Ferritins, bacterial virulence and plant defence

The enterobacterial pathogen Erwinia chrysanthemi causes soft rot diseases on a wide range of plants, including the model plant Arabidopsis thaliana. This bacterium proliferates in the host by secreting a set of pectin degrading enzymes responsible for symptom development. In addition, survival of this bacterium in planta requires two high-affinity iron acquisition systems mediated by siderophores and protective systems against oxidative damages, suggesting the implication by both partners of accurate mechanisms controlling their iron homeostasis under conditions of infection. In this review, we address this question and we show that ferritins both from the pathogen and the host are subtly implicated in the control of this interplay.     

Ferritins: iron/oxygen biominerals in protein nanocages

Ferritin protein nanocages that form iron oxy biominerals in the central nanometer cavity are nature’s answer to managing iron and oxygen; gene deletions are lethal in mammals and render bacteria more vulnerable to host release of antipathogen oxidants. The multifunctional, multisubunit proteins couple iron with oxygen (maxi-ferritins) or hydrogen peroxide (mini-ferritins) at catalytic sites that are related to di-iron sites oxidases, ribonucleotide reductase, methane monooxygenase and fatty acid desaturases, and synthesize mineral precursors. Gated pores, distributed symmetrically around the ferritin cages, control removal of iron by reductants and chelators. Gene regulation of ferritin, long known to depend on iron and, in animals, on a noncoding messenger RNA (mRNA) structure linked in a combinatorial array to functionally related mRNA of iron transport, has recently been shown to be linked to an array of proteins for antioxidant responses such as thioredoxin and quinone reductases. Ferritin DNA responds more to oxygen signals, and ferritin mRNA responds more to iron signals. Ferritin genes (DNA and RNA) and protein function at the intersection of iron and oxygen chemistry in biology.     

Ferritins,iron uptake and storage from the bacterioferritin viewpoint

Ferritins constitute a broad superfamily of iron storage proteins, widespread in all domains of life, in aerobic or anaerobic organisms. Ferritins isolated from bacteria may be haem-free or contain a haem. In the latter case they are called bacterioferritins. The primary function of ferritins inside cells is to store iron in the ferric form. A secondary function may be detoxification of iron or protection against O(2) and its radical products. Indeed, for bacterioferritins this is likely to be their primary function. Ferritins and bacteroferritins have essentially the same architecture, assembling in a 24mer cluster to form a hollow, roughly spherical construction. In this review, special emphasis is given to the structure of the ferroxidase centres with native iron-containing sites, since oxidation of ferrous iron by molecular oxygen takes place in these sites. Although present in other ferritins, a specific entry route for iron, coupled with the ferroxidase reaction, has been proposed and described in some structural studies. Electrostatic calculations on a few selected proteins indicate further ion channels assumed to be an entry route in the later mineralization processes of core formation.     

Differential effects of basolateral and apical iron supply on iron transport in Caco-2 cells

Iron homeostasis in the human body is maintained primarily through regulation of iron absorption in the duodenum. The liver peptide hepcidin plays a central role in this regulation. Additionally, expression and functional control of certain components of the cellular iron transport machinery can be influenced directly by the iron status of enterocytes. The significance of this modulation, relative to the effects of hepcidin, and the comparative effects of iron obtained directly from the diet and/or via the bloodstream are not clear. The studies described here were performed using Caco-2 cell monolayers as a model of intestinal epithelium, to compare the effects of iron supplied in physiologically relevant forms to either the apical or basolateral surfaces of the cells. Both sources of iron provoked increased cellular ferritin content, indicating iron uptake from both sides of the cells. Supply of basolateral transferrin-bound iron did not affect subsequent iron transport across the apical surface, but reduced iron transport across the basolateral membrane. In contrast, the apical iron supply led to subsequent reduction in iron transport across the apical cell membrane without altering iron export across the basolateral membrane. The apical and basolateral iron supplies also elicited distinct effects on the expression and subcellular distribution of iron transporters. These data suggest that, in addition to the effects of cellular iron status on the expression of iron transporter genes, different modes and direction of iron supply to enterocytes can elicit distinct functional effects on iron transport.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0463-5) contains supplementary material, which is available to authorized users.     

