UBC Theses and Dissertations
The roles of J domain co-chaperones in the fungal pathogen Cryptococcus neoformans Horianopoulos, Linda Catherine
The opportunistic fungal pathogen, Cryptococcus neoformans, must survive within a human host to cause disease. Stress responsive proteins such as heat shock proteins facilitate adaptation to the host environment mitigating proteotoxic stress induced by elevated temperature and the immune system. We undertook the characterization of J domain protein (JDP) co-chaperones with the prediction that JDPs would contribute to processes required for virulence. To this end, deletion mutants were generated for three type III JDPs, which lack glycine phenylalanine rich and zinc finger domains. The in vitro and in vivo phenotypes of these mutants were characterized to gain insights regarding their functions. These JDPs were tagged to interrogate their localization using fluorescent microscopy and identify binding partners using affinity purification-mass spectrometry. This approach allowed the characterization of Mrj1 as a mitochondrial JDP which supports mitochondrial respiration by facilitating electron flow through complexes III and IV of the electron transport chain. Mitochondrial function supported by Mrj1 also contributed to maintenance of cell wall architecture and elaboration of capsule. In a mouse model of cryptococcosis, mutants lacking MRJ1 were avirulent. The ER co-chaperone, Dnj1, was identified as a JDP which facilitates the elaboration and secretion of virulence factors at host temperature. Accordingly, a mutant lacking DNJ1 proliferated more slowly compared with the wild type in the lungs and had decreased dissemination to the brain in a mouse model of cryptococcosis. Dnj4 was characterized as a nuclear co-chaperone which interacts with histones 3 and 4. Deletion mutants lacking DNJ4 were hypersensitive to the DNA damaging agent hydroxyurea, prompting characterization of the transcriptional response to hydroxyurea in the wild type and mutant. This analysis revealed a role for Dnj4 in up-regulation of DNA repair genes in response to DNA damage. Mutants lacking DNJ4 were also impaired in the in vitro elaboration of key virulence factors but had no defect in virulence in a mouse model of cryptococcosis. Taken together, this study of JDPs demonstrates that co-chaperones contribute to distinct biological processes in C. neoformans. Importantly, Mrj1 and Dnj1, which have divergent amino acid sequences from human JDPs, represent promising targets for antifungal drug development.
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