Environment-sensing and tip-steering by the hyphae of Candida albicans

Lead Research Organisation: University of Aberdeen
Department Name: College of Life Sci and Med Graduate Sch

Abstract

Most species of fungi grow as long filamentous threads, or hyphae, which penetrate their surroundings in search of nutrients. As they grow, hyphae often meet solid objects or toxic zones in their path, which they avoid by changing direction and growing around them. Although fungi seem such simple organisms, we don!
t know much about how they do this. This question is more important than it first appears, for two reasons. Firstly, there are lots of other cells that grow in the same way as fungi, including neurons in the human brain. Fungi are much easier to manipulate experimentally and what we find in fungi often translates generically to more complex organisms, including humans. Secondly, some filamentous fungi, such as Candida albicans, can cause fatal infections in hospital patients whose immune system is suppressed by cancer treatments or drugs associated with organ-transplants. Candida cells that normally live amongst the bacteria in our gut can sometimes escape into the bloodstream. Without a healthy immune system to mop them up, the cells can become lodged in the blood vessels of internal organs. Once attached, hyphae grow and penetrate into the tissue below to form fungal masses, but we don!
t know what signals tell them to do this. Because human biology is quite similar to that of fungi, it has proved difficult to develop new antifungal drugs that target C. albicans without also harming the patient. This research project will therefore help us to understand the disease process of C. albicans.

The aim is to investigate how fungal hyphae navigate and steer their tips as they grow and find out which environmental signals are important to them. Some significant discoveries have already been made. We know that calcium is an important signal in fungi, and might tell the hyphal tip which way to grow. We have also identified some proteins whose position has to be locked or unlocked so that the hyphal tip can turn. The next step is to observe whether hyphae that lack specific proteins can still steer normally when they are grown on surfaces with different chemistries and elasticity. If we can find out how fungi sense their environment and which molecules are important for tip-turning, we can also investigate whether these processes help C. albicans to cause disease. One day, this research might help us to tell new or damaged neurons which way we want them to grow, or diagnose diseases that are caused by faulty orientation responses of cells. Additionally, we may be able to save lives by finding new drugs that stop the hyphal tips of Candida albicans from penetrating and invading human tissue during bloodstream infections.

Technical Summary

Filamentous fungi penetrate their surroundings in search of specific targets or nutrients. Fundamental to this lifestyle is the ability to regulate the direction of tip growth in response to environmental cues. Despite the importance of environment-sensing and tip regulation in fungi and other polarised cells, including pollen tubes, root hairs and neurons, the underlying molecular processes are not well understood. During systemic infection in humans by the opportunistic pathogen, Candida albicans, morbidity is associated with the absorption and penetration of endothelial layers followed by the invasion of internal organs, but it is not known whether tropic tip growth is involved in this process. C. albicans hyphae exhibit tropic behaviour in response to several environmental cues in vitro, including contact, electric fields and substrate rigidity and topography. My studies have shown that they are all dependent on calcium influx from the environment. In addition, the plasma-membrane calcium-channels that mediate influx appear to also act as the environment-sensor. Mid1, a stretch-activated calcium channel, is primarily required for contact-sensing, while Cch1, a voltage-gated calcium channel, is required for germination towards the cathode in electric fields. The mechanism by which the calcium signal activates tip-turning is a subject for investigation in this research. I have shown that the Ras-like and Rho-like small GTPases, Rsr1 and Cdc42, which are involved in polarity site-determination and polarised growth, are required for the response to calcium signalling. Interestingly, loss of normal GTP-cycling by either of these proteins severely reduces tropic tip responses but in markedly different ways. The rsr1 null mutant was tested for virulence in a mouse-model of systemic infection. The erratic tip orientation of the mutant hyphae correlated with a reduction in tissue penetration, lesion size and virulence, suggesting that normal tip regulation is important for pathogenesis. The aims of the proposed research are to understand the molecular processes that direct hyphal tip orientation in response to environmental cues and to assess the contribution of regulated tip-growth to disease progression during infection by C. albicans. The novel approach of this study is to combine microbiology-based reverse genetics with the observation of hyphal tropic growth in highly-defined environments that have been generated using surface engineering technology. This will give important insights into the interplay between cellular processes within the hyphal tip and properties of the micro-environment that influence tip behaviour.

