EastBio: Targeting proteotoxic stress and aneuploidy in Crytococcus neoformans

Cryptococcus neoformans is an important human fungal pathogen with a fascinating life cycle, forming polyploid titan cells during infection in a bid to evade the immune system. It kills hundreds of thousands of immune-compromised AIDS patients each year, mainly in Sub-Saharan Africa.

We study mitosis and chromosome segregation, with a particular focus on the spindle assembly checkpoint (SAC) and error correction pathways. The SAC monitors interactions between chromosomes and the mitotic spindle and delays mitotic progression until all pairs of sister chromatids are attached appropriately to spindle microtubules. The SAC provides time for the error-correction machinery (Aurora B and the chromosomal passenger complex) to resolve inappropriate kinetochore-microtubule attachments.

We have found that Mps1 overexpression is sufficient to activate the Cryptococcus SAC, to recruit Bub1 to kinetochores and to arrest cells in metaphase through inhibition of Cdc20-APC/C [https://doi.org/10.1371/journal.pgen.1011302]. Cryptococcus Bub1 acts as a central SAC scaffold, interacting with the whole signalling pathway from kinetochores (via Spc105) to the APC/C via Cdc20 [https://doi.org/10.1101/2022.09.21.508923].

Whilst the SAC is not essential in Cryptococcus, we have found that deletion strains of the central SAC kinases, Mps1 and Bub1, are both aneuploid. Aneuploidy (an incorrect number of chromosomes) typically leads to proteotoxic stress and we have found that the ubiquitin system and the proteosome are both overexpressed in mps1 and bub1 deletion strains. Presumably they are actively degrading excess proteins to reduce/buffer proteotoxic stress in these aneuploid mutants. Our working hypothesis is that these, and other, aneuploid strains will be sensitised to proteasome inhibition. Chromosome 1 disomy is frequently found in drug-resistant clinical isolates of Cryptococcus neoformans.

Aim 1: you will purify, through tandem affinity purification, the proteosome from wild-type and aneuploid strains of Cryptococcus neoformans. The purified complex will be analysed by mass-spectrometry and cryo-electron microscopy.

Aim 2: you will screen for novel inhibitors of the Cryptococcus neoformans proteosome, in the Drug Discovery Unit of the University of Dundee.

Aim 3: you will test whether proteasome inhibitors act effectively in combination therapy, alongside inhibitors of mitotic kinases such as Mps1.

In this way we hope to generate novel drugs for treatment of Cryptococcus infections.