveal the mode of action of such compounds.
In following this line of investigation, our intention is to develop a new therapeutic strategy to accelerate fungal clearance in patients. This strategy is based around combining existing antifungal regimes with new compounds, identified from our screen, which will induce the expulsion of intracellular cryptococci, rendering them susceptible to the antifungal agents. Our approach, focusing on a drug-redeployment screen within current FDA-approved compounds, is likely to identify agents that are safe in patients and can be rapidly deployed into clinical settings. In addition, we are mindful of the fact that analogous pathogen expulsion processes have recently been identified in a range of infectious organisms. Thus findings we make as part of this proposal are likely to have far-reaching consequences both in terms of fundamental microbiology and translational approaches for dealing with intracellular infections.
Here we propose a programme of work to both investigate the intracellular behaviour of cryptococci and identify agents that manipulate this behaviour. Specifically, we will:
a) identify the molecular steps that lead to cryptococcal expulsion
b) determine how differing execution of these steps leads to inter-strain variation in these infectious species
c) identify compounds that induce vomocytosis, and
d) reveal the mode of action of such compounds.
In following this line of investigation, our intention is to develop a new therapeutic strategy to accelerate fungal clearance in patients. This strategy is based around combining existing antifungal regimes with new compounds, identified from our screen, which will induce the expulsion of intracellular cryptococci, rendering them susceptible to the antifungal agents. Our approach, focusing on a drug-redeployment screen within current FDA-approved compounds, is likely to identify agents that are safe in patients and can be rapidly deployed into clinical settings. In addition, we are mindful of the fact that analogous pathogen expulsion processes have recently been identified in a range of infectious organisms. Thus findings we make as part of this proposal are likely to have far-reaching consequences both in terms of fundamental microbiology and translational approaches for dealing with intracellular infections.
Planned Impact
The major beneficiaries of our research will ultimately be cryptococcosis patients, particularly those for whom best-practice antifungal treatment does not lead to a rapid rate of fungal clearance and who are thus at the highest risk of death. As with any novel therapeutic approach, it will take time for our findings to be adopted in clinical practice. However, by focusing our drug-screening approach on existing FDA-approved compounds, we aim to identify lead molecules that can be rapidly and cheaply 'rolled out'; certainly on a much shorter timescale than the 17 years that MRC has previously identified as the average time taken for research findings to enter the clinic.
More broadly, a substantial fraction of this proposal is dedicated to a detailed molecular investigation of intracellular parasitism by cryptococci. The information arising from this section impacts not only on cryptococcal disease, but also provides detailed information on intracellular parasitism and phagocyte biology that will be of value to immunologists, cell biologists and microbiologists. Specifically, non-lytic expulsion processes, similar to the one we propose to study, have now been identified in a diverse range of human pathogens. Hence both clinical and non-clinical colleagues working on other infections (and thus, ultimately, their patients) will benefit from the data we produce.
Finally, the processes we propose to investigate are tightly linked to host cell vesicle trafficking and exocytosis. Thus we will be generating both data and reagents of value to a wide range of individuals, ranging from academic cell biologists to commercial microscope companies.
More broadly, a substantial fraction of this proposal is dedicated to a detailed molecular investigation of intracellular parasitism by cryptococci. The information arising from this section impacts not only on cryptococcal disease, but also provides detailed information on intracellular parasitism and phagocyte biology that will be of value to immunologists, cell biologists and microbiologists. Specifically, non-lytic expulsion processes, similar to the one we propose to study, have now been identified in a diverse range of human pathogens. Hence both clinical and non-clinical colleagues working on other infections (and thus, ultimately, their patients) will benefit from the data we produce.
Finally, the processes we propose to investigate are tightly linked to host cell vesicle trafficking and exocytosis. Thus we will be generating both data and reagents of value to a wide range of individuals, ranging from academic cell biologists to commercial microscope companies.
