Metals at the Host-Fungal Pathogen Battleground.

Fungal infections continue to represent a major threat to public health, particularly with the emergence of multidrug-resistant fungal pathogens. As part of the innate immune response, the host modulates the availability of metals as armament against pathogenic microbes, including fungi. The transition metals Fe, Cu, Zn, and Mn are essential micronutrients for all life forms, but when present in excess, these same metals are potent toxins. The host exploits the double-edged sword of these metals, and will either withhold metal micronutrients from pathogenic fungi or attack them with toxic doses. In response to these attacks, fungal pathogens cleverly adapt by modulating metal transport, metal storage, and usage of metals as cofactors for enzymes. Here we review the current state of understanding on Fe, Cu, Zn, and Mn at the host-fungal pathogen battleground and provide perspectives for future research, including a hope for new antifungals based on metals.

SOD1 integrates signals from oxygen and glucose to repress respiration.

Cu/Zn superoxide dismutase (SOD1) is an abundant enzyme that has been best studied as a regulator of antioxidant defense. Using the yeast Saccharomyces cerevisiae, we report that SOD1 transmits signals from oxygen and glucose to repress respiration. The mechanism involves SOD1-mediated stabilization of two casein kinase 1-gamma (CK1γ) homologs, Yck1p and Yck2p, required for respiratory repression. SOD1 binds a C-terminal degron we identified in Yck1p/Yck2p and promotes kinase stability by catalyzing superoxide conversion to peroxide. The effects of SOD1 on CK1γ stability are also observed with mammalian SOD1 and CK1γ and in a human cell line. Therefore, in a single circuit, oxygen, glucose, and reactive oxygen can repress respiration through SOD1/CK1γ signaling. Our data therefore may provide mechanistic insight into how rapidly proliferating cells and many cancers accomplish glucose-mediated repression of respiration in favor of aerobic glycolysis.

The role of manganese in morphogenesis and pathogenesis of the opportunistic fungal pathogen Candida albicans.

Metals such as Fe, Cu, Zn, and Mn are essential trace nutrients for all kingdoms of life, including microbial pathogens and their hosts. During infection, the mammalian host attempts to starve invading microbes of these micronutrients through responses collectively known as nutritional immunity. Nutritional immunity for Zn, Fe and Cu has been well documented for fungal infections; however Mn handling at the host-fungal pathogen interface remains largely unexplored. This work establishes the foundation of fungal resistance against Mn associated nutritional immunity through the characterization of NRAMP divalent metal transporters in the opportunistic fungal pathogen, Candida albicans. Here, we identify C. albicans Smf12 and Smf13 as two NRAMP transporters required for cellular Mn accumulation. Single or combined smf12Δ/Δ and smf13Δ/Δ mutations result in a 10-80 fold reduction in cellular Mn with an additive effect of double mutations and no losses in cellular Cu, Fe or Zn. As a result of low cellular Mn, the mutants exhibit impaired activity of mitochondrial Mn-superoxide dismutase 2 (Sod2) and cytosolic Mn-Sod3 but no defects in cytosolic Cu/Zn-Sod1 activity. Mn is also required for activity of Golgi mannosyltransferases, and smf12Δ/Δ and smf13Δ/Δ mutants show a dramatic loss in cell surface phosphomannan and in glycosylation of proteins, including an intracellular acid phosphatase and a cell wall Cu-only Sod5 that is key for oxidative stress resistance. Importantly, smf12Δ/Δ and smf13Δ/Δ mutants are defective in formation of hyphal filaments, a deficiency rescuable by supplemental Mn. In a disseminated mouse model for candidiasis where kidney is the primary target tissue, we find a marked loss in total kidney Mn during fungal invasion, implying host restriction of Mn. In this model, smf12Δ/Δ and smf13Δ/Δ C. albicans mutants displayed a significant loss in virulence. These studies establish a role for Mn in Candida pathogenesis.

Copper in infectious disease: Using both sides of the penny.

