Sre1p, a regulator of oxygen sensing and sterol homeostasis, is required for virulence in Cryptococcus neoformans

Cryptococcus neoformans is an environmental pathogen requiring atmospheric levels of oxygen for optimal growth. Upon inhalation, C. neoformans disseminates to the brain and causes meningoencephalitis, but the mechanisms by which the pathogen adapts to the low-oxygen environment in the brain have not been investigated. We found that SRE1, a homologue of the mammalian sterol regulatory element-binding protein (SREBP), functions in an oxygen-sensing pathway. Low oxygen decreased sterol synthesis in C. neoformans and triggered activation of membrane-bound Sre1p by the cleavage-activating protein, Scp1p. Microarray and Northern blot analysis demonstrated that under low oxygen, Sre1p activates genes required for ergosterol biosynthesis and iron uptake. Consistent with these regulatory functions, sre1Delta cells were hypersensitive to azole drugs and failed to grow under iron-limiting conditions. Importantly, sre1Delta cells failed to produce fulminating brain infection in mice. Our in vitro data support a model in which Sre1p is activated under low oxygen leading to the upregulation of genes required for sterol biosynthesis and growth in a nutrient-limiting environment. Animal studies confirm the importance of SRE1 for C. neoformans to adapt to the host environment and to cause fatal meningoencephalitis, thereby identifying the SREBP pathway as a therapeutic target for cryptococcosis.     

4-Methyl sterols regulate fission yeast SREBP-Scap under low oxygen and cell stress

In fission yeast, orthologs of mammalian SREBP and Scap, called Sre1 and Scp1, monitor oxygen-dependent sterol synthesis as a measure of cellular oxygen supply. Under low oxygen conditions, sterol synthesis is inhibited, and Sre1 cleavage is activated. However, the sterol signal for Sre1 activation is unknown. In this study, we characterized the sterol signal for Sre1 activation using a combination of Sre1 cleavage assays and gas chromatography sterol analysis. We find that Sre1 activation is regulated by levels of the 4-methyl sterols 24-methylene lanosterol and 4,4-dimethylfecosterol under conditions of low oxygen and cell stress. Both increases and decreases in the level of these ergosterol pathway intermediates induce Sre1 proteolysis in a Scp1-dependent manner. The SREBP ortholog in the pathogenic fungus Cryptococcus neoformans is also activated by high levels of 4-methyl sterols, suggesting that this signal for SREBP activation is conserved among unicellular eukaryotes. Finally, we provide evidence that the sterol-sensing domain of Scp1 is important for regulating Sre1 proteolysis. The conserved mutations Y247C, L264F, and D392N in Scp1 that render Scap insensitive to sterols cause constitutive Sre1 activation. These findings indicate that unlike Scap, fission yeast Scp1 responds to 4-methyl sterols and thus shares properties with mammalian HMG-CoA reductase, a sterol-sensing domain protein whose degradation is regulated by the 4-methyl sterol lanosterol.     

A link between virulence and homeostatic responses to hypoxia during infection by the human fungal pathogen Cryptococcus neoformans

Fungal pathogens of humans require molecular oxygen for several essential biochemical reactions, yet virtually nothing is known about how they adapt to the relatively hypoxic environment of infected tissues. We isolated mutants defective in growth under hypoxic conditions, but normal for growth in normoxic conditions, in Cryptococcus neoformans, the most common cause of fungal meningitis. Two regulatory pathways were identified: one homologous to the mammalian sterol-response element binding protein (SREBP) cholesterol biosynthesis regulatory pathway, and the other a two-component-like pathway involving a fungal-specific hybrid histidine kinase family member, Tco1. We show that cleavage of the SREBP precursor homolog Sre1-which is predicted to release its DNA-binding domain from the membrane-occurs in response to hypoxia, and that Sre1 is required for hypoxic induction of genes encoding for oxygen-dependent enzymes involved in ergosterol synthesis. Importantly, mutants in either the SREBP pathway or the Tco1 pathway display defects in their ability to proliferate in host tissues and to cause disease in infected mice, linking for the first time to our knowledge hypoxic adaptation and pathogenesis by a eukaryotic aerobe. SREBP pathway mutants were found to be a hundred times more sensitive than wild-type to fluconazole, a widely used antifungal agent that inhibits ergosterol synthesis, suggesting that inhibitors of SREBP processing could substantially enhance the potency of current therapies.