Quantitative analysis of septin Cdc10 & Cdc3-associated proteome during stress response in the fungal pathogen...

Cryptococcus neoformans is a pathogenic basidiomycetous yeast that primarily infects immunocompromised individuals. Fatal outcome of cryptococcosis depends on the ability of C. neoformans to sense and adapt to 37°C. A complex of conserved filament forming GTPases, called septins, composed of Cdc3, Cdc10, Cdc11, and Cdc12, assembles at the mother-bud neck in C. neoformans. Septins Cdc3 and Cdc12 are essential for proliferation of C. neoformans at 37°C and for virulence in the Galleria mellonella model of infection, presumably due to their requirement for septin complex formation, and the involvement in cytokinesis. However, how exactly Cdc3, and Cdc12 contribute to C. neoformans growth at 37°C remains unknown. Based on studies investigating roles of septins in Saccharomyces cerevisiae, septin complex at the mother-bud neck of C. neoformans is predicted to interact with proteins involved in cell cycle control, morphogenesis, and cytokinesis, but the septin-associated proteome in C. neoformans has not been investigated. Here, we utilized tandem mass spectrometry to define C. neoformans proteins that associate with either Cdc3 or Cdc10 at ∼25°C or after the shift to 37°C. Our findings unveil a diverse array of septin-associated proteins, highlighting potential roles of septins in cell division, and stress response. Two proteins, identified as associated with both Cdc3 and Cdc10, the actin-binding protein profilin, which was detected at both temperatures, and ATP-binding multi-drug transporter Afr1, which was detected exclusively at 37°C, were further confirmed by co-immunoprecipitation. We also confirmed that association of Cdc3 with Afr1 was enhanced at 37°C. Upon shift to 37°C, septins Cdc3 and Cdc10 exhibited altered localization and Cdc3 partially co-localized with Afr1. In addition, we also investigated changes to levels of individual C. neoformans proteins upon shift from ∼25 to 37°C in exponentially grown culture and when cells entered stationary phase at ∼25°C. Our study reveals changes to C. neoformans proteome associated with heat and nutrient deprivation stresses and provides a landscape of septin-associated C. neoformans proteome, which will facilitate elucidating the biology of septins and mechanisms of stress response in this fungal pathogen.