The transition metal Cu is an essential micronutrient that serves as a co-factor for numerous enzymes involved in redox and oxygen chemistry. However, Cu is also a potentially toxic metal, especially to unicellular microbes that are in direct contact with their environment. Since 400 BCE, Cu toxicity has been leveraged for its antimicrobial properties and even today, Cu based materials are being explored as effective antimicrobials against human pathogens spanning bacteria, fungi, and viruses, including the SARS-CoV-2 agent of the 2019-2020 pandemic. Given that Cu has the double-edged property of being both highly toxic and an essential micronutrient, it plays an active and complicated role at the host-pathogen interface. Humans have evolved methods of incorporating Cu into innate and adaptive immune processes and both sides of the penny (Cu toxicity and Cu as a nutrient) are employed. Here we review the evolution of Cu in biology and its multi-faceted roles in infectious disease, from the viewpoints of the microbial pathogens as well as the animal hosts they infect.

Cdc42 regulates reactive oxygen species production in the pathogenic yeast Candida albicans.

Across eukaryotes, Rho GTPases such as Rac and Cdc42 play important roles in establishing cell polarity, which is a key feature of cell growth. In mammals and filamentous fungi, Rac targets large protein complexes containing NADPH oxidases (NOX) that produce reactive oxygen species (ROS). In comparison, Rho GTPases of unicellular eukaryotes were believed to signal cell polarity without ROS, and it was unclear whether Rho GTPases were required for ROS production in these organisms. We document here the first example of Rho GTPase-mediated post-transcriptional control of ROS in a unicellular microbe. Specifically, Cdc42 is required for ROS production by the NOX Fre8 of the opportunistic fungal pathogen Candida albicans. During morphogenesis to a hyphal form, a filamentous growth state, C. albicans FRE8 mRNA is induced, which leads to a burst in ROS. Fre8-ROS is also induced during morphogenesis when FRE8 is driven by an ectopic promoter; hence, Fre8 ROS production is in addition controlled at the post-transcriptional level. Using fluorescently tagged Fre8, we observe that the majority of the protein is associated with the vacuolar system. Interestingly, much of Fre8 in the vacuolar system appears inactive, and Fre8-induced ROS is only produced at sites near the hyphal tip, where Cdc42 is also localized during morphogenesis. We observe that Cdc42 is necessary to activate Fre8-mediated ROS production during morphogenesis. Cdc42 regulation of Fre8 occurs without the large NOX protein complexes typical of higher eukaryotes and therefore represents a novel form of ROS control by Rho GTPases.

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.

Expanded role of the Cu-sensing transcription factor Mac1p in Candida albicans.

As part of the innate immune response, the host withholds metal micronutrients such as Cu from invading pathogens, and microbes respond through metal starvation stress responses. With the opportunistic fungal pathogen Candida albicans, the Cu-sensing transcription factor Mac1p governs the cellular response to Cu starvation by controlling Cu import. Mac1p additionally controls reactive oxygen species (ROS) homeostasis by repressing a Cu-containing superoxide dismutase (SOD1) and inducing Mn-containing SOD3 as a non-Cu alternative. We show here that C. albicans Mac1p is essential for virulence in a mouse model for disseminated candidiasis and that the cellular functions of Mac1p extend beyond Cu uptake and ROS homeostasis. Specifically, mac1∆/∆ mutants are profoundly deficient in mitochondrial respiration and Fe accumulation, both Cu-dependent processes. Surprisingly, these deficiencies are not simply the product of impaired Cu uptake; rather mac1∆/∆ mutants appear defective in Cu allocation. The respiratory defect of mac1∆/∆ mutants was greatly improved by a sod1∆/∆ mutation, demonstrating a role for SOD1 repression by Mac1p in preserving respiration. Mac1p downregulates the major Cu consumer SOD1 to spare Cu for respiration that is essential for virulence of this fungal pathogen. The implications for such Cu homeostasis control in other pathogenic fungi are discussed.

Exploiting the vulnerable active site of a copper-only superoxide dismutase to disrupt fungal pathogenesis.