Limited effects of combined dietary copper deficiency/iron overload on oxidative stress parameters in rat liver and plasma

Copper (Cu) deficiency decreases the activity of Cu-dependent antioxidant enzymes such as Cu,zinc-superoxide dismutase (Cu,Zn-SOD) and may be associated with increased susceptibility to oxidative stress. Iron (Fe) overload represents a dietary oxidative stress relevant to overuse of Fe-containing supplements and to hereditary hemochromatosis. In a study to investigate oxidative stress interactions of dietary Cu deficiency with Fe overload, weanling male Long–Evans rats were fed one of four sucrose-based modified AIN-93G diets formulated to differ in Cu (adequate 6 mg/kg diet vs. deficient 0.5 mg/kg) and Fe (adequate 35 mg/kg vs. overloaded 1500 mg/kg) in a 2×2 factorial design for 4 weeks prior to necropsy. Care was taken to minimize oxidation of the diets prior to feeding to the rats. Liver and plasma Cu content and liver Cu,Zn-SOD activity declined with Cu deficiency and liver Fe increased with Fe overload, confirming the experimental dietary model. Liver thiobarbituric acid reactive substances were significantly elevated with Fe overload (pooled across Cu treatments, 0.80±0.14 vs. 0.54±0.08 nmol/mg protein; P<.0001) and not affected by Cu deficiency. Liver cytosolic protein carbonyl content and the concentrations of several oxidized cholesterol species in liver tissue did not change with these dietary treatments. Plasma protein carbonyl content decreased in Cu-deficient rats and was not influenced by dietary Fe overload. The various substrates (lipid, protein and cholesterol) appeared to differ in their susceptibility to the in vivo oxidative stress induced by dietary Fe overload, but these differences were not exacerbated by Cu deficiency.     

Systemic iron status

Iron is one of the essential micronutrients, and as such, is required for growth, development, and normal cellular functioning. In contrast to some other micronutrients such as water-soluble vitamins, there is a significant danger of toxicity if excessive amounts of iron accumulate in the body. A finely tuned feedback control system functions to limit this excessive accumulation by limiting absorption of iron. This chapter will discuss systemic and brain iron homeostasis.     

Tocopherol content and enzymatic antioxidant activities in chloroplasts from NaCl-stressed tomato plants

Changes in tocopherol, chlorophyll and TBARS levels and the activities of antioxidant enzymes i.e., GSH-Px, GST, and SOD in chloroplasts of tomato plants subjected to moderate (50 mM) and severe (150 mM) NaCl stress were determined. Increase in tocopherol content around the second day under both stresses did not correlate with the chlorophyll degradation while such correlation was observed from the fifth day of severe stress. The activities of GSH-Px and GST as well as TBARS content showed NaCl-induced enhancement which was dose- and time-dependent. However, chloroplastic SOD was rather not involved in the response of tomato plants to NaCl stress. The obtained results suggest that under the moderate stress similarly as in the early phase of severe stress tocopherol functions as a typical antioxidant, while in the late phase of the latter it may be involved in senescence signaling pathway and enables the recovery and recycling of the compounds significant for a plant organism.     

Impact of iron and vitamin C-containing supplements on preterm human milk: in vitro

Stress due to reactive oxygen species (ROS) may lead to neonatal diseases, such as necrotizing enterocolitis and respiratory distress. Enteral supplements for premature infants (PREM) added to human milk (HM) to increase nutrient content may induce lipid oxidation due to free radical formation via Fenton chemistry. We hypothesized that ferrous iron and vitamin C-containing supplements added to HM in vitro cause oxidation of milk fats, affect intracellular redox balance, and induce DNA damage. Lipid peroxidation in HM was measured by FOX-2 and TBARS assays; fatty acid composition of supplemented HM was measured by gas chromatography. Two cell culture bioassays were used for assessing either intracellular oxidative stress or DNA damage: the former involved Caco-2BBe cells, a secondary differentiated cell line, and the latter utilized FHS-74 Int cells, a primary fetal small intestinal culture. Lipid oxidation products of HM increased after the addition of iron alone, iron and vitamin C, or iron and a vitamin C-containing supplement (Trivisol, TVS). A reduced content of mono and polyunsaturated fatty acids in HM was also observed. Iron, not iron+vitamin C, but iron+TVS induced significant intracellular oxidative stress in FHS-74 Int cells. In contrast, iron, either alone or in combination with TVS or vitamin C, increased DNA damage in Caco-2BBE cells. Iron supplementation may increase oxidative stress in PREM infants and should be given separately from vitamin C-containing supplements.     

