Biochemical Analysis of CaurSOD4, a Potential Therapeutic Target for the Emerging Fungal Pathogen Candida auris.

Candida auris is an emerging multidrug-resistant fungal pathogen. With high mortality rates, there is an urgent need for new antifungals to combat C. auris. Possible antifungal targets include Cu-only superoxide dismutases (SODs), extracellular SODs that are unique to fungi and effectively combat the superoxide burst of host immunity. Cu-only SODs are essential for the virulence of diverse fungal pathogens; however, little is understood about these enzymes in C. auris. We show here that C. auris secretes an enzymatically active Cu-only SOD (CaurSOD4) when cells are starved for Fe, a condition mimicking host environments. Although predicted to attach to cell walls, CaurSOD4 is detected as a soluble extracellular enzyme and can act at a distance to remove superoxide. CaurSOD4 selectively binds Cu and not Zn, and Cu binding is labile compared to bimetallic Cu/Zn SODs. Moreover, CaurSOD4 is susceptible to inhibition by various metal-binding drugs that are without effect on mammalian Cu/Zn SODs. Our studies highlight CaurSOD4 as a potential antifungal target worthy of consideration.

Copper-only superoxide dismutase enzymes and iron starvation stress in Candida fungal pathogens.

Copper (Cu)-only superoxide dismutases (SOD) represent a newly characterized class of extracellular SODs important for virulence of several fungal pathogens. Previous studies of the Cu-only enzyme SOD5 from the opportunistic fungal pathogen Candida albicans have revealed that the active-site structure and Cu binding of SOD5 strongly deviate from those of Cu/Zn-SODs in its animal hosts, making Cu-only SODs a possible target for future antifungal drug design. C. albicans also expresses a Cu-only SOD4 that is highly similar in sequence to SOD5, but is poorly characterized. Here, we compared the biochemical, biophysical, and cell biological properties of C. albicans SOD4 and SOD5. Analyzing the recombinant proteins, we found that, similar to SOD5, Cu-only SOD4 can react with superoxide at rates approaching diffusion limits. Both SODs were monomeric and they exhibited similar binding affinities for their Cu cofactor. In C. albicans cultures, SOD4 and SOD5 were predominantly cell wall proteins. Despite these similarities, the SOD4 and SOD5 genes strongly differed in transcriptional regulation. SOD5 was predominantly induced during hyphal morphogenesis, together with a fungal burst in reactive oxygen species. Conversely, SOD4 expression was specifically up-regulated by iron (Fe) starvation and controlled by the Fe-responsive transcription factor SEF1. Interestingly, Candida tropicalis and the emerging fungal pathogen Candida auris contain a single SOD5-like SOD rather than a pair, and in both fungi, this SOD was induced by Fe starvation. This unexpected link between Fe homeostasis and extracellular Cu-SODs may help many fungi adapt to Fe-limited conditions of their hosts.

Chemical Warfare at the Microorganismal Level: A Closer Look at the Superoxide Dismutase Enzymes of Pathogens.

Superoxide anion radical is generated as a natural byproduct of aerobic metabolism but is also produced as part of the oxidative burst of the innate immune response design to kill pathogens. In living systems, superoxide is largely managed through superoxide dismutases (SODs), families of metalloenzymes that use Fe, Mn, Ni, or Cu cofactors to catalyze the disproportionation of superoxide to oxygen and hydrogen peroxide. Given the bursts of superoxide faced by microbial pathogens, it comes as no surprise that SOD enzymes play important roles in microbial survival and virulence. Interestingly, microbial SOD enzymes not only detoxify host superoxide but also may participate in signaling pathways that involve reactive oxygen species derived from the microbe itself, particularly in the case of eukaryotic pathogens. In this Review, we will discuss the chemistry of superoxide radicals and the role of diverse SOD metalloenzymes in bacterial, fungal, and protozoan pathogens. We will highlight the unique features of microbial SOD enzymes that have evolved to accommodate the harsh lifestyle at the host-pathogen interface. Lastly, we will discuss key non-SOD superoxide scavengers that specific pathogens employ for defense against host superoxide.

Candida albicans FRE8 encodes a member of the NADPH oxidase family that produces a burst of ROS during fungal morphogenesis.

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.