Copper-only superoxide dismutases (SODs) represent a new class of SOD enzymes that are exclusively extracellular and unique to fungi and oomycetes. These SODs are essential for virulence of fungal pathogens in pulmonary and disseminated infections, and we show here an additional role for copper-only SODs in promoting survival of fungal biofilms. The opportunistic fungal pathogen Candida albicans expresses three copper-only SODs, and deletion of one of them, SOD5, eradicated candidal biofilms on venous catheters in a rodent model. Fungal copper-only SODs harbor an irregular active site that, unlike their Cu,Zn-SOD counterparts, contains a copper co-factor unusually open to solvent and lacks zinc for stabilizing copper binding, making fungal copper-only SODs highly vulnerable to metal chelators. We found that unlike mammalian Cu,Zn-SOD1, C. albicans SOD5 indeed rapidly loses its copper to metal chelators such as EDTA, and binding constants for Cu(II) predict that copper-only SOD5 has a much lower affinity for copper than does Cu,Zn-SOD1. We screened compounds with a variety of indications and identified several metal-binding compounds, including the ionophore pyrithione zinc (PZ), that effectively inhibit C. albicans SOD5 but not mammalian Cu,Zn-SOD1. We observed that PZ both acts as an ionophore that promotes uptake of toxic metals and inhibits copper-only SODs. The pros and cons of a vulnerable active site for copper-only SODs and the possible exploitation of this vulnerability in antifungal drug design are discussed.

Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence.

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells' oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells' hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.

Eukaryotic copper-only superoxide dismutases (SODs): A new class of SOD enzymes and SOD-like protein domains.

The copper-containing superoxide dismutases (SODs) represent a large family of enzymes that participate in the metabolism of reactive oxygen species by disproportionating superoxide anion radical to oxygen and hydrogen peroxide. Catalysis is driven by the redox-active copper ion, and in most cases, SODs also harbor a zinc at the active site that enhances copper catalysis and stabilizes the protein. Such bimetallic Cu,Zn-SODs are widespread, from the periplasm of bacteria to virtually every organelle in the human cell. However, a new class of copper-containing SODs has recently emerged that function without zinc. These copper-only enzymes serve as extracellular SODs in specific bacteria (i.e. Mycobacteria), throughout the fungal kingdom, and in the fungus-like oomycetes. The eukaryotic copper-only SODs are particularly unique in that they lack an electrostatic loop for substrate guidance and have an unusual open-access copper site, yet they can still react with superoxide at rates limited only by diffusion. Copper-only SOD sequences similar to those seen in fungi and oomycetes are also found in the animal kingdom, but rather than single-domain enzymes, they appear as tandem repeats in large polypeptides we refer to as CSRPs (copper-only SOD-repeat proteins). Here, we compare and contrast the Cu,Zn versus copper-only SODs and discuss the evolution of copper-only SOD protein domains in animals and fungi.

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.

Role of Calprotectin in Withholding Zinc and Copper from Candida albicans.

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 host-pathogen interface and CP acts early in infection to restrict metal nutrients from C. albicans.

A role for Candida albicans superoxide dismutase enzymes in glucose signaling.

The Saccharomyces cerevisiae and Candida albicans yeasts have evolved to differentially use glucose for fermentation versus respiration. S. cerevisiae is Crabtree positive, where glucose represses respiration and promotes fermentation, while the opportunistic fungal pathogen C. albicans is Crabtree negative and does not repress respiration with glucose. We have previously shown that glucose control in S. cerevisiae involves the antioxidant enzyme Cu/Zn superoxide dismutase (SOD1), where H2O2 generated by SOD1 stabilizes the casein kinase YCK1 for glucose sensing. We now demonstrate that C. albicans SODs also participate in glucose regulation. C. albicans expresses two cytosolic SODs, Cu/Zn SOD1 and Mn containing SOD3, and both complemented a S. cerevisiae sod1Δ mutant in stabilizing YCK1. Moreover, in C. albicans cells, both SODs functioned to repress glucose transporter genes in response to glucose. However, the action of SODs in glucose control has diverged in the two yeasts. In S. cerevisiae, SOD1 specifically functions in the glucose sensing pathway involving YCK1 and the RGT1 repressor, but the analogous YCK/RGT1 pathway in C. albicans shows no control by SOD enzymes. Instead C. albicans SODs work in the glucose repression pathway involving the MIG1 transcriptional repressor. In C. albicans, the SODs repress glucose uptake, while in S. cerevisiae, SOD1 activates glucose uptake, in accordance with the divergent modes for glucose utilization in these two distantly related yeasts.