Ferritins: a family of molecules for iron storage, antioxidation and more

Ferritins are characterized by highly conserved three-dimensional structures similar to spherical shells, designed to accommodate large amounts of iron in a safe, soluble and bioavailable form. They can have different architectures with 12 or 24 equivalent or non-equivalent subunits, all surrounding a large cavity. All ferritins readily interact with Fe(II) to induce its oxidation and deposition in the cavity in a mineral form, in a reaction that is catalyzed by a ferroxidase center. This is an anti-oxidant activity that consumes Fe(II) and peroxides, the reagents that produce toxic free radicals in the Fenton reaction. The mechanism of ferritin iron incorporation has been characterized in detail, while that of iron release and recycling has been less thoroughly studied. Generally ferritin expression is regulated by iron and by oxidative damage, and in vertebrates it has a central role in the control of cellular iron homeostasis. Ferritin is mostly cytosolic but is found also in mammalian mitochondria and nuclei, in plant plastids and is secreted in insects. In vertebrates the cytosolic ferritins are composed of H and L subunit types and their assembly in a tissues specific ratio that permits flexibility to adapt to cell needs. The H-ferritin can translocate to the nuclei in some cell types to protect DNA from iron toxicity, or can be actively secreted, accomplishing various functions. The mitochondrial ferritin is found in mammals, it has a restricted tissue distribution and it seems to protect the mitochondria from iron toxicity and oxidative damage. The various functions attributed to the cytosolic, nuclear, secretory and mitochondrial ferritins are discussed.     

The role of iron in Campylobacter gene regulation,metabolism and oxidative stress defense

Enteric Campylobacter species cause gastrointestinal diseases in humans. Like almost all organisms, campylobacters have an absolute requirement for iron, but are faced with variable availability of iron in the environment and host tissues. Campylobacters have developed mechanisms to scavenge sufficient iron for metabolism and growth. However, iron also participates in the formation of reactive oxygen species, and this forces pathogens to maintain intracellular iron homeostasis and to cope with oxidative stresses. The presence of two separate, but possibly overlapping iron-responsive regulatory systems, which regulate iron acquisition and oxidative stress defense, and the presence of genes encoding multiple iron acquisition and detoxification systems in Campylobacter indicate the central role that iron plays in Campylobacter gene regulation and virulence.     

Progressive iron overload enhances chemically mediated tumor promotion in murine skin

Iron overload has been shown to enhance chemically mediated cutaneous tumor promotion in animals. However, the majority of these animal studies have used high concentrations of iron before initiating tumor development. The current study was designed to evaluate the effect of small doses of iron on the promotion stage of chemically mediated cutaneous carcinogenesis. We found an increased tumor response in mice initiated with dimethylbenz(a)anthracene (DMBA) when iron at the dose levels of 0.5, 1.0, and 1.5mg/mouse was injected (intramuscularly) once a week into mice at the promotion stage of skin carcinogenesis, employing 12-O-tetradecanoyl phorbol-13-acetate (TPA)/benzoyl peroxide (BPO) as tumor promoter. The appearance of first papilloma and the number of tumors/mouse were recorded weekly. When compared to the control (non-iron-treated) group, the iron-treated groups showed an augmented incidence of tumors and number of tumors/mouse. In iron-treated mice, tumors appeared earlier than in the control group. TPA/BPO treatment resulted in a significant decrease in the activities of antioxidant enzymes and depletion in the level of epidermal reduced glutathione (GSH). TPA treatment in non-iron-treated mice resulted in approximately 20-40% decrease in GSH level and in the activities of antioxidant enzymes, whereas 1.5-mg iron treatment along with TPA treatment resulted in about approximately 30-70% decrease in GSH level and in the activities of antioxidant enzymes. Similarly, treatment of iron along with BPO treatment resulted in a dose-dependent higher depletion of GSH and the antioxidant enzymes as compared to non-iron-treated animals treated with BPO. Further, TPA/BPO-mediated induction in ornithine decarboxylase activity and [3H]thymidine incorporation in cutaneous DNA was approx two- to threefold higher in mice treated with iron as compared to non-iron-treated mice. Cutaneous lipid peroxidation and iron levels were also higher in mice treated with iron as compared to non-iron-treated mice. These data suggest that progressive iron overload can enhance the tumor promotion ability of TPA/BPO in DMBA-initiated murine skin.     

