Proteomic analysis of iron acquisition,metabolic and regulatory responses of <Emphasis Type="Italic">Yersinia pestis</Emphasis> to iron starvation

Background  

The Gram-negative bacterium Yersinia pestis is the causative agent of the bubonic plague. Efficient iron acquisition systems are critical to the ability of Y. pestis to infect, spread and grow in mammalian hosts, because iron is sequestered and is considered part of the innate host immune defence against invading pathogens. We used a proteomic approach to determine expression changes of iron uptake systems and intracellular consequences of iron deficiency in the Y. pestis strain KIM6+ at two physiologically relevant temperatures (26°C and 37°C).     

PfeT,a P1B4‐type ATPase,effluxes ferrous iron and protects Bacillus subtilis against iron intoxication

Iron is an essential element for nearly all cells and limited iron availability often restricts growth. However, excess iron can also be deleterious, particularly when cells expressing high affinity iron uptake systems transition to iron rich environments. Bacillus subtilis expresses numerous iron importers, but iron efflux has not been reported. Here, we describe the B. subtilis PfeT protein (formerly YkvW/ZosA) as a P_1B4‐type ATPase in the PerR regulon that serves as an Fe(II) efflux pump and protects cells against iron intoxication. Iron and manganese homeostasis in B. subtilis are closely intertwined: a pfeT mutant is iron sensitive, and this sensitivity can be suppressed by low levels of Mn(II). Conversely, a pfeT mutant is more resistant to Mn(II) overload. In vitro, the PfeT ATPase is activated by both Fe(II) and Co(II), although only Fe(II) efflux is physiologically relevant in wild‐type cells, and null mutants accumulate elevated levels of intracellular iron. Genetic studies indicate that PfeT together with the ferric uptake repressor (Fur) cooperate to prevent iron intoxication, with iron sequestration by the MrgA mini‐ferritin playing a secondary role. Protection against iron toxicity may also be a key role for related P_1B4‐type ATPases previously implicated in bacterial pathogenesis.     

Smad7 deficiency decreases iron and haemoglobin through hepcidin up‐regulation by multilayer compensatory mechanisms

To maintain iron homoeostasis, the iron regulatory hormone hepcidin is tightly controlled by BMP‐Smad signalling pathway, but the physiological role of Smad7 in hepcidin regulation remains elusive. We generated and characterized hepatocyte‐specific Smad7 knockout mice (Smad7^Alb/Alb), which showed decreased serum iron, tissue iron, haemoglobin concentration, up‐regulated hepcidin and increased phosphor‐Smad1/5/8 levels in both isolated primary hepatocytes and liver tissues. Increased levels of hepcidin lead to reduced expression of intestinal ferroportin and mild iron deficiency anaemia. Interestingly, we found no difference in hepcidin expression or phosphor‐Smad1/5/8 levels between iron‐challenged Smad7^Alb/Alb and Smad7^flox/flox, suggesting other factors assume the role of iron‐induced hepcidin regulation in Smad7 deletion. We performed RNA‐seq to identify differentially expressed genes in the liver. Significantly up‐regulated genes were then mapped to pathways, revealing TGF‐β signalling as one of the most relevant pathways, including the up‐regulated genes Smad6, Bambi and Fst (Follistatin). We found that Smad6 and Bambi—but not Follistatin—are controlled by the iron‐BMP–Smad pathway. Overexpressing Smad6, Bambi or Follistatin in cells significantly reduced hepcidin expression. Smad7 functions as a key regulator of iron homoeostasis by negatively controlling hepcidin expression, and Smad6 and Smad7 have non‐redundant roles. Smad6, Bambi and Follistatin serve as additional inhibitors of hepcidin in the liver.     

Transmission electron microscopy X-ray microanalysis of two algae of the generaScenedesmus andSiderocelis

Summary Mineral deposits have been found in the cell walls of two newly described green algae,Siderocelis minor Crawford andScenedesmus granulatus West et West f.salina Crawford. A morphological account using transmission electron microscopy coupled with X-ray micro-analysis has shown that the deposits consist chiefly of iron and manganese though other elements were also found. Several possible explanations have been suggested for the variation in iron/manganese ratio—the ecological conditions pertaining in the natural habitat being considered particularly relevant.     