Publications

10 25 50
 
Description British Mycological Society Summer Student Bursary
Amount £2,400 (GBP)
Organisation British Mycological Society 
Sector Learned Society
Country United Kingdom
Start 06/2016 
End 08/2016
 
Description MRC Centenary Award
Amount £79,986 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 06/2013
 
Description Royal Society URF Equipment fund
Amount £13,508 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 01/2011 
End 01/2013
 
Description Summer Studentship
Amount £1,200 (GBP)
Organisation British Mycological Society 
Sector Learned Society
Country United Kingdom
Start 06/2011 
End 08/2011
 
Description Wellcome Institutional Strategic Support Fund
Amount £19,902 (GBP)
Funding ID ISSF_2017_SCO1 
Organisation Wellcome Trust 
Department Wellcome Trust Strategic Award
Sector Charity/Non Profit
Country United Kingdom
Start 05/2016 
End 01/2018
 
Description Wellcome Trust ISSF
Amount £20,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2015 
End 03/2016
 
Title Calcium reporter 
Description A genomically-encoded reporter for intracellular calcium fluxes. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact As-yet unpublished. 
 
Title Mutant cell polarity C. albicans strains 
Description Two strains generated with mutated proteins that cause altered interactions in the cell polarity complex 
Type Of Material Cell line 
Provided To Others? No  
Impact Manuscript published 2014. Presented at 4 conferences. 
 
Title Transposon mutagenesis system 
Description Agrobacterium-based transposon mutagenesis system for use in environmental fungi and in Candida albicans. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact As-yet unpublished - seeking funding to use the tool to make the tDNA library in C. albicans and screen for phenotypes. Will be used by BBSRC student for an environmental fungus. 
 
Description Calcium signalling in Candida dubliniensis 
Organisation Duke University Medical Centre
Department Center for Microbial Pathogenesis
Country United States 
Sector Academic/University 
PI Contribution We assayed the mutant strains for changes in directional growth responses, and advised on the manuscript.
Collaborator Contribution The partner generated mutant strains and wrote the published manuscript.
Impact A published paper. This collaboration was between laboratories that work on different species of fungal pathogen.
Start Year 2010
 
Description Composition analysis of abiotic surfaces 
Organisation University of Oxford
Department Department of Materials
Country United Kingdom 
Sector Academic/University 
PI Contribution We designed the experiments, generated the samples and wrote the paper.
Collaborator Contribution Partners used XPS to analyse and validate modified abiotic surfaces prepared by us, and helped with data analysis.
Impact Manuscript published in Cellular Microbiology. This was a multi-disciplinary collaboration between the Oxford Materials Department who use various methods to analyse surface chemistry and the Aberdeen Fungal Group who specialise in fungal pathogenesis.
Start Year 2011
 
Description Modification of medical silicones by fungal biofilms 
Organisation Queen's University Belfast
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution We have designed the experiments to test samples provided by our collaborators.
Collaborator Contribution Partner in Belfast has provided and validated silicones with grafted surface modification. Partner in Reading is supplying 'fuzzy' molecules for grafting and mechanical analysis by partner in Belfast.
Impact Early-stage collaboration. We have been awarded funding from the MRC Centenary fund and SULSA for this project. Our MRC DPFS outline application was recently rejected. A PhD student has been identified in Aberdeen to continue sample analysis. Data for a microbiology and a chemistry paper has been generated and these papers are being drafted.
Start Year 2011
 
