MRes in Medical Mycology and Fungal Immunology

Project 1
Do Reactive Oxygen Species influence Genome Plasticity in the Fungal Pathogen Cryptococcus neoformans?
Cryptococcus neoformans is a globally distribiuted human fungal pathogen that causes an estimated 1 million infections each year. Mortality can be as highas 70%, with global deaths ranging from 250,000 to 600,000 annually. During infection, haploid yeast in the host lung disseminate to the organs and are translocated to the central nervous system and brain, where they proliferate and cause cryptococcal meningitis. Successful clearance required long term antifungal therapy (>1 year total treatment time) and is associated with toxicity for the patient and drug resistance on the part of the fungus. One major mechanism of azole resistance in C. neoformans is aneuploidy- changes in copy number of drug efflux pump genes via disomy (duplication) of their respective chromosomes. The capacity of C. neoformans specifically, and fungi in general, to generate and tolerate aneuploidy (a phenomenon known as genome plasticity) is a major unanswered research question with implications for therapeutic regimens and human health.
We have previously demonstrated that genome plasticity in C. neoformans is mediated in part by the Rac/Nox complex. Cells deficient in Nox proteins have altered ploidy compared to wild-type cells, and Rac proteins can play a regulatory role in genome maintanence. We have shown that the C. neoformans Rac/Nox complex is responsible for the generation of endogenous Reactive Oxygen Species (ROS) within the fungal cell. This project will extend this investigation by measuring genome plasticity in racA/noxA deficient strains; probing the role of the Rac/Nox complex in nuclear dynamics and chromosome segregation, for example using a LifeAct-mCherry fluorescent reported for actin dynamics; and investigating the interaction of racA/noxA strains with host immune cells. Using these approaches, the student will test hypotheses regarding the role of the Rac/Nox complex in genome plasticity, drug resistance, and fungal pathogenesis. The project will provide training in MIC testing, flow cytometric approaches to fungal cell analysis, live cell imaging including epi-fluorescent and confocal approaches, and fungal-mammalian tissue culture techniques.

Project 2
Interaction of CGD murine bone marrow-derived macrophages with A. fumigatus and A. nidulans
Chronic granulomatous disease (CGD) is an inherited immune deficiency condition characterised by an NADPH disorder. Patients often encounter recurring episodes of bacterial and/or fungal infections, which are often life-threatening. Normally a rare pathogen, A. nidulans displays an anomalous affinity for the CGD host; invasive aspergillosis episodes involving A. nidulans are relatively more aggressive and often lethal.
We have developed a well-characterised experimental murine model of invasive aspergillosis in CGD mice and are now aiming to look at specific antifungal immune responses of various subsets of cell types to further unravel this particular host-fungus interaction.
In this project, we will investigate specifically the immune response of healthy (wild-type) and CGD (gp91-/-) murine bone-marrow derived macrophages (BMDM0s) to the two most common Aspergillus strains encountered in CGD: A. nidulans and A. fumigatus. Bone marrow will be harvested from murine femurs and cultured in L292-treated media for 7-11 days to obtain macrophages. The macrophages will be stimulated with A. fumigatus or A. nidulans conidia in certain cell:conidia ratios and for defined periods of time. Antifungal killing will be assessed by using the XTT-assay (a metabolic assay) and confocal microscopy for live-cell imaging will be performed by using the labelled Aspergillus conidia (incl. fluorescent Aspergillus reporter conidia). Cytokine production will be measured by using commercial ELISA's. In addition, we will assess the effect of priming, pretreatment of the macrophages w IFNy