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.

Ceruloplasmin as a source of Cu for a fungal pathogen.

Copper is an essential metal for virtually all organisms, yet little is known about the extracellular sources of this micronutrient. In serum, the most abundant extracellular Cu-binding molecule is the multi­copper oxidase ceruloplasmin (Cp). Cp levels increase during infection and inflammation, and pathogens can be exposed to high Cp at sites of infection. It is not known whether Cp might serve as a Cu source for microbial pathogens and we tested this using the opportunistic fungal pathogen Candida albicans. We find that C. albicans can use whole serum as a Cu source and that this Cu is sensed by the transcription factor protein Mac1. Mac1 activates expression of Mn-SOD3 superoxide dismutase and represses Cu/Zn-SOD1 during Cu starvation and both responses are regulated by serum Cu. We also show that purified human Cp can act as a sole source of Cu for the fungus and likewise modulates the Mac1 Cu stress response. To investigate whether Cp is a Cu source in serum, we compared the ability of C. albicans to use serum from wild type versus Cp-/- mutant mice. We find that serum lacking Cp is deficient in its ability to trigger the Mac1 Cu response. C. albicans did accumulate Cu from Cp-/- serum, but this Cu was not efficiently sensed by Mac1. We conclude that Cp and non-Cp Cu sources are not equivalent and are handled differently by the fungal cell. Overall, these studies are the first to show that Cp is a preferred source of Cu for a pathogen.

Antimicrobial action of calprotectin that does not involve metal withholding.

Calprotectin is a potent antimicrobial that inhibits the growth of pathogens by tightly binding transition metals such as Mn and Zn, thereby preventing their uptake and utilization by invading microbes. At sites of infection, calprotectin is abundantly released from neutrophils, but calprotectin is also present in non-neutrophil cell types that may be relevant to infections. We show here that in patients infected with the Lyme disease pathogen Borreliella (Borrelia) burgdorferi, calprotectin is produced in neutrophil-free regions of the skin, in both epidermal keratinocytes and in immune cells infiltrating the dermis, including CD68 positive macrophages. In culture, B. burgdorferi's growth is inhibited by calprotectin, but surprisingly, the mechanism does not involve the classical withholding of metal nutrients. B. burgdorferi cells exposed to calprotectin cease growth with no reduction in intracellular Mn and no loss in activity of Mn enzymes including the SodA superoxide dismutase. Additionally, there is no obvious loss in intracellular Zn. Rather than metal depletion, we find that calprotectin inhibits B. burgdorferi growth through a mechanism that requires physical association of calprotectin with the bacteria. By comparison, calprotectin inhibited E. coli growth without physically interacting with the microbe, and calprotectin effectively depleted E. coli of intracellular Mn and Zn. Our studies with B. burgdorferi demonstrate that the antimicrobial capacity of calprotectin is complex and extends well beyond simple withholding of metal micronutrients.

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.

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.

Revisiting and re-engineering the classical zinc finger peptide: consensus peptide-1 (CP-1).

Zinc plays key structural and catalytic roles in biology. Structural zinc sites are often referred to as zinc finger (ZF) sites, and the classical ZF contains a Cys2His2 motif that is involved in coordinating Zn(II). An optimized Cys2His2 ZF, named consensus peptide 1 (CP-1), was identified more than 20 years ago using a limited set of sequenced proteins. We have reexamined the CP-1 sequence, using our current, much larger database of sequenced proteins that have been identified from high-throughput sequencing methods, and found the sequence to be largely unchanged. The CCHH ligand set of CP-1 was then altered to a CAHH motif to impart hydrolytic activity. This ligand set mimics the His2Cys ligand set of peptide deformylase (PDF), a hydrolytically active M(II)-centered (M = Zn or Fe) protein. The resultant peptide [CP-1(CAHH)] was evaluated for its ability to coordinate Zn(II) and Co(II) ions, adopt secondary structure, and promote hydrolysis. CP-1(CAHH) was found to coordinate Co(II) and Zn(II) and a pentacoordinate geometry for Co(II)-CP-1(CAHH) was implicated from UV-vis data. This suggests a His2Cys(H2O)2 environment at the metal center. The Zn(II)-bound CP-1(CAHH) was shown to adopt partial secondary structure by 1-D (1)H NMR spectroscopy. Both Zn(II)-CP-1(CAHH) and Co(II)-CP-1(CAHH) show good hydrolytic activity toward the test substrate 4-nitrophenyl acetate, exhibiting faster rates than most active synthetic Zn(II) complexes.