People |
ORCID iD |
Robin May (Principal Investigator) |
Publications
Bojarczuk A
(2016)
Cryptococcus neoformans Intracellular Proliferation and Capsule Size Determines Early Macrophage Control of Infection.
in Scientific reports
Brown GD
(2017)
Editorial overview: Host-microbe interactions: fungi.
in Current opinion in microbiology
Dambuza IM
(2018)
The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis.
in PLoS pathogens
Evans RJ
(2015)
Cryptococcal phospholipase B1 is required for intracellular proliferation and control of titan cell morphology during macrophage infection.
in Infection and immunity
Evans RJ
(2017)
Using Flow Cytometry to Analyze Cryptococcus Infection of Macrophages.
in Methods in molecular biology (Clifton, N.J.)
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
Garelnabi M
(2018)
Quantifying donor-to-donor variation in macrophage responses to the human fungal pathogen Cryptococcus neoformans.
in PloS one
Gilbert AS
(2017)
Vomocytosis of live pathogens from macrophages is regulated by the atypical MAP kinase ERK5.
in Science advances
Head MG
(2014)
Systematic analysis of funding awarded for mycology research to institutions in the UK, 1997-2010.
in BMJ open
Johnston SA
(2013)
Cryptococcus interactions with macrophages: evasion and manipulation of the phagosome by a fungal pathogen.
in Cellular microbiology
Kocurek KI
(2017)
Top-Down LESA Mass Spectrometry Protein Analysis of Gram-Positive and Gram-Negative Bacteria.
in Journal of the American Society for Mass Spectrometry
Kwon-Chung KJ
(2017)
The Case for Adopting the "Species Complex" Nomenclature for the Etiologic Agents of Cryptococcosis.
in mSphere
May RC
(2016)
Custom-Made Quorum Sensing for a Eukaryote.
in Developmental cell
May RC
(2016)
Cryptococcus: from environmental saprophyte to global pathogen.
in Nature reviews. Microbiology
Ropars J
(2018)
Gene flow contributes to diversification of the major fungal pathogen Candida albicans.
in Nature communications
Sabiiti W
(2014)
Efficient phagocytosis and laccase activity affect the outcome of HIV-associated cryptococcosis.
in The Journal of clinical investigation
Samantaray S
(2016)
Novel cell-based in vitro screen to identify small-molecule inhibitors against intracellular replication of Cryptococcus neoformans in macrophages.
in International journal of antimicrobial agents
Seoane PI
(2020)
Vomocytosis: What we know so far.
in Cellular microbiology
Silva VKA
(2020)
Pyrifenox, an ergosterol inhibitor, differentially affects Cryptococcus neoformans and Cryptococcus gattii.
in Medical mycology
Simm C
(2019)
Zinc and Iron Homeostasis: Target-Based Drug Screening as New Route for Antifungal Drug Development.
in Frontiers in cellular and infection microbiology
Smith LM
(2015)
The fungal pathogen Cryptococcus neoformans manipulates macrophage phagosome maturation.
in Cellular microbiology
Smith LM
(2013)
Mechanisms of microbial escape from phagocyte killing.
