Characterization of Cryptococcus neoformans PDK1 ortholog PKH2-02

Currently, antifungal therapy for C. neoformans is very limited and can be problematic in developing countries.  Recent chemical genetics-guided mechanistic studies in our laboratory demonstrated that mammalian PDK1 inhibitors also target fungal PDK1 orthologs as part of their mode of action, revealing possible new drug targets.  In C. neoformans PKH2-01 and PKH2-02 are orthologous to mammalian PDK1 kinases.  These protein kinases have been well characterized in S. cerevisiae, but not in pathogenic yeast.  Here, we characterize the role of the C. neoformans protein kinase PKH2-02 in its ability to tolerate cell wall and oxidative stress, and its role in the host-pathogen interaction.  Our data indicated that pkh2-02Δ grows slower at 30°C and is temperature sensitive at 37°C.  Phenotypic characterization of pkh2-02Δ demonstrated that it is very sensitive to cell wall stressors caffeine, calcofluor white and Congo Red.  To determine if C. neoformans PKH2-02 plays a role in the Pkc1-MAPK pathway the cellular Pkc1 activity and activation of MPK1were assayed. The level of Pkc1 activity and MPK1 phosphorylation were diminished in pkh2-02Δ compared to WT.  To determine if pkh2-02Δ had virulence effects separate from the high temperature growth defect at 37°C, we assayed its virulence using the Galleria mellonella model at 30^oC and found it is avirulent. In addition, pkh2-02Δ is not able to neutralize ROS, survive within macrophages and it induces higher levels of the pro-inflammatory cytokine TNFα. Taken all together this data suggests that C. neoformans PKH2-02 mediated signaling is important to stress tolerance and host-pathogen interactions.