Hemochromatosis: Evaluation of the dietary iron model and regulation of hepcidin

Our knowledge of iron homeostasis has increased steadily over the last two decades; much of this has been made possible through the study of animal models of iron-related disease. Analysis of transgenic mice with deletions or perturbations in genes known to be involved in systemic or local regulation of iron metabolism has been particularly informative. The effect of these genes on iron accumulation and hepcidin regulation is traditionally compared with wildtype mice fed a high iron diet, most often a 2% carbonyl iron diet. Recent studies have indicated that a very high iron diet could be detrimental to the health of the mice and could potentially affect homeostasis of other metals, for example zinc and copper. We analyzed mice fed a diet containing either 0.25%, 0.5%, 1% or 2% carbonyl iron for two weeks and compared them with mice on a control diet. Our results indicate that a 0.25% carbonyl iron diet is sufficient to induce maximal hepatic hepcidin response. Importantly these results also demonstrate that in a chronic setting of iron administration, the amount of excess hepatic iron may not further influence hepcidin regulation and that expression of hepcidin plateaus at lower hepatic iron levels. These studies provide further insights into the regulation of this important hormone.     

Drosophila multicopper oxidase 3 is a potential ferroxidase involved in iron homeostasis

Multicopper oxidases (MCOs) are a specific group of enzymes that contain multiple copper centers through which different substrates are oxidized. Main members of MCO family include ferroxidases, ascorbate oxidases, and laccases. MCO type of ferroxidases is key to iron transport across the plasma membrane. In Drosophila, there are four potential multicopper oxidases, MCO1–4. No convincing evidence has been presented so far to indicate any of these, or even any insect multicopper oxidase, to be a ferroxidase. Here we show Drosophila MCO3 (dMCO3) is highly likely a bona fide ferroxidase. In vitro activity assay with insect-cell-expressed dMCO3 demonstrated it has potent ferroxidase activity. Meanwhile, the ascorbate oxidase and laccase activities of dMCO3 are much less significant. dMCO3 expression in vivo, albeit at low levels, appears mostly extracellular, reminiscent of mammalian ceruloplasmin in the serum. A null dMCO3 mutant, generated by CRISPR/Cas9 technology, showed disrupted iron homeostasis, evidenced by increased iron level and reduced metal importer Mvl expression. Notably, dMCO3-null flies phenotypically are largely normal at normal or iron stressed-conditions. We speculate the likely existence of a similar iron efflux apparatus as the mammalian ferroportin/ferroxidase in Drosophila. However, its importance to fly iron homeostasis is greatly minimized, which is instead dominated by another iron efflux avenue mediated by the ZIP13-ferritin axis along the ER/Golgi secretion pathway.     

The role of active oxygen in iron tolerance of rice (Oryza sauva L.)

Summary Iron tolerance of rice (Oryza sativa L.) was investigated using an oxygen depleted hydroculture system. Treatment with high concentrations of Fe²⁺ induced yellowing and bronzing symptoms as well as iron coatings at the root surface. Root and shoot growth were inhibited by increasing iron concentration in the medium. All symptoms were more pronounced in an iron sensitive cultivar (IR 64) compared to an iron tolerant one (IR 9764-45-2). Superoxide dismutase and peroxidase activity of root extracts of IR 97 were about twice that of IR 64 in untreated control plants. No significant increase of peroxidase activity was detected with increasing iron concentration in the medium. Catalase activity of IR 64 was slightly higher than that of IR 97, independent of iron concentration.Abbreviations SOD Superoxide dismutase (EC 1.15.1.1) - POD peroxidase (EC 1.11.1.7) - EDTA ethylenediamintetraacetic acid - fwt fresh weight - Hepes (N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]) - BSA bovine serum albumin - IR 97 IR 9764-45-2 an iron tolerant rice cultivar - IR 64 iron sensitive rice cultivar - PM plasma membrane     

