Conservation of the Sterol Regulatory Element-Binding Protein Pathway and Its Pathobiological Importance in Cryptococcus neoformans

The mammalian sterol regulatory element-binding protein (SREBP) homolog, Sre1, is important for adaptation and growth of Cryptococcus neoformans in the mouse brain, where oxygen concentration and nutritional conditions are suboptimal for fungal growth. The extent of conservation of the SREBP pathway in C. neoformans or in any other fungi, however, has not been investigated. We generated mutants susceptible to low oxygen and identified six genes that play a role in the SREBP pathway. Three of these genes (SFB2, KAP123, and GSK3) are not known to be involved in the SREBP pathway in other fungi. Furthermore, we show that C. neoformans contains an additional gene, DAM1, which functions in the SREBP pathway but is yet to be described. Mutants associated with the steps prior to formation of the nuclear Sre1 form dramatically reduced accumulation of the nuclear form under low-oxygen conditions. Concurrently, two mutant strains, scp1Δ and stp1Δ, and the previously isolated sre1Δ strain showed reduction in ergosterol levels, hypersensitivity to several chemical agents, including azole antifungals, CoCl₂, and compounds producing reactive oxygen or nitrogen species, and most importantly, reduced virulence in mice. Mutants affecting genes involved in later steps of the Sre1 pathway, such as those required for import and phosphorylation of proteins in the nucleus, showed less compelling phenotypes. These findings suggest that the SREBP pathway is highly conserved in C. neoformans and it serves as an important link between sterol biosynthesis, oxygen sensing, CoCl₂ sensitivity, and virulence in C. neoformans.Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningoencephalitis, primarily in immunocompromised patients (22). Cryptococcosis is initiated by inhalation of airborne C. neoformans cells, which spread to the central nervous system by hematogenous dissemination. Upon entry into the brain, the sterol regulatory element-binding protein (SREBP) homolog, Sre1, is required for cells to adapt to the host environment and cause fulminating meningoencephalitis (3, 5). Mutations in SRE1 and the gene encoding a homolog of the SREBP cleavage-activating protein (SCAP), SCP1, resulted in reduced growth under low-oxygen conditions in vitro. The sre1 mutant was either significantly reduced in virulence or unable to cause fatal CNS infection in mice, depending on the strain''s genetic background (3, 5).SREBPs, a family of membrane-bound transcription factors, are involved in the control of cholesterol and lipid metabolism in mammalian systems (7, 34), and they are known to directly enhance the transcription of more than 30 genes required in these processes (15). These unique transcription factors are themselves subject to positive and negative feedback regulation at the transcriptional, translational, and posttranslational levels (34). SREBPs are synthesized as inactive precursors with two transmembrane helices and reside in the endoplasmic reticulum (ER) membrane. The N-terminal domain of SREBP is a basic helix-loop-helix leucine zipper transcription factor. The C terminus forms a tight complex with the tryptophan-aspartate repeat (WD) domain of SCAP, which functions as a sensor for membrane cholesterol levels. In sterol-replete cells, SCAP binds to cholesterol in the ER membrane and assumes a conformation that promotes its binding to the ER-resident protein Insig (for insulin-induced gene) (33). In sterol-depleted cells, this binding to Insig is disrupted and SREBP-SCAP is sorted from the ER to Golgi complex via coat protein complex II-coated transport vesicles, which contain Sar1, Sec23, and Sec24 (8, 12, 35, 38). In the Golgi complex, the N-terminal transcription factor domain of SREBP is released from the membrane by two sequential proteolytic cleavage events mediated by the site 1 and site 2 proteases (S1P and S2P) (12). The released SREBP is transported into the nucleus as a dimer by importin β through interactions with the helix-loop-helix domain (24, 30). In the nucleus, SREBP executes its transcriptional regulatory function. Turnover of SREBP involves phosphorylation by Gsk3, whose activity is inhibited by insulin signaling (10, 21), followed by its binding to the E3 ubiquitin ligase SCF^Fbw7, which leads to rapid degradation of SREBP (39).In fungi, the SREBP pathway appears to be diverse among different species. For example, SREBP and SCAP homologs exist in C. neoformans and Schizosaccharomyces pombe but are absent in both Saccharomyces cerevisiae and Candida albicans, while Aspergillus fumigatus contains the SREBP but not SCAP homolog (3, 16, 43). Moreover, no gene other than homologs of SREBP, SCAP, and S2P has been identified to play a role in the fungal SREBP pathway (3, 5).We used a genetic approach to identify C. neoformans genes involved in adaptation to low-oxygen conditions by screening insertional mutants under low oxygen at 37°C. We found a class of mutants showing sensitivity to low oxygen due to mutations in the genes that are homologous to mammalian genes in the SREBP pathway. Most of these mutants showed sensitivity to CoCl₂, azole antifungals, and various reactive oxygen species (ROS)-generating chemicals. Interestingly, all but one of these mutants showed reduced virulence compared to the wild type in a murine model. We also identified the gene Dam1, which functions in the Sre1 pathway but had not been described previously. Our findings show that the SREBP pathway is well conserved in C. neoformans, and they underscore its importance in the pathobiology of C. neoformans.