The Route to Infection: The Role of Fungal Paxillin in Tissue Invasion

* DEADLINE FOR APPLICATIONS EXTENDED TO MONDAY 11 DECEMBER 2017 *

Supervisors: Dr Alex Brand (Aberdeen), Dr Stewart Smith (Edinburgh) & Professor Ken Sawin (Edinburgh)

Project overview and aims:

Fungal infections caused by Candida albicans and Aspergillus fumigatus kill half a million people a year by forming highly invasive filaments that colonise internal organs. These filaments, called hyphae, have evolved steering mechanisms that are essential for the fungus to penetrate and disrupt host tissue [1]. We have discovered a dynamic Paxillin-like protein complex (Pxl1) in fungi that is vital for normal hyphal steering. The aims of the project are to identify the regulators and effectors of Pxl1 and examine their role during interactions with host cells. We hypothesise that fungal Pxl1 acts as an adapter protein that directs the delivery of specific cell-wall modifiers via the actin cytoskeleton. We will use new biophysical tools coupled with live-cell imaging and proteomics to investigate the role of fungal Pxl1 in the cell steering system that is so important for C. albicans and A. fumigatus during host tissue invasion.

Objectives:

Hyphal sensing of the physical world is a dynamic process. For example, in C. albicans, tip contact causes polarity proteins to swiftly translocate to the touch zone to drive directional responses such as contour-following and gap penetration [2, 3 & unpublished data]. Deletion of Pxl1 results in loss of these responses in C. albicans but its role in A. fumigatus is not known. The questions this project will answer are: 1. Is Pxl1 important for hyphal steering in A. fumigatus? We will answer this by characterising the real-time growth behaviours of wild-type and Pxl1-deleted cells in microfabricated chambers for the first time. Expertise in novel chamber design will be provided by Dr Stewart Smith (School of Engineering, University of Edinburgh). 2. Which proteins interact with C. albicans Pxl1? We will use proteomics to identify interacting effectors and Pxl1 phosphorylation regulators using the Proteomics Core Facility at Aberdeen and the Phosphoproteomics capacity of Prof Ken Sawin, (Wellcome Centre for Cell Biology, University of Edinburgh). This will enable us to build a model of Pxl1 function and regulation. 3. How does Pxl1 influence interactions between C. albicans/A. fumigatus with human cell layers? We will use a new endothelialised cell model and fluorescent hyphae to quantify the translocation of mutant cells from the bloodstream into solid organ tissue below.

Research training provided:

The world-renowned Aberdeen Fungal Group specialises in host-pathogen interactions (Brand, Aberdeen). The student will learn fungal and tissue-culture techniques, along with molecular methods including PCR, cloning, gene synthesis, sequencing and CRISPR-Cas9 technology. Real-time fluorescence microscopy and quantitative image analysis will be used for cell and protein tracking. Training in customised microfluidics chamber design and manufacture will be given for the development of novel methods for studying contact-dependent cell responses in real time (Smith, Edinburgh). Proteomic analysis from pull-down assays and phosphoprotein enrichment techniques will be used to identify Pxl1 interactors and regulators (Sawin, Edinburgh). Together, these skills will provide the student with a solid grounding in the skills and technologies required for a wide choice of future careers in the field of eukaryotic cell biology.