A cysteine-rich domain of the Cryptococcus neoformans Cuf1 transcription factor is required for high copper stress sensing and fungal virulence

The ability to sense, import and detoxify copper (Cu) has been shown to be crucial for microbial pathogens to survive within an infected host. Previous studies conducted with the opportunistic human fungal pathogen Cryptococcus neoformans ( Cn ) have revealed two extreme Cu environments encountered during infection: a high Cu environment within the lung and a low Cu environment within the brain. However, how Cn senses these different host Cu microenvironments, and the consequences of a blunted Cu stress adaptation for pathogenesis are not well understood. In contrast to ascomycete model fungi, the basidiomycete Cn has a single transcription factor (TF), Cn Cuf1, to regulate adaptive responses to both high- and low- Cu stress. Sequence comparison with other fungal Cu-responsive TFs identified three conserved cysteine (Cys)-rich motifs located within the Cn Cuf1 N-terminal domain, which were therefore predicted to play a role in Cu sensing. Mutation of these conserved Cys-rich motifs demonstrated that the 1 ^st Cys-rich motif is functionally relevant for Cn Cuf1 transcriptional activity during high Cu stress, while it is dispensable for low Cu stress adaptation. An inhalation model of murine infection showed that strains with defective high Cu stress regulation present a distinct and anatomically constrained pattern of yeast distribution within the infected lungs compared to a more widespread infection observed in lungs infected with the wild-type strain. Based on these findings, we hypothesize that Cuf1-driven high Cu responses modulate not absolute fitness but containment of Cn cells at the initial site of infection within the lung.