Chaperones: mediators of the switch from commensal to pathogen?

In the 1980s fragments of fungal heat shock protein 90s (Hsp90) were discovered in the blood of patients who survived systemic Candida infection (candidemia), but were absent from patients who succumbed to this illness. This suggests that fungal Hsp90 may be essential to the hosts ability to fight candidemia.
Candidemia is most often caused by the ascomycetous yeast species Candida albicans and Candida glabrata. Both are found commensally on humans and commonly cause ailments such as thrush and urinary tract infections. Healthy patients are able to control these infections; however in patients with compromised immunity Candida species can become opportunistic pathogens, causing serious conditions like candidemia.
Candidemia has a mortality rate of >50%, killing ~700 people and costing over £16million in treatments per year in the UK alone. One reason candidemia survival rates are low is because of increased resistance to anti-fungal drugs due to selection pressure from therapeutic use. This is aggravated by C. glabratas intrinsic resistance to the most common family of anti-fungal drugs.
Drug resistance and virulence in C. albicans depend upon functional Hsp90. Hsp90 is a chaperone that assists in correct protein folding and stabilises proteins during environmental stress. Although Hsp90 inhibitors are effective anti-fungal agents in vitro, in experimental settings they caused host toxicity. An attractive drug target would be an Hsp90-interacting molecule that is less conserved between species.
Previously, an Hsp90 interaction network in C. albicans and a cryo-electron microscopy (cryoEM) structure of a human Hsp90-kinase complex have been produced, however little is known about Hsp90 in C. glabrata. Therefore the broad aim of this PhD is to discover genetic interactions of Hsp90 in C. glabrata and characterise Hsp90s physical interactions with host proteins in C. albicans and C. glabrata. This will be achieved in three aims:
Firstly, a global map of genetic interactions of Hsp90 in C. glabrata will be constructed by screening knockout strains for lack of growth in response to Hsp90 inhibition. Using the concept of synthetic lethality, genes found to prohibit viability when knocked out are likely to be involved in the same pathway or complexes as Hsp90.
Secondly, a bipartite approach will be undertaken in order to identify human proteins which bind fungal Hsp90. A two-hybrid screen will use a human epithelial cell cDNA prey library to find protein-protein interactions. In complement with this, purified fungal Hsp90 will be added to human epithelial cell culture and complexes will be analysed by mass spectrometry. These protein complexes will be subjected to tomography and high resolution cryo-EM, with the aim of producing atomic-resolution structures.
Thirdly, to investigate which sites are essential for these protein interactions, CRISPR-cas9 genome editing will be utilised to modify the binding sites of Candida Hsp90.
Together, the results of these objectives will provide considerable new targets for rational design of novel anti-fungal drugs.

This project fits securely into the remit of the MRCs infection and immunity science area by providing possible new targets for anti-fungal drugs, elucidating potential resistance mechanisms and providing structural information about host-pathogen interactions.