The route to fungal infection - how physical cues guide tissue invasion

Growing hyphal filaments of the fungal pathogen, Candida albicans, are able to sense contact with a surface, which directs growth behaviours such as asymmetric cell shape, contour-following, gap penetration and trajectory maintenance. How contact is sensed is not known and the early stages are technically difficult to study at the molecular level.

However, directed polarisation can be studied using applied electric fields and live-cell imaging of fluorescent reporter proteins. There are several signalling pathways involved in sensing in cell-wall perturbation that may also function in sensing external contact.

The aim of this project is to examine the role of three signalling systems – calcium influx, the cell-wall integrity pathway and the hyper-osmolarity rescue pathway - in cell polarisation and contact-induced growth responses.

The approach will be to generate mutant and fluorescent reporter strains and to observe changes in specific growth responses using fluorescence microscopy, micro-fabricated chambers, electric fields and live-cell imaging.

Background and objectives

C. albicans is a fungus that causes a variety of infections in humans by forming invasive polarised cells called hyphae, whose ability to respond to directional cues is essential for tissue invasion and disease [1]. Using strains carrying fluorescently-tagged polarity proteins, we showed that polarity complexes change their position in response to external cues such as touch and small electric fields, and that constitutive activation of specific proteins can completely reverse the direction of polarisation [2,3 & unpublished data]. Our hypothesis is that external cues are sensed by cell-integrity sensing systems that influence the positioning of polarity protein activity. To test this hypothesis, the objectives of the project are:
1.Generate gene deletion mutants and strains expressing fluorescent reporter proteins as markers for polarity complexes and ion flux using GFP labelling.
2.Adapt existing electric field and hyphal contact assays into novel microfluidic formats to study directional polarisation and hyphal tip responses.
3.Use live-cell imaging and image analysis software to quantify changes in polarity protein localisation during cell polarisation, steering and growth.

Research training provided:

The student will be based in the world-renowned MRC Centre for Medical Mycology (~ 80 people) which specialises in the biology of human pathogens, fungal immunology and anti-fungal drug discovery. Basic mycology and cell culture techniques will be learned, along with molecular methods including PCR, cloning, gene synthesis, sequencing and expression analysis.

The student will use real-time fluorescence microscopy and analyse imaging outputs using a variety of software packages. Training in customised microfluidics chamber design and manufacture will be given for the development of novel methods for studying the localisation of polarity proteins during galvanotropic cell polarisation in real time (Brand, Aberdeen & Smith, Edinburgh). Together, these skills will provide the student with a strong background in the tools and technology required to underpin a range of career choices in the field of eukaryotic cell biology.