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

The ability to sense, import but also detoxify copper (Cu) has been shown to be crucial for microbial pathogens to survive within the 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 adaption for pathogenesis, are not well understood. In contrast to other fungi, Cn has a single transcription factor, Cuf1, to regulate adaptive responses to both high- and low-Cu stress. Sequence analysis of Cn Cuf1 identified three conserved cysteine (Cys)-rich regions that may play a role in Cu sensing. We mutated the 1st Cys-rich region within the CUF1 gene to investigate its role for Cn high Cu stress sensing. Subsequent analysis of Cuf1 transcriptional activity and target gene promoter binding demonstrated that the 1st Cys-rich region is required for Cuf1 transcriptional activity in high Cu stress. We performed an inhalational murine infection to analyze the effects of a blunted high Cu stress response on pathogenesis. No significant differences in lung fungal burden were observed based on variable Cuf1 activity. However, strains with defective high Cu stress regulation induced a markedly altered immune response in mice. Based on these findings, we hypothesize that Cuf1-driven high Cu responses are not required for initial survival but instead modulate immune recognition and inflammation within the mouse lung. ImportanceCopper 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 for 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 region within the copper responsive transcription factor Cuf1 to be essential for high copper stress sensing. Mutation of this region led to an impaired high copper stress adaptation, which did not affect fitness of the yeast but did impact immune recognition and inflammation inside the host lung.