In vivo growth of Staphylococcus lugdunensis is facilitated by the concerted function of heme and non-heme iron acquisition mechanisms

Acquisition of iron underpins the ability of pathogens to cause disease and Staphylococcus lugdunensis has increasingly been recognized as a pathogen that can cause serious infection. In this study, we sought to address the knowledge gap that exists regarding the iron acquisition mechanisms employed by S. lugdunensis, especially during infection of the mammalian host. Here we show that S. lugdunensis utilizes diverse genome encoded iron acquisition mechanisms to satisfy its need for this nutrient. Indeed, S. lugdunensis can usurp hydroxamate siderophores, and staphyloferrin A and B from S. aureus, using the fhuC ATPase-encoding gene. Acquisition of catechol siderophores and catecholamine stress hormones necessitates the presence of the sst-1 transporter-encoding locus, but not the sst-2 locus. Iron-dependent growth in acidic culture conditions necessitates the feoAB locus. Heme iron is acquired via expression of the iron-regulated surface determinant (isd) locus. During systemic infection of mice we demonstrate that while S. lugdunensis does not cause overt illness, it does colonize and proliferate to high numbers in the kidneys. By combining mutations in the various iron acquisition loci, we further demonstrate that only a strain mutated for all of isd, fhuC, sst-1, and feo, versus combination mutants carrying wild type copies of any one of those loci, was attenuated in its ability to proliferate to high numbers in kidneys. Taken together our data reveal that S. lugdunensis requires a repertoire of both heme and non-heme iron acquisition mechanisms to proliferate during systemic infection of mammals

Aging is associated with increased brain iron through cortex-derived hepcidin expression

Iron is an essential molecule for biological processes, but its accumulation can lead to oxidative stress and cellular death. Due to its oxidative effects, iron accumulation is implicated in the process of aging and neurodegenerative diseases. However, the mechanism for this increase in iron with aging, and whether this increase is localized to specific cellular compartment(s), are not known. Here, we measured the levels of iron in different tissues of aged mice, and demonstrated that while cytosolic non-heme iron is increased in the liver and muscle tissue, only the aged brain cortex exhibits an increase in both the cytosolic and mitochondrial non-heme iron. This increase in brain iron is associated with elevated levels of local hepcidin mRNA and protein in the brain. We also demonstrate that the increase in hepcidin is associated with increased ubiquitination and reduced levels of the only iron exporter, ferroportin-1 (FPN1). Overall, our studies provide a potential mechanism for iron accumulation in the brain through increased local expression of hepcidin, and subsequent iron accumulation due to decreased iron export. Additionally, our data support that aging is associated with mitochondrial and cytