A cysteine-rich domain of the <i>Cryptococcus neoforman</i> s Cuf1 transcription factor is required for high copper stress sensing and fungal virulence

ABSTRACT 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 1st 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 the containment of Cn cells at the initial site of infection within the lung. IMPORTANCE Copper is an essential micronutrient required for survival in all kingdoms of life, as it is used as a catalytic cofactor for many essential processes in the cell. In turn, this reactivity of copper ions makes elevated levels of free copper toxic to the cell. This dual nature of copper—essential for life but toxic at elevated levels—is used by our innate immune system in a process called nutritional immunity to combat and kill invading pathogens. In this work, we explore how the fungal human pathogen Cryptococcus neoformans senses high copper stress, a copper microenvironment encountered within the host lung. We identified a specific cysteine-rich motif within the copper-responsive transcription factor Cuf1 to be essential for high copper stress sensing. Mutation of this motif led to an impaired high copper stress adaptation, which did not affect the fitness of the yeast but did impact the containment and distribution of yeast cells inside the host lung.