The Phylogeny and Active Site Design of Eukaryotic Copper-only Superoxide Dismutases.

In eukaryotes the bimetallic Cu/Zn superoxide dismutase (SOD) enzymes play important roles in the biology of reactive oxygen species by disproportionating superoxide anion. Recently, we reported that the fungal pathogen Candida albicans expresses a novel copper-only SOD, known as SOD5, that lacks the zinc cofactor and electrostatic loop (ESL) domain of Cu/Zn-SODs for substrate guidance. Despite these abnormalities, C. albicans SOD5 can disproportionate superoxide at rates limited only by diffusion. Here we demonstrate that this curious copper-only SOD occurs throughout the fungal kingdom as well as in phylogenetically distant oomycetes or "pseudofungi" species. It is the only form of extracellular SOD in fungi and oomycetes, in stark contrast to the extracellular Cu/Zn-SODs of plants and animals. Through structural biology and biochemical approaches we demonstrate that these copper-only SODs have evolved with a specialized active site consisting of two highly conserved residues equivalent to SOD5 Glu-110 and Asp-113. The equivalent positions are zinc binding ligands in Cu/Zn-SODs and have evolved in copper-only SODs to control catalysis and copper binding in lieu of zinc and the ESL. Similar to the zinc ion in Cu/Zn-SODs, SOD5 Glu-110 helps orient a key copper-coordinating histidine and extends the pH range of enzyme catalysis. SOD5 Asp-113 connects to the active site in a manner similar to that of the ESL in Cu/Zn-SODs and assists in copper cofactor binding. Copper-only SODs are virulence factors for certain fungal pathogens; thus this unique active site may be a target for future anti-fungal strategies.

The Yin and Yang of copper during infection.

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.

Cu/Zn superoxide dismutase and the proton ATPase Pma1p of Saccharomyces cerevisiae.

In eukaryotes, the Cu/Zn containing superoxide dismutase (SOD1) plays a critical role in oxidative stress protection as well as in signaling. We recently demonstrated a function for Saccharomyces cerevisiae Sod1p in signaling through CK1γ casein kinases and identified the essential proton ATPase Pma1p as one likely target. The connection between Sod1p and Pma1p was explored further by testing the impact of sod1Δ mutations on cells expressing mutant alleles of Pma1p that alter activity and/or post-translational regulation of this ATPase. We report here that sod1Δ mutations are lethal when combined with the T912D allele of Pma1p in the C-terminal regulatory domain. This "synthetic lethality" was reversed by intragenic suppressor mutations in Pma1p, including an A906G substitution that lies within the C-terminal regulatory domain and hyper-activates Pma1p. Surprisingly the effect of sod1Δ mutations on Pma1-T912D is not mediated through the Sod1p signaling pathway involving the CK1γ casein kinases. Rather, Sod1p sustains life of cells expressing Pma1-T912D through oxidative stress protection. The synthetic lethality of sod1Δ Pma1-T912D cells is suppressed by growing cells under low oxygen conditions or by treatments with manganese-based antioxidants. We now propose a model in which Sod1p maximizes Pma1p activity in two ways: one involving signaling through CK1γ casein kinases and an independent role for Sod1p in oxidative stress protection.

Superoxide triggers an acid burst in Saccharomyces cerevisiae to condition the environment of glucose-starved cells.