Genetic and biochemical analysis of high iron toxicity in yeast: iron toxicity is due to the accumulation of cytosolic iron and occurs under both aerobic and anaerobic conditions

Iron storage in yeast requires the activity of the vacuolar iron transporter Ccc1. Yeast with an intact CCC1 are resistant to iron toxicity, but deletion of CCC1 renders yeast susceptible to iron toxicity. We used genetic and biochemical analysis to identify suppressors of high iron toxicity in Δccc1 cells to probe the mechanism of high iron toxicity. All genes identified as suppressors of high iron toxicity in aerobically grown Δccc1 cells encode organelle iron transporters including mitochondrial iron transporters MRS3, MRS4, and RIM2. Overexpression of MRS3 suppressed high iron toxicity by decreasing cytosolic iron through mitochondrial iron accumulation. Under anaerobic conditions, Δccc1 cells were still sensitive to high iron toxicity, but overexpression of MRS3 did not suppress iron toxicity and did not result in mitochondrial iron accumulation. We conclude that Mrs3/Mrs4 can sequester iron within mitochondria under aerobic conditions but not anaerobic conditions. We show that iron toxicity in Δccc1 cells occurred under both aerobic and anaerobic conditions. Microarray analysis showed no evidence of oxidative damage under anaerobic conditions, suggesting that iron toxicity may not be solely due to oxidative damage. Deletion of TSA1, which encodes a peroxiredoxin, exacerbated iron toxicity in Δccc1 cells under both aerobic and anaerobic conditions, suggesting a unique role for Tsa1 in iron toxicity.     

Augmentation of hepatic and renal oxidative stress and disrupted glucose homeostasis by monocrotophos in streptozotocin-induced diabetic rats

Several recent studies have demonstrated that organophosphorus insecticides (OPI) possess the potential to disrupt glucose homeostasis leading to hyperglycemia in experimental animals. The propensity of OPI to induce hyperglycemia along with oxidative stress may have far-reaching consequences on diabetic outcomes and associated complications. The primary objective of this study was to assess the potential of monocrotophos (MCP), an extensively used OPI, on hepatic and renal oxidative stress markers and dysregulation of hepatic glucose homeostasis in experimentally induced diabetic rats. Rats rendered diabetic by a single dose of streptozotocin (60 mg/kg b.w) were orally administered MCP (0.9 mg/kg b.w/d for 5 d). Monocrotophos per se caused only a marginal increase in blood glucose levels but significantly elevated the blood glucose levels and also disrupted glucose homeostasis by depleting liver glycogen content and increasing the gluconeogenetic enzyme activities in diabetic rats. Experimentally induced diabetes was also associated with alterations in antioxidant enzymes in liver and kidney. MCP markedly enhanced lipid peroxidation in kidney and altered the enzymatic antioxidant defense mechanisms in both liver and kidney of diabetic rats. Collectively our data provides evidence that MCP has the propensity to augment the oxidative stress and further disrupt glucose homeostasis in diabetic rats.     

Heme-dependent metalloregulation by the iron response regulator (Irr) protein in Rhizobium and other Alpha-proteobacteria

Perception and response to nutritional iron by bacteria is essential for viability, and for the ability to adapt to the environment. The iron response regulator (Irr) is part of a novel regulatory scheme employed by Rhizobium and other Alpha-Proteobacteria to control iron-dependent gene expression. Bradyrhizobium japonicum senses iron through the status of heme biosynthesis to regulate gene expression, thus it responds to an iron-dependent process rather than to iron directly. Irr mediates this response by interacting directly with ferrochelatase, the enzyme that catalyzes the final step in heme biosynthesis. Irr is expressed under iron limitation to both positively and negatively modulate gene expression, but degrades in response to direct binding to heme in iron-sufficient cells. Studies with Rhizobium reveal that the regulation of iron homeostasis in bacteria is more diverse than has been generally assumed.