Copper is an essential micronutrient for both pathogens and the animal hosts they infect. However, copper can also be toxic in cells due to its redox properties and ability to disrupt active sites of metalloproteins, such as Fe-S enzymes. Through these toxic properties, copper is an effective antimicrobial agent and an emerging concept in innate immunity is that the animal host intentionally exploits copper toxicity in antimicrobial weaponry. In particular, macrophages can attack invading microbes with high copper and this metal is also elevated at sites of lung infection. In addition, copper levels in serum rise during infection with a wide array of pathogens. To defend against this toxic copper, the microbial intruder is equipped with a battery of copper detoxification defenses that promote survival in the host, including copper exporting ATPases and copper binding metallothioneins. However, it is important to remember that copper is also an essential nutrient for microbial pathogens and serves as important cofactor for enzymes such as cytochrome c oxidase for respiration, superoxide dismutase for anti-oxidant defense and multi-copper oxidases that act on metals and organic substrates. We therefore posit that the animal host can also thwart pathogen growth by limiting their copper nutrients, similar to the well-documented nutritional immunity effects for starving microbes of essential zinc, manganese and iron micronutrients. This review provides both sides of the copper story and evaluates how the host can exploit either copper-the-toxin or copper-the-nutrient in antimicrobial tactics at the host-pathogen battleground.

The opportunistic fungal pathogen Candida albicans acquires essential metals from the host, yet the host can sequester these micronutrients through a process known as nutritional immunity. How the host withholds metals from C. albicans has been poorly understood; here we examine the role of calprotectin (CP), a transition metal binding protein. When CP depletes bioavailable Zn from the extracellular environment, C. albicans strongly upregulates ZRT1 and PRA1 for Zn import and maintains constant intracellular Zn through numerous cell divisions. We show for the first time that CP can also sequester Cu by binding Cu(II) with subpicomolar affinity. CP blocks fungal acquisition of Cu from serum and induces a Cu starvation stress response involving SOD1 and SOD3 superoxide dismutases. These transcriptional changes are mirrored when C. albicans invades kidneys in a mouse model of disseminated candidiasis, although the responses to Cu and Zn limitations are temporally distinct. The Cu response progresses throughout 72 h, while the Zn response is short-lived. Notably, these stress responses were attenuated in CP null mice, but only at initial stages of infection. Thus, Zn and Cu pools are dynamic at the hostpathogen interface and CP acts early in infection to restrict metal nutrients from C. albicans.

Until recently, NADPH oxidase (NOX) enzymes were thought to be a property of multicellularity, where the reactive oxygen species (ROS) produced by NOX acts in signaling processes or in attacking invading microbes through oxidative damage. We demonstrate here that the unicellular yeast and opportunistic fungal pathogen Candida albicans is capable of a ROS burst using a member of the NOX enzyme family, which we identify as Fre8. C. albicans can exist in either a unicellular yeast-like budding form or as filamentous multicellular hyphae or pseudohyphae, and the ROS burst of Fre8 begins as cells transition to the hyphal state. Fre8 is induced during hyphal morphogenesis and specifically produces ROS at the growing tip of the polarized cell. The superoxide dismutase Sod5 is co-induced with Fre8 and our findings are consistent with a model in which extracellular Sod5 acts as partner for Fre8, converting Fre8-derived superoxide to the diffusible H2O2 molecule. Mutants of fre8Δ/Δ exhibit a morphogenesis defect in vitro and are specifically impaired in development or maintenance of elongated hyphae, a defect that is rescued by exogenous sources of H2O2. A fre8Δ/Δ deficiency in hyphal development was similarly observed in vivo during C. albicans invasion of the kidney in a mouse model for disseminated candidiasis. Moreover C. albicans fre8Δ/Δ mutants showed defects in a rat catheter model for biofilms. Together these studies demonstrate that like multicellular organisms, C. albicans expresses NOX to produce ROS and this ROS helps drive fungal morphogenesis in the animal host.