Cryptococcus neoformans Iron-Sulfur Protein Biogenesis Machinery Is a Novel Layer of Protection against Cu Stress

Copper (Cu) ions serve as catalytic cofactors to drive key biochemical processes, and yet Cu levels that exceed cellular homeostatic control capacity are toxic. The underlying mechanisms for Cu toxicity are poorly understood. During pulmonary infection by the fungal pathogen Cryptococcus neoformans, host alveolar macrophages compartmentalize Cu to the phagosome, and the ability to detoxify Cu is critical for its survival and virulence. Here, we report that iron-sulfur (Fe-S) clusters are critical targets of Cu toxicity in both Saccharomyces cerevisiae and C. neoformans in a manner that depends on the accessibility of Cu to the Fe-S cofactor. To respond to this Cu-dependent Fe-S stress, C. neoformans induces the transcription of mitochondrial ABC transporter Atm1, which functions in cytosolic-nuclear Fe-S protein biogenesis in response to Cu and in a manner dependent on the Cu metalloregulatory transcription factor Cuf1. As Atm1 functions in exporting an Fe-S precursor from the mitochondrial matrix to the cytosol, C. neoformans cells depleted for Atm1 are sensitive to Cu even while the Cu-detoxifying metallothionein proteins are highly expressed. We provide evidence for a previously unrecognized microbial defense mechanism to deal with Cu toxicity, and we highlight the importance for C. neoformans of having several distinct mechanisms for coping with Cu toxicity which together could contribute to the success of this microbe as an opportunistic human fungal pathogen.IMPORTANCEC. neoformans is an opportunistic pathogen that causes lethal meningitis in over 650,000 people annually. The severity of C. neoformans infections is further compounded by the use of toxic or poorly effective systemic antifungal agents as well as by the difficulty of diagnosis. Cu is a natural potent antimicrobial agent that is compartmentalized within the macrophage phagosome and used by innate immune cells to neutralize microbial pathogens. While the Cu detoxification machinery of C. neoformans is essential for virulence, little is known about the mechanisms by which Cu kills fungi. Here we report that Fe-S cluster-containing proteins, including members of the Fe-S protein biogenesis machinery itself, are critical targets of Cu toxicity and therefore that this biosynthetic process provides an important layer of defense against high Cu levels. Given the role of Cu ionophores as antimicrobials, understanding how Cu is toxic to microorganisms could lead to the development of effective, broad-spectrum antimicrobials. Moreover, understanding Cu toxicity could provide additional insights into the pathophysiology of human diseases of Cu overload such as Wilson's disease.