Although yeast cells grown in abundant glucose tend to acidify their extracellular environment, they raise the pH of the environment when starved for glucose or when grown strictly with non-fermentable carbon sources. Following prolonged periods in this alkaline phase, Saccharomyces cerevisiae cells will switch to producing acid. The mechanisms and rationale for this "acid burst" were unknown. Herein we provide strong evidence for the role of mitochondrial superoxide in initiating the acid burst. Yeast mutants lacking the mitochondrial matrix superoxide dismutase (SOD2) enzyme, but not the cytosolic Cu,Zn-SOD1 enzyme, exhibited marked acceleration in production of acid on non-fermentable carbon sources. Acid production is also dramatically enhanced by the superoxide-producing agent, paraquat. Conversely, the acid burst is eliminated by boosting cellular levels of Mn-antioxidant mimics of SOD. We demonstrate that the acid burst is dependent on the mitochondrial aldehyde dehydrogenase Ald4p. Our data are consistent with a model in which mitochondrial superoxide damage to Fe-S enzymes in the tricarboxylic acid (TCA) cycle leads to acetate buildup by Ald4p. The resultant expulsion of acetate into the extracellular environment can provide a new carbon source to glucose-starved cells and enhance growth of yeast. By triggering production of organic acids, mitochondrial superoxide has the potential to promote cell population growth under nutrient depravation stress.

A manganese-rich environment supports superoxide dismutase activity in a Lyme disease pathogen, Borrelia burgdorferi.

The Lyme disease pathogen Borrelia burgdorferi represents a novel organism in which to study metalloprotein biology in that this spirochete has uniquely evolved with no requirement for iron. Not only is iron low, but we show here that B. burgdorferi has the capacity to accumulate remarkably high levels of manganese. This high manganese is necessary to activate the SodA superoxide dismutase (SOD) essential for virulence. Using a metalloproteomic approach, we demonstrate that a bulk of B. burgdorferi SodA directly associates with manganese, and a smaller pool of inactive enzyme accumulates as apoprotein. Other metalloproteins may have similarly adapted to using manganese as co-factor, including the BB0366 aminopeptidase. Whereas B. burgdorferi SodA has evolved in a manganese-rich, iron-poor environment, the opposite is true for Mn-SODs of organisms such as Escherichia coli and bakers' yeast. These Mn-SODs still capture manganese in an iron-rich cell, and we tested whether the same is true for Borrelia SodA. When expressed in the iron-rich mitochondria of Saccharomyces cerevisiae, B. burgdorferi SodA was inactive. Activity was only possible when cells accumulated extremely high levels of manganese that exceeded cellular iron. Moreover, there was no evidence for iron inactivation of the SOD. B. burgdorferi SodA shows strong overall homology with other members of the Mn-SOD family, but computer-assisted modeling revealed some unusual features of the hydrogen bonding network near the enzyme's active site. The unique properties of B. burgdorferi SodA may represent adaptation to expression in the manganese-rich and iron-poor environment of the spirochete.

Species-specific activation of Cu/Zn SOD by its CCS copper chaperone in the pathogenic yeast Candida albicans.

Candida albicans is a pathogenic yeast of important public health relevance. Virulence of C. albicans requires a copper and zinc containing superoxide dismutase (SOD1), but the biology of C. albicans SOD1 is poorly understood. To this end, C. albicans SOD1 activation was examined in baker's yeast (Saccharomyces cerevisiae), a eukaryotic expression system that has proven fruitful for the study of SOD1 enzymes from invertebrates, plants, and mammals. In spite of the 80% similarity between S. cerevisiae and C. albicans SOD1 molecules, C. albicans SOD1 is not active in S. cerevisiae. The SOD1 appears incapable of productive interactions with the copper chaperone for SOD1 (CCS1) of S. cerevisiae. C. albicans SOD1 contains a proline at position 144 predicted to dictate dependence on CCS1. By mutation of this proline, C. albicans SOD1 gained activity in S. cerevisiae, and this activity was independent of CCS1. We identified a putative CCS1 gene in C. albicans and created heterozygous and homozygous gene deletions at this locus. Loss of CCS1 resulted in loss of SOD1 activity, consistent with its role as a copper chaperone. C. albicans CCS1 also restored activity to C. albicans SOD1 expressed in S. cerevisiae. C. albicans CCS1 is well adapted for activating its partner SOD1 from C. albicans, but not SOD1 from S. cerevisiae. In spite of the high degree of homology between the SOD1 and CCS1 molecules in these two fungal species, there exists a species-specific barrier in CCS-SOD interactions which may reflect the vastly different lifestyles of the pathogenic versus the noninfectious yeast.