The F-Box Protein Fbp1 Regulates Sexual Reproduction and Virulence in Cryptococcus neoformans

Abstract

Cryptococcus neoformans is the leading cause of fungal meningitis in immunocomprised populations. Although extensive studies have been conducted on signal transduction pathways important for fungal sexual reproduction and virulence, how fungal virulence is regulated during infection is still not understood. In this study, we identified the F-box protein Fbp1, which contains a putative F-box domain and 12 leucine-rich repeats (LRR). Although fbp1 mutants showed normal growth and produced normal major virulence factors, such as melanin and capsule, Fbp1 was found to be essential for fungal virulence, as fbp1 mutants were avirulent in a murine systemic-infection model. Fbp1 is also important for fungal sexual reproduction. Basidiospore production was blocked in bilateral mating between fbp1 mutants, even though normal dikaryotic hyphae were observed during mating. In vitro assays of stress responses revealed that fbp1 mutants are hypersensitive to SDS, but not calcofluor white (CFW) or Congo red, indicating that Fbp1 may regulate cell membrane integrity. Fbp1 physically interacts with Skp1 homologues in both Saccharomyces cerevisiae and C. neoformans via its F-box domain, suggesting it may function as part of an SCF (Skp1, Cullins, F-box proteins) E3 ligase. Overall, our study revealed that the F-box protein Fbp1 is essential for fungal sporulation and virulence in C. neoformans, which likely represents a conserved novel virulence control mechanism that involves the SCF E3 ubiquitin ligase-mediated proteolysis pathway.

The Casein Kinase I Protein Cck1 Regulates Multiple Signaling Pathways and Is Essential for Cell Integrity and Fungal Virulence in Cryptococcus neoformans

“…Our previous study has demonstrated that the expression of FBP1 was negatively regulated by glucose (21). Therefore, we checked the expression of Cck1 under glucose induction or repression conditions using qRT-PCR.…”

“…Assays for melanin and capsule production were performed as previously described (21). In brief, melanin production was tested on niger seed agar and incubated at 30°C or 37°C for 2 days, and pigmentation of fungal colonies was assessed and photographed.…”

“…Plates were incubated at both 30°C and 37°C for 2 days and photographed. Trypan blue staining was performed as previously described (11,21). The percentages of cells able to take up dye were calculated by counting at least 500 yeast cells for each strain.…”

“…Fungal SCF complexes have been reported to regulate a variety of cellular functions (17). Our previous studies identified an Fbox protein, Fbp1, that is essential for fungal virulence despite its dispensability for the development of several classical virulence factors, including the production of melanin, capsule formation, and growth at 37°C (21). We hypothesize that Fbp1 may be part of a novel virulence mechanism and could have potential as a drug target.…”

“…Two casein kinase I proteins in S. cerevisiae, Yck1 and Yck2, play important roles in the phosphorylation of the Grr1 substrates that are involved in glucose sensing (Mth1 and Std1) (27), amino acid signaling (Stp1, Stp2, and Ptr3) (22,36), pheromone sensing (Ste2) (14), and uracil uptake (Fur4) (24,25). Fbp1 is a homolog of Grr1, and its expression is also regulated by glucose (21). Therefore, we predict that a similar substrate phosphorylation mechanism exists to activate Fbp1-mediated protein turnover and that the casein kinase I family in C. neoformans is involved in the phosphorylation of Fbp1 substrates for degradation.…”