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UBC Theses and Dissertations

Analysis of the roles of a monothiol glutaredoxin and glutathione synthetase in the virulence of the AIDS-associated pathogen Cryptococcus neoformans Attarian, Rodgoun


Cryptococcus neoformans is a fungal pathogen and the causative agent of meningoencephalitis in immunocompromised and AIDS patients. Iron acquisition and the maintenance of iron homeostasis in C. neoformans are important aspects of virulence. Here, the monothiol glutaredoxin Grx4 was identified as a binding partner of Cir1, the master regulator of iron-responsive genes and virulence factor expression in C. neoformans. We confirmed that Grx4 binds Cir1 in vitro and in a yeast two-hybrid assay. RNA-Seq performed on the grx4 and cir1 mutants, and the WT strain, under low or high iron conditions identified genes involved in iron metabolism and expanded the concept that Grx4 plays a central role in iron homeostasis. A grx4 mutant, with deletion of the glutaredoxin domain, displayed iron-related phenotypes similar to those of a cir1 mutant, including elevated activity for cell surface reductases, sensitivity to high iron levels and increased susceptibility to phleomycin. Importantly it was shown that a grx4 mutant was avirulent in a mouse model of infection. We also observed that after 48 hour of starvation grx4 mutant had a growth defect in an iron-supplemented media. Interestingly, the growth defect of the grx4 mutant was rescued by exogenous GSH. We therefore further investigated the role of GSH in C. neoformans. Deletion of the GSH2 gene encoding the second enzyme in GSH biosynthesis resulted in a significant decrease in intracellular GSH levels in gsh2 mutants compared to the WT. This also resulted in loss of elaboration of major virulence factors including production of capsule and melanin, growth at host body temperature, and enhanced susceptibility to antifungal drugs. In conclusion GSH is required for expression of major virulence factors and defense against iron starvation-induced stress. GSH also influenced cell wall integrity. These results indicate that GSH plays a major role in the virulence of C. neoformans, and suggest a crosstalk between Grx4 and GSH as components of iron homeostasis and virulence expression. Overall, our findings provide further understanding of the regulation of virulence in C. neoformans and suggest novel drug targets for anticryptococcal therapy.

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