in Biochemical Society transactions
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 | 'Life Under a Lens', UK Fungus Day |
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 | Took part in an interactive activity day to promote UK Fungus Day at the Birmingham ThinkTank (science museum). Introduced nearly 300 children and their families to fungi-related activities together with my Lab (MayLab) and the wider Host Pathogen Interaction (HAPI) Lab. |
Year(s) Of Engagement Activity | 2015 |
URL | http://www.biosciences-labs.bham.ac.uk/may/Home.html |
Description | 'Meet the Scientist' (University of Birmingham) |
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 | Approximately 300 children attended a 'Meet the Scientist' Biology Spring Series for Schoolchildren, in which I gave a talk about microbiology and science. This took place at my organisation, the School of Biosciences, University of Birmingham. This sparked questions and a discussion, and the schools involved reported increased interest amongst their pupils in the subject areas. |
Year(s) Of Engagement Activity | 2015 |
Description | 'Meet the Scientist' School Visit (Derby) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | I gave talk about microbiology and science to around 100 pupils at Chellaston Academy, Derby. This sparked questions and discussion afterwards, and the school reported an increased interest in related subject areas. |
Year(s) Of Engagement Activity | 2015 |
Description | 'Meet the Scientist' School Visit (Rugby) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | Gave a talk to approximately 100 school pupils at Ashlawn School Rugby re microbiology and science. This sparked questions and discussion afterwards, and the school reported an increased interest in related subject areas. |
Year(s) Of Engagement Activity | 2015 |
Description | 'The Human Zoo' (ASE Annual Conference Lecture) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | 'The Human Zoo: discovering your hidden microbes & their unexpected influences on your life', talk to 50 school Science teachers at The Association for Science Education (ASE) Annual Conference 2019. This sparked questions and discussion afterwards and lots of positive feedback was given, for e.g.. 'This was the highlight of the day for me! Excellent presentation, informative, engaging and highly enjoyable!' |
Year(s) Of Engagement Activity | 2019 |
URL | http://www.ase.org.uk/annual-conference |
Description | Annual Microbiology Lecture for Schools |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Schools |
Results and Impact | Gave a talk 'The Human Zoo', to 350 school children interested in studying Medicine, Biomedical or Biological Sciences at University at the Annual Microbiology Lecture for Schools at Aberdeen University. This sparked questions and discussion afterwards and increased interest in Microbiology from many of those who attended. |
Year(s) Of Engagement Activity | 2015 |
URL | http://www.abdn.ac.uk/events/8578/ |
Description | Cafe Scientifique at the Pure Bar, Birmingham City Centre |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Talk sparked questions and discussion afterwards. I have been invited to talk to MENSA. |
Year(s) Of Engagement Activity | 2015 |
URL | http://www.cafescientifique.org/index.php?option=com_contentbuilder&title=uk-birmingham&controller=d... |
Description | Horizons Series Lecture (UOB School) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | Lecture to 100 pupils at University of Birmingham's School which sparked questions and discussion afterward, and the school reported increased interest in related subject areas. |
Year(s) Of Engagement Activity | 2018 |
Description | Meet Your Inner Neanderthal Public Talk (UOB) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | A public lecture part of UOB's College of Life and Environmental Sciences Lecture Series which sparked questions and discussion afterwards. |
Year(s) Of Engagement Activity | 2018 |
Description | Microbes interview (Daily Mail February 2019) |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Interview with a Daily Mail journalist regarding microbes led to media coverage across 9 different paper and web-based newspapers in the UK and Ireland. |
Year(s) Of Engagement Activity | 2019 |
URL | https://www.dailymail.co.uk/health/article-6693033/The-perils-sharing-popcorn-not-mention-earphones-... |
Description | Public Lecture (Birmingham Natural History Society) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | A talk for 100 members of Birmingham's Natural History Society which sparked questions and discussion afterwards. |
Year(s) Of Engagement Activity | 2017 |
Description | Research Pop-Up Shop, Birmingham City Centre |
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 | Open-door exhibition in Birmingham shopping centre. Around 100 children and adults dropped in during the day to learn more about current research in microbiology at Birmingham. None at present; awaiting formal feedback |
Year(s) Of Engagement Activity | 2014 |
Description | School Talk (Sutton Coldfield) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | Talk to 150 pupils at Sutton Coldfield Grammar School, which sparked questions and discussion afterwards and the school reported increased interest in related subject areas. |
Year(s) Of Engagement Activity | 2017 |
Description | School Workshop - What Is A Microbe? (Beoley) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | 100 school children took part in an afternoon's workshop re 'What is a Microbe', which sparked questions and discussion afterwards, and the school reported increased interest in related subject areas. |
Year(s) Of Engagement Activity | 2018 |