A Link between Virulence and Homeostatic Responses to Hypoxia during Infection by the Human Fungal Pathogen Cryptococcus neoformans

Abstract

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.

Author Summary

Opportunistic environmental pathogens adapt to hostile conditions within the host to cause disease. We describe two pathways in the pathogenic fungus Cryptococcus neoformans that are both necessary for adaptation to hypoxia and required for its virulence. One pathway uses a pathway homologous to the mammalian sterol-response element binding protein (SREBP) pathway to activate genes involved in sterol biosynthesis in response to low oxygen levels, while the other pathway involves the two-component hybrid histidine kinase protein Tco1. Mutant strains containing deletions of genes encoding components in either of these pathways were found to be less virulent in experimental mouse models. This study suggests that this pathogenic fungus experiences low levels of oxygen in the mammalian host, and that adaptation to these conditions is important for infection. Targeting components of the hypoxia response could yield more effective treatments for C. neoformans infections, which cause a large fraction of HIV/AIDS-related deaths worldwide. Notably, we find that mutants in the SREBP-like pathway are a hundred times more sensitive than wild-type cells to the widely used antifungal drug fluconazole.