Unravelling of the novel melanin biosynthesis-signalling networks in Cryptococcus neoformans

Melanin is an antioxidant polyphenol pigment, which is required for the pathogenicity of many human and plant fungal pathogens. Nevertheless, its comprehensive regulatory mechanism still remain elusive. In this study, we systematically analyzed melanin-regulating signaling pathways in Cryptococcus neoformans. Here we identified four core melanin-regulating TFs, Bzp4, Usv101, Mbs1, and Hob1, which are required for induction of the laccase gene (LAC1). Bzp4, Usv101 and Mbs1 independently regulate LAC1 induction while Hob1 controls Bzp4 and Usv101 expression. Both Bzp4 and Usv101 are translocated from the cytoplasm into the nucleus upon nutrient starved condition, while Mbs1 is constitutively localized in the nucleus. Notably, the cAMP pathway is not involved in regulation of the four TFs, while the HOG pathway negatively regulates induction of BZP4 and LAC1. As potential kinases upstream of the core TFs, we identified the nine core kinases, whose deletion leads to defective melanin production and LAC1 induction. Deletion of GSK3 or KIC1 abolishes induction of LAC1 and BZP4, and also perturbs nuclear translocation of Bzp4. Notably, Gsk3 also regulates the expression of HOB1, USV101, and MBS1, indicating that it is a critical melanin-regulating kinase. Finally, the RNA-sequencing based transcriptome analysis of the wild-type and bzp4?, usv101?, hob1?, and mbs1? strains under nutrient rich and starved conditions reveals that the core melanin-regulating TFs govern redundant and distinct classes of genes, which are involved in a variety of biological processes. Overall design: SET1 (WT and bzp4? mutant) and SET2 (WT and hob1, mbs1, usv101 mutants) strains were inoculated into 50 ml of YPD liquid medium and cultured overnight at 30°C in a shaking incubator. Cells were subcultured in 80 ml fresh YPD medium until OD600 indicated 0.6 to 0.8. 40 ml of the culture was placed in a liquid nitrogen tank as a basal sample (nutrient-rich samples) and remaining 40 ml was spun down, washed three times with phosphate-buffered saline, and resuspended in 40 ml of YNB medium containing ammonium sulfate without glucose. After resuspension, cells were further incubated at 30°C in a shaking incubator for 2 h (nutrient-starved samples). Incubated cells were spun down, frozen in liquid nitrogen and lyophilized overnight to extract the total RNA. R1, R2, R3 indicated the biological replicates.