Mitochondrial Morphology, Oxidative Stress Resistance, and Pathogenesis in Cryptococcus neoformans

Cryptococcus neoformans is an important pathogen that annually kills 200,000 people worldwide. It survives in the environment as a yeast or spore and can also proliferate within host macrophages after being inhaled into the lungs. In conditions of immunocompromise, cryptococcal cells can escape from the lungs to the brain, where they cause a deadly meningoencephalitis that is both difficult and expensive to treat. Cryptococcal adaptation to the harsh lung environment is a critical first step in its pathogenesis, and consequently a compelling topic of study. This adaptation is mediated by a complex transcriptional program that integrates cellular responses to environmental stimuli. Although several key regulators in this process have been examined, one important protein complex that modulates transcription yet remains understudied in C. neoformans is the Mediator complex. In other organisms, this complex promotes transcription of specific genes by increasing the assembly of the RNA Polymerase II pre-initiation complex. We have focused on the Kinase Module of the Mediator complex, which consists of cyclin C (Ssn801), cyclin-dependent kinase 8 (Cdk8), and two other proteins. This module provides important inhibitory control of Mediator complex assembly and activity. The Kinase Module has also been found to directly enhance mitochondrial fission in model organisms via protein-protein interactions. Mitochondria are essential organelles that normally reside as a complex reticular network that undergoes fusion and fission, mediated by dynamin-related proteins (DRPs). Mitochondrial morphology has been implicated in virulence in multiple fungi, as with the association between virulence and increased mitochondrial fusion in Cryptococcus gattii. We first used gene deletion of key DRPs to study their role in mitochondrial morphology and perform an in-depth study of the pathogenesis of these deletion strains. We then probed the function of the Kinase Module in C. neoformans. We discovered that it does not directly regulate mitochondrial morphology in C. neoformans. However, we discovered that Cdk8 and Ssn801 do regulate cryptococcal functions relevant to pathogenesis, including the response to oxidative stress, at the transcriptional level. For example, deletion of CDK8 resulted in the dysregulation of genes involved in oxidation-reduction processes, such that this strain exhibited increased susceptibility to oxidative stress. This resulted in an inability of mutant cells to proliferate within phagocytes, decreased lung burdens, and attenuated virulence in in vivo studies. These findings increase our understanding of cryptococcal adaptation to the host environment and its regulation of oxidative stress resistance and virulence