Iron acquisition by Streptococcus species: An updated review

Streptococcus is a genus of spherical Gram-positive bacteria responsible for many cases of meningitis, bacterial pneumonia, endocarditis, erysipelas, and necrotizing fasciitis. To survive in the host environment with limited free iron available, Streptococcus species have developed various mechanisms to uptake iron as an essential nutrient. They can directly extract the metal ions from host iron-containing proteins such as ferritin, transferrin, lactoferrin, and hemoproteins. Other iron-uptake strategies, which are broadly distributed in the strains, include the employment of specialized secreted hemophores to acquire heme and the usage of small molecules called siderophores as high-affinity ferric chelators. This review intends to discuss the most recent discoveries of these iron acquisition systems and their relevant regulators in Streptococcus species.     

Genomic insights into the iron uptake mechanisms of the biomining microorganism Acidithiobacillus ferrooxidans

Commercial bioleaching of copper and the biooxidation of gold is a cost-effective and environmentally friendly process for metal recovery. A partial genome sequence of the acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans is available from two public sources. This information has been used to build preliminary models that describe how this microorganism confronts unusually high iron loads in the extremely acidic conditions (pH 2) found in natural environments and in bioleaching operations. A. ferrooxidans contains candidate genes for iron uptake, sensing, storage, and regulation of iron homeostasis. Predicted proteins exhibit significant amino acid similarity with known proteins from neutrophilic organisms, including conservation of functional motifs, permitting their identification by bioinformatics tools and allowing the recognition of common themes in iron transport across distantly related species. However, significant differences in amino acid sequence were detected in pertinent domains that suggest ways in which the periplasmic and outer membrane proteins of A. ferrooxidans maintain structural integrity and relevant protein-protein contacts at low pH. Unexpectedly, the microorganism also contains candidate genes, organized in operon-like structures that potentially encode at least 11 siderophore systems for the uptake of Fe(III), although it does not exhibit genes that could encode the biosynthesis of the siderophores themselves. The presence of multiple Fe(III) uptake systems suggests that A. ferrooxidans can inhabit aerobic environments where iron is scarce and where siderophore producers are present. It may also help to explain why it cannot tolerate high Fe(III) concentrations in bioleaching operations where it is out-competed by Leptospirillum species.

     

Polymorphisms in calpastatin and mu‐calpain genes are associated with beef iron content

The objective of this study was to assess the association of markers in the calpastatin and mu‐calpain loci with iron in beef cattle muscle. The population consisted of 259 cross‐bred steers from Beefmaster, Brangus, Bonsmara, Romosinuano, Hereford and Angus sires. Total iron and heme iron concentrations were measured. Markers in the calpastatin (referred to as CAST) and mu‐calpain (referred to as CAPN4751) genes were used to assess their association with iron levels. The mean and standard error for iron and heme iron content in the population was 35.6 ± 1.3 μg and 27.1 ± 1.4 μg respectively. Significant associations (P < 0.01) of markers were observed for both iron and heme iron content. For CAST, animals with the CC genotype had higher levels of iron and heme iron in longissimus dorsi muscle. For CAPN4751, individuals with the TT genotype had higher concentrations of iron and heme iron than did animals with the CC and CT genotypes. Genotypes known to be associated with tougher meat were associated with higher levels of iron concentration.     

