Cryptococcus gattii is a resurgent fungal pathogen that primarily infects immunocompetent hosts. Thus, it poses an increasingly significant impact on global public health; however, the mechanisms underlying its pathogenesis remain largely unknown. We conducted a detailed characterization of the deubiquitinase Ubp5 in the biology and virulence of C. gattii using the hypervirulent strain R265, and defined its properties as either distinctive or shared with C. neoformans. Deletion of the C. gattii Ubp5 protein by site-directed disruption resulted in a severe growth defect under both normal and stressful conditions (such as high temperature, high salt, cell wall damaging agents, and antifungal agents), similar to the effects observed in C. neoformans. However, unlike C. neoformans, the C. gattii ubp5Δ mutant displayed a slight enhancement of capsule and melanin production, indicating the evolutionary convergence and divergence of Ubp5 between these two sibling species. Attenuated virulence of the Cg-ubp5Δ mutant was not solely due to its reduced thermotolerance at 37°C, as shown in both worm and mouse survival assays. In addition, the assessment of fungal burden in mammalian organs further indicated that Ubp5 was required for C. gattii pulmonary survival and, consequently, extrapulmonary dissemination. Taken together, our work highlights the importance of deubiquitinase Ubp5 in the virulence composite of both pathogenic cryptococcal species, and it facilitates a better understanding of C. gattii virulence mechanisms.

Ubiquitin is a highly conserved protein required for various fundamental cellular processes in eukaryotes. Herein, we first report the contribution of the ubiquitin fusion protein Ubi1 (a ubiquitin monomer fused with the ribosome protein L40e, Rpl40e) in the growth and pathogenicity of Cryptococcus neoformans. UBI1 deletion resulted in severe growth restriction of C. neoformans, whose growth rate was positively correlated with UBI1 expression level. The growth defect of the ubi1 strain could be closely associated with its morphological abnormalities, such as its reduced ribosome particles. In addition, the ubi1 mutant also displayed increased cell ploidy, cell cycle arrest, and decreased intracellular survival inside macrophages. All these phenotypes were reversed by the reconstitution of the full-length UBI1 gene or RPL40a domain. Mouse survival and fungal burden assays further revealed a severely attenuated pathogenicity for the ubi1 mutant, which is probably associated with its reduced stress tolerance and the induction of T-helper 1-type immune response. Taken together, Ubi1 is required for maintaining the vegetative growth, morphological homeostasis, cell cycle progression, and pathogenicity in vivo of C. neoformans. The pleiotropic roles of Ubi1 are dependent on the presence of Rpl40e and associated with its regulation of cryptococcal ribosome biogenesis.

Cryptococcus neoformans is an important invasive fungal pathogen that causes life-threatening meningoencephalitis in humans. Its biological and pathogenic regulatory mechanisms remain largely unknown, particularly due to the presence of those core transcription factors (TFs). Here, we conducted a detailed characterization of the TF Liv4 in the biology and virulence of C. neoformans. Deletion of TF Liv4 protein resulted in growth defect under both normal and stress conditions (such as high temperature and cell wall/membrane damaging agents), drastic morphological damage and also attenuated virulence in C. neoformans. These phenotypic changes might be contributed to transcriptional abnormality in the liv4Δ mutant, in which several cryptococcal genes involved in energy metabolism and cell wall integrity were downregulated. Furthermore, ChIP-seq and ChIP-qPCR assays suggested TF Liv4 might exert its regulatory function in transcription by its activation of RBP1 in C. neoformans. Taken together, our work highlights the importance of TF Liv4 in the growth and virulence of C. neoformans, and it facilitates a better understanding of cryptococcal pathogenesis mechanisms.