Description Modification of medical silicones by fungal biofilms 
Organisation School of Pharmacy; School of Biological Sciences Reading
Country United Kingdom 
Sector Academic/University 
PI Contribution We have designed the experiments to test samples provided by our collaborators.
Collaborator Contribution Partner in Belfast has provided and validated silicones with grafted surface modification. Partner in Reading is supplying 'fuzzy' molecules for grafting and mechanical analysis by partner in Belfast.
Impact Early-stage collaboration. We have been awarded funding from the MRC Centenary fund and SULSA for this project. Our MRC DPFS outline application was recently rejected. A PhD student has been identified in Aberdeen to continue sample analysis. Data for a microbiology and a chemistry paper has been generated and these papers are being drafted.
Start Year 2011
 
Description Trace element analysis of fungi 
Organisation University of Aberdeen
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We designed the project, applied for funding, generated the samples and wrote the paper.
Collaborator Contribution Our partners have determined the tracel element composition of fungal samples and helped with data analysis and manuscript preparation.
Impact Manuscript in preparation but post-doc has left due to child-care commitments. Multi-disciplinary study involving the TESLA Chemistry group who determine the trace element composition and protein associations of metals and inorganic ions, and the Aberdeen Fungal Group who specialise in fungal pathogenesis.
Start Year 2011
 
Description Tropic responses of C. albicans dfi1 mutant 
Organisation Tufts University
Department Sackler School of Graduate Biomedical Sciences
Country United States 
Sector Academic/University 
PI Contribution My research team has analysed the tropic responses of the mutant strain generated by the Collaborator.
Collaborator Contribution The partners generated the strain and will include my research team as co-authors in their publication.
Impact No outcomes as yet. This is not a multi-disciplinary collaboration.
Start Year 2013
 
Description Autumn exhibition at Duff House 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact An exhibit on my work and career was featured as part of a Royal Society celebration of the scientific background of previous owners of Duff House. My exhibit was featured as an example of scientific endeavour in the present day. It described how important the growth patterns of filamentous fungi are in supporting plant growth and causing human disease.

Duff House is run by Historic Scotland and the National Galleries of Scotland. Although no enquiries have been forthcoming from the public concerning this exhibition, Duff House is a well-known heritage site and attracts 28,000 visitors every year.
Year(s) Of Engagement Activity 2010
 
Description Departmental Seminar, University of Sheffield 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I gave a seminar to staff and students in the Department of Molecular Biology and Biotechnology, University of Sheffield.

I discussed a new cross-disciplinary collaboration with a physicist at the University.
Year(s) Of Engagement Activity 2014
 
Description Public workshop on pathogenic microbes 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 60 members of the public attended a presentation called 'The war within us', with interviews of scientists streamed live from our microbiology lab.

Brownie group requested talk for 'Science' badge.
Year(s) Of Engagement Activity 2012
 
Description Radio broadcast 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Interview given to 'The Naked Scientist', aired as a pod-cast on the Naked Scientist website and as a article on Radio 5 Live.

Responses from the fungal community but not the public.
Year(s) Of Engagement Activity 2011
URL http://www.thenakedscientists.com/HTML/content/interviews/interview/1783/
 
Description Radio interview by Tina Bedekovic, Prague 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact After giving a talk at the Society of Experimental Biology conference, my PhD student was chosen by the radio media in Prague to give an interview about our work.
Year(s) Of Engagement Activity 2015
 
Description Royal Society 'The Next Big Thing', Hay Festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The Royal Society 'The Next Big Thing' event at the Hay Festival for the Arts consisted of a panel of 4 RS-funded scientists describing to the public audience (300 people) why their research is so important.

After short talks by a panel of scientists, the floor was thrown open to the public for 45 minutes of questions.
Year(s) Of Engagement Activity 2014
URL https://royalsociety.org/events/2014/05/the-next-big-thing/http://www.hayfestival.com/p-8222-liz-tun...
 
Description Schools visit to Aberdeen Fungal Group 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Senior school pupils spent a week in the Aberdeen Fungal Group labs and animal house facilities
Year(s) Of Engagement Activity 2015