The Casein Kinase I Protein Cckl Regulates Multiple Signaling Pathways And Is Essential For Cell Integrity And Fungal Virulence In Cryptococcus Neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen most commonly observed in immunocompromised individuals. C.neoformans spores or desiccated yeast cells are inhaled into the lungs and lodge in the alveoli. In immunocompromised patients, C.neoformans is able to disseminate from the lungs to cause a systemic infection. At late stages of infection, C.neoformans penetrates the blood brain barrier (BBB) and infects the central nervous system (CNS) to cause meningitis.Besides its medical importance, C. neoformans has also been developed as a model organism to study fungal genetics and pathogenesis; it is a fast-growing haploid yeast with a defined sexual cycle and the availability of robust animal models to study its virulence. As an important pathogen with genetic tractability, extensive studies have been conducted in C. neoformans.Although signaling pathways that are important for fungal sexual reproduction and virulence have also been identified and extensively studied. These studies revealed that fungal virulence is a complex trait and virulence determining mechanisms remain to be explored.Casein kinases regulate a wide range of cellular function in eukaryotes, including phosphorylation of proteins that are substrates for degradation via the ubiquitin-proteasome system (UPS). Our previous study demonstrated that Fbpl, a component of the SCFFBP1E3ligase complex, was essential for Cryptococcus virulence. Because the Saccharomyces cerevisiae homolog of Fbpl, Grrl, requires casein kinase I (Yckl and Yck2) to phosphorylate its substrates, we investigated the function of casein kinase I in C. neoformans. In this report, we identified a C. neoformans casein kinase I protein homolog Cckl. Similar to Fbpl, the expression of Cckl is negatively regulated by glucose and during mating, cckl null mutants showed significant virulence attenuation in a murine systemic infection model, but Cckl was dispensable for the development of classical virulence factors (capsule, melanin, and growth at37℃). cckl mutants were hypersensitive to SDS treatment indicating that Cckl is required for cell integrity. The functional overlap between Cckl and Fbpl suggests that Cckl may be required for the phosphorylation of Fbpl substrates. Interestingly, the cckl mutant also showed increased sensitivity to osmotic stress and oxidative stress, suggesting Cckl regulates both cell integrity and the cellular stress response. Our results show that Cck1regulates the phosphorylation of both Mpkl and Hog1mitogen-activated protein kinases (MAPKs), demonstrating that Cck1regulates cell integrity via the Mpkl pathway, and regulates the cell adaptation to stresses via the Hogl pathway. Overall, our study revealed that Cckl plays important roles in regulating multiple signaling pathways and is required for fungal pathogenicity.