Mechanisms of neuroprotection by hemopexin: modeling the control of heme and iron homeostasis in brain neurons in inflammatory states

Hemopexin provides neuroprotection in mouse models of stroke and intracerebral hemorrhage and protects neurons in vitro against heme or reactive oxygen species (ROS) toxicity via heme oxygenase‐1 (HO1) activity. To model human brain neurons experiencing hemorrhages and inflammation, we used human neuroblastoma cells, heme–hemopexin complexes, and physiologically relevant ROS, for example, H₂O₂ and HOCl, to provide novel insights into the underlying mechanism whereby hemopexin safely maintains heme and iron homeostasis. Human amyloid precursor protein (hAPP), needed for iron export from neurons, is induced ~twofold after heme–hemopexin endocytosis by iron from heme catabolism via the iron‐regulatory element of hAPP mRNA. Heme–hemopexin is relatively resistant to damage by ROS and retains its ability to induce the cytoprotective HO1 after exposure to tert‐butylhydroperoxide, although induction is impaired, but not eliminated, by exposure to high concentrations of H₂O₂ in vitro. Apo‐hemopexin, which predominates in non‐hemolytic states, resists damage by H₂O₂ and HOCl, except for the highest concentrations likely in vivo. Heme–albumin and albumin are preferential targets for ROS; thus, albumin protects hemopexin in biological fluids like CSF and plasma where it is abundant. These observations provide strong evidence that hemopexin will be neuroprotective after traumatic brain injury, with heme release in the CNS, and during the ensuing inflammation. Hemopexin sequesters heme, thus preventing unregulated heme uptake that leads to toxicity; it safely delivers heme to neuronal cells; and it activates the induction of proteins including HO1 and hAPP that keep heme and iron at safe levels in neurons.     

Age-related changes in iron homeostasis in mouse ferroxidase mutants

Disorders of iron metabolism are a significant problem primarily in young and old populations. In this study, We compared 1-year-old C57BL6/J mice on iron deficient, iron overload, or iron sufficient diets with two similarly aged genetic models of disturbed iron homeostasis, the sla (sex-linked anemia), and the ceruloplasmin knockout mice (Cp ^−/−) on iron sufficient diet. We found tissue specific changes in sla and nutritional iron deficiency including decreased liver Hamp1 expression and increased protein expression of the enterocyte basolateral iron transport components, hephaestin and ferroportin. In contrast, the Cp ^−/− mice did not show significantly increased Hamp1 expression despite increased liver iron suggesting that regulation is independent of liver iron levels. Together, these results suggest that older mice have a distinct response to alterations in iron metabolism and that age must be considered in future studies of iron metabolism.     

Superoxide dismutase 1 modulates expression of transferrin receptor

Copper–zinc superoxide dismutase (SOD1) plays a protective role against the toxicity of superoxide, and studies in Saccharomyces cerevisiae and in Drosophila have suggested an additional role for SOD1 in iron metabolism. We have studied the effect of the modulation of SOD1 levels on iron metabolism in a cultured human glial cell line and in a mouse motoneuronal cell line. We observed that levels of the transferrin receptor and the iron regulatory protein 1 were modulated in response to altered intracellular levels of superoxide dismutase activity, carried either by wild-type SOD1 or by an SOD-active amyotrophic lateral sclerosis (ALS) mutant enzyme, G93A-SOD1, but not by a superoxide dismutase inactive ALS mutant, H46R-SOD1. Ferritin expression was also increased by wild-type SOD1 overexpression, but not by mutant SOD1s. We propose that changes in superoxide levels due to alteration of SOD1 activity affect iron metabolism in glial and neuronal cells from higher eukaryotes and that this may be relevant to diseases of the nervous system.     

Iron homeostasis in <Emphasis Type="Italic">Brucella abortus</Emphasis>: the role of bacterioferritin