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.

Neural Zinc Finger Factor/Myelin Transcription Factor Proteins: Metal Binding, Fold, and Function.

Zinc finger (ZF) proteins make up a large family of metalloproteins that contain discrete domains with amino acid ligands (cysteine and histidine) that serve to coordinate zinc in a tetrahedral geometry. Upon zinc coordination, the domains adopt three-dimensional structure. The most well-studied ZFs are the "classical" ZFs, which use a Cys2His2 motif to bind zinc and adopt an antiparallel ß sheet/α helical fold. In addition to the classical ZF class, at least 13 other ZF classes, collectively termed nonclassical ZFs, have been identified. These other classes are distinguished by the combination and order of the cysteine and histidine ligands within each domain, the spacing between each ligand (i.e., number and type of amino acid), and the structural architecture that the domain adopts in the presence of zinc. One class of nonclassical ZFs, the neural zinc finger/myelin transcription factor (NZF/MyT) class, contains ZF domains with a Cys2His2Cys ligand set, adopts a fold that consists of a series of loops in the presence of zinc, and functions as transcription factors by binding to and regulating genes that are critical for the development of the central nervous system. This Current Topic focuses on our understanding of the NZF/MyT class of nonclassical ZFs and presents current hypotheses regarding this class' unique mechanism of metal-mediated folding and function.

A role for hydrogen bonding in DNA recognition by the non-classical CCHHC type zinc finger, NZF-1.

The non-classical zinc finger protein, Neural Zinc Finger Factor-1, contains six Cys2His2Cys domains. All three cysteines and the second histidine directly bind Zn(II). Using a combination of mutagenesis, metal coordination and DNA binding studies, we report that the first histidine is involved in a functionally important hydrogen bonding interaction.

Random and site-specific mutagenesis of the Helicobacter pylori ferric uptake regulator provides insight into Fur structure-function relationships.

The ferric uptake regulator (Fur) of Helicobacter pylori is a global regulator that is important for colonization and survival within the gastric mucosa. H. pylori Fur is unique in its ability to activate and repress gene expression in both the iron-bound (Fe-Fur) and apo forms (apo-Fur). In the current study we combined random and site-specific mutagenesis to identify amino acid residues important for both Fe-Fur and apo-Fur function. We identified 25 mutations that affected Fe-Fur repression and 23 mutations that affected apo-Fur repression, as determined by transcriptional analyses of the Fe-Fur target gene amiE, and the apo-Fur target gene, pfr. In addition, eight of these mutations also significantly affected levels of Fur in the cell. Based on regulatory phenotypes, we selected several representative mutations to characterize further. Of those selected, we purified the wild-type (HpFurWT) and three mutant Fur proteins (HpFurE5A, HpFurA92T and HpFurH134Y), which represent mutations in the N-terminal extension, the regulatory metal binding site (S2) and the structural metal binding site (S3) respectively. Purified proteins were evaluated for secondary structure by circular dichroism spectroscopy, iron-binding by atomic absorption spectrophotometry, oligomerization in manganese-substituted and apo conditions by in vitro cross-linking assays, and DNA binding to Fe-Fur and apo-Fur target sequences by fluorescence anisotropy. The results showed that the N-terminal, S2 and S3 regions play distinct roles in terms of Fur structure-function relationships. Overall, these studies provide novel information regarding the role of these residues in Fur function, and provide mechanistic insight into how H. pylori Fur regulates gene expression in both the iron-bound and apo forms of the protein.

Switching metal ion coordination and DNA Recognition in a Tandem CCHHC-type zinc finger peptide.

