Pleiotropic Effects Of Deubiquitinating Enzymes On Growth And Pathogenesis Of Cryptococcus Neoformans

Cryptococcus neoformans is an important human pathogen, which can causelife-threatening CNS infections primarily within the immunocompromised population.Due to its well-defined sexual cycle, robust animal models and molecular biology tools, C.neoformans has become an excellent model for the study of fungal pathogenesis.Previous studies have confirmed the fact that fungal virulence is a very complicatedcomposite. Identification of the essential regulatory mechanisms in control of thecryptococcal virulence composite and its networks should be helpful in understandingtargets to interrupt this yeast's ability to produce disease.Ubiquitination is a ubiquitously reversible protein modification in eukaryotic cells,which involved in various cellular processes such as cell cycle, signaling transduction,sexual reproduction, transcriptional regulation, and also stress response. Deubiquitinatingenzymes, the proteins responsible for the removal of ubiquitin, act as essential regulators tomaintain ubiquitin homeostasis and to exquisitely regulate protein degradation ormodification via the ubiquitination pathway.In order to understand the possible influence deubiquitinating enzymes have ongrowth and virulence of the model pathogenic yeast Cryptococcus neoformans, wegenerated deletion mutants of7putative deubiquitinase genes in this study. Compared towild-type strain and other deubiquitinating enzyme mutants, a ubp5mutant exhibitedseverely attenuated virulence and many distinct phenotypes, including slower growth rate,decreased capsule formation, hypomelanization, defective sporulation, and elevatedsensitivity to several external stressors (such as high temperature, oxidative and nitrosativestresses, high salts, and antifungal agents). The ubp5mutant produced capsule butmight have a defect in extracellular assembly of the capsule on the cell surface. Ubp5might regulate melanization by its influence on growth rate and copper ion metabolism.The sporulation defect of ubp5mutant occurred in the stage of cell fusion, which mightpartially be contributed to its down-regulation of α pheromone expression during mating.Ubp5appears to be the major deubiquitinating enzyme in C. neoformans for maintaining apool of free ubiquitin for stress responses. Deletion of Ubp5led to decrease of cellularfree ubiquitin in Cryptococcus and thus increase of its demand on polyubiquitin precursorUbi4. Meantime, down-regulation of polyubiquitin in wild-type strain contributed tosimilar phenotypic changes as in ubp5Δ mutant, such as slower growth rate, enhanced stress susceptibility, sporulation defect and attenuated virulence, which furtherdemonstrates that Ubp5may have a linear relationship with Ubi4and a distinct andirreplaceable role in the ubiquitin cycle compared to the other DUBs in C. neoformans.Furthermore, deletion of Ubp5altered the transcriptional patterns of Cryptococcus in both30°C and37°C. A group of genes involved in nutritional metabolism, stress response andseveral other biological processes such as DNA replication, transcriptional regulation andcell cycle, exhibited differential expression in the ubp5Δ mutant compared to WT strain.Deletions in either the UBP5or DOA4led to dramatic attenuation in virulence in bothmodels and there were no significant virulence differences between ubp5and doa4mutants in either model, although disruption of UBP5did yield more altered in vitrophenotypes under several conditions. And deubiquitinase Otu1had a negative regulationin cryptococcal virulence. Their regulatory mechanisms remained to be further explored.Besides, like ubp5, the doa4mutant also showed decreased resistance to high sodiumions,0.02%SDS and0.1%caffeine, and both doa4and ubp13mutants are moresensitive to fluconazole and caspofungin, demonstrating some functional overlap anddiversity of the deubiquitinating enzymes in C. neoformans.Our study highlights the crucial importance of deubiquitination in cryptococcalpathogenesis, and illustrates the evolutionary divergence of Cryptococcus spp. from themodel yeast S. cerevisiae, which provides an important paradigm for understanding thepotential role of deubiquitination in virulence by other pathogenic fungi such as Candidaalbicans and Aspergillus fumigatus. Indeed, inhibitors of some deubiquitinating enzymesincluding Usp7(Ubp5's homolog in human) are current viable targets for anticancertherapy.