Brucella abortus is the etiological agent of bovine brucellosis, an infectious disease of humans and cattle. Its pathogenesis is mainly based on its ability to survive and multiply inside macrophages. It has been demonstrated that if B. abortus ferrochelatase cannot incorporate iron into protoporphyrin IX to synthesize heme, the intracellular replication and virulence in mice is highly attenuated. Therefore, it can be hypothesized that the unavailability of iron could lead to the same attenuation in B. abortus pathogenicity. Thus, the purpose of this work was to obtain a B. abortus derivative unable to keep an internal iron pool and test its ability to replicate under iron limitation. To achieve this, we searched for iron-storage proteins in the genome of brucellae and found bacterioferritin (Bfr) as the sole ferritin encoded. Then, a B. abortus bfr mutant was built up and its capacity to store iron and replicate under iron limitation was investigated. Results indicated that B. abortus Bfr accounts for 70% of the intracellular iron content. Under iron limitation, the bfr mutant suffered from enhanced iron restriction with respect to wild type according to its growth retardation pattern, enhanced sensitivity to oxidative stress, accelerated production of siderophores, and altered expression of membrane proteins. Nonetheless, the bfr mutant was able to adapt and replicate even inside eukaryotic cells, indicating that B. abortus responds to internal iron starvation before sensing external iron availability. This suggests an active role of Bfr in controlling iron homeostasis through the availability of Bfr-bound iron.     

Phylogenetic and sequence analyses of insect transferrins suggest that only transferrin 1 has a role in iron homeostasis

Iron is essential to life,but surprisingly little is known about how iron is managed in nonvertebrate animals.In mammals,the well-characterized transferrins bind iron and are involved in iron transport or immunity,whereas other members of the transferrin family do not have a role in iron homeostasis.In insects,the functions of transferrins are still poorly understood.The goals of this project were to identify the transferrin genes in a diverse set of insect species,resolve the evolutionary relationships among these genes,and predict which of the transferrins are likely to have a role in iron homeostasis.Our phylogenetic analysis of transferrins from 16 orders of insects and two orders of noninsect hexapods demonstrated that there are four orthologous groups of insect transferrins.Our analysis suggests that transferrin 2 arose prior to the origin of insects,and transferrins/,i,and 4 arose early in insect evolution.Primary sequence analysis of each of the insect transferrins was used to predict signal peptides,carboxyl-terminal transmembrane regions,GPI-anchors,and iron binding.Based on this analysis,we suggest that transferrins 2,and 4 are unlikely to play a major role in iron homeostasis.In contrast,the transferrin 1 orthologs are predicted to be secreted,soluble,iron-binding proteins.We conclude that transferrin 1 orthologs are the most likely to play an important role in iron homeostasis.Interestingly,it appears that the louse,aphid,and thrips lineages have lost the transferrin 1 gene and,thus,have evolved to manage iron without transferrins.     

Targeting intracellular transport combined with efficient uptake and storage significantly increases grain iron and zinc levels in rice

Rice, a staple food for more than half of the world population, is an important target for iron and zinc biofortification. Current strategies mainly focus on the expression of genes for efficient uptake, long‐distance transport and storage. Targeting intracellular iron mobilization to increase grain iron levels has not been reported. Vacuole is an important cell compartment for iron storage and the NATURAL RESISTANCE ASSOCIATED MACROPHAGE PROTEIN (NRAMP) family of transporters export iron from vacuoles to cytosol when needed. We developed transgenic Nipponbare rice lines expressing AtNRAMP3 under the control of the UBIQUITIN or rice embryo/aleurone‐specific 18‐kDa Oleosin (Ole18) promoter together with NICOTIANAMINE SYNTHASE (AtNAS1) and FERRITIN (PvFER), or expressing only AtNRAMP3 and PvFER together. Iron and zinc were increased close to recommended levels in polished grains of the transformed lines, with maximum levels when AtNRAMP3, AtNAS1 and PvFER were expressed together (12.67 μg/g DW iron and 45.60 μg/g DW zinc in polished grains of line NFON16). Similar high iron and zinc levels were obtained in transgenic Indica IR64 lines expressing the AtNRAMP3, AtNAS1 and PvFER cassette (13.65 μg/g DW iron and 48.18 μg/g DW zinc in polished grains of line IR64_1), equalling more than 90% of the recommended iron increase in rice endosperm. Our results demonstrate that targeting intracellular iron stores in combination with iron and zinc transport and endosperm storage is an effective strategy for iron biofortification. The increases achieved in polished IR64 grains are of dietary relevance for human health and a valuable nutrition trait for breeding programmes.