Pleiotropic roles of LAMMER kinase, Lkh1 in stress responses and virulence of Cryptococcus neoformans

To investigate the pleiotropic roles of Lkh1 in stress response and virulence, we constructed lkh1? mutant strains. In this study, we found that Tor1 was an upstream regulator of Lkh1 in C. neoformans. We then performed gene expression profiling analysis to elucidate signaling circuitry downstream of CnLkh1 in the TOR1-Lkh1 pathway using data obtained from RNA seq of 2 different strains (WT of lkh1? mutant) with or without rapamycin treatment. Overall design: To investigate downstream genes regulated by TOR1-Lkh1 pathway, total RNA was isolated from wild-type (H99) and lkh1? mutant. Strains were grown in 30ml in a liquid YPD medium for 16hr at 30?. Then, overnight culture was inoculated into 100 ml fresh YPD medium and adjusted to OD600=0.2. The cells were further incubated until OD600 reached approximately 0.6. 50 ml of cells were harvested for the zero-time sample and rapamycin (3 ng/ml) were treated to the remaining cells. After rapamycin treatment, cells were further incubated at 30? for 60 mins.