Neural Zinc Finger Factor-1 (NZF-1) and Myelin Transcription Factor 1 (MyT1) are two homologous nonclassical zinc finger (ZF) proteins that are involved in the development of the central nervous system (CNS). Both NZF-1 and MyT1 contain multiple ZF domains, each of which contains an absolutely conserved Cys2His2Cys motif. All three cysteines and the second histidine have been shown to coordinate Zn(II); however, the role of the first histidine remains unresolved. Using a functional form of NZF-1 that contains two ZF domains (NZF-1-F2F3), mutant proteins in which each histidine was sequentially mutated to a phenylalanine were prepared to determine the role(s) of the histidine residues in DNA recognition. When the first histidine is mutated, the protein binds Zn(II) in an analogous manner to the native protein. Surprisingly, this mutant does not bind to target DNA (ß-RAR), suggesting that the noncoordinating histidine is critical for sequence selective DNA recognition. The first histidine will coordinate Zn(II) when the second histidine is mutated; however, the overall fold of the protein is perturbed leading to abrogation of DNA binding. NZF-1-F2F3 selectively binds to a specific DNA target sequence (from ß-RAR) with high affinity (nM); while its homologue MyT1 (MyT1-F2F3), which is 92% identical to NZF-1-F2F3, binds to this same DNA sequence nonspecifically. A single, nonconserved amino acid residue in NZF-1-F2F3 is shown to be responsible for this high affinity DNA binding to ß-RAR. When this residue (arginine) is engineered into the MyT1-F2F3 sequence, the affinity for ß-RAR DNA increases.

Cysteine and histidine shuffling: mixing and matching cysteine and histidine residues in zinc finger proteins to afford different folds and function.

Zinc finger proteins utilize zinc for structural purposes: zinc binds to a combination of cysteine and histidine ligands in a tetrahedral coordination geometry facilitating protein folding and function. While much is known about the classical zinc finger proteins, which utilize a Cys(2)His(2) ligand set to coordinate zinc and fold into an anti-parallel beta sheet/alpha helical fold, there are thirteen other families of 'non-classical' zinc finger proteins for which relationships between metal coordination and protein structure/function are less defined. This 'Perspective' article focuses on two classes of these non-classical zinc finger proteins: Cys(3)His type zinc finger proteins and Cys(2)His(2)Cys type zinc finger proteins. These proteins bind zinc in a tetrahedral geometry, like the classical zinc finger proteins, yet they adopt completely different folds and target different oligonucleotides. Our current understanding of the relationships between ligand set, metal ion, fold and function for these non-classical zinc fingers is discussed.

Classical Cys2His2 zinc finger peptides are rapidly oxidized by either H2O2 or O2 irrespective of metal coordination.

ZIF268, a member of the classical zinc finger protein family, contains three Cys(2)His(2) zinc binding domains that together recognize the DNA sequence 5'-AGCGTGGGCGT-3'. These domains can be fused to an endonuclease to make a chimeric protein to target and cleave specific DNA sequences. A peptide corresponding to these domains, named ZIF268-3D, has been prepared to determine if the zinc finger domain itself can promote DNA cleavage when a redox active metal ion, Fe(II), is coordinated. The UV-vis absorption spectrum of Fe(II)-ZIF268-3D is indicative of Fe(II) coordination. Using fluorescence anisotropy, we demonstrate that Fe(II)-ZIF268-3D binds selectively to its target DNA in the same manner as Zn(II)-ZIF268-3D. In the presence of added oxidant, H(2)O(2) or O(2), DNA cleavage is not observed by Fe(II)-ZIF268-3D. Instead, the peptide itself is rapidly oxidized. Similarly, Zn(II)-ZIF268-3D and apo-ZIF268-3D are rapidly oxidized by H(2)O(2) or O(2), and we propose that ZIF268-3D is highly susceptible to oxidation.

Functional characterization of iron-substituted neural zinc finger factor 1: metal and DNA binding.

Neural zinc finger factor 1 (NZF-1) is a nonclassical zinc finger protein involved in neuronal development. NZF-1 contains multiple copies of a unique CCHHC zinc-binding domain that recognize a promoter element in the beta-retinoic acid receptor gene termed beta-retinoic acid receptor element (beta-RARE). Previous studies have established that a two-domain fragment of NZF-1 bound with zinc is sufficient for specific DNA binding. Proper functioning of the nervous system relies heavily on iron and misregulation of this highly redox active metal has serious consequences. Several classes of zinc finger proteins have been shown to bind other metal ions, including iron. To determine if ferrous iron can coordinate to the metal-binding sites of NZF-1 and assess the functional consequences of such coordination, a fragment of NZF-1 that contains two zinc-binding domains, NZF-1 double finger (NZF-1-DF), was prepared. UV-vis spectroscopy experiments demonstrated that Fe(II) is capable of binding to NZF-1-DF. Upon reconstitution with either Fe(II) or Zn(II), NZF-1-DF binds selectively and tightly (nanomolar affinity) to its target beta-RARE DNA sequence, whereas apo-NZF-1-DF does not bind to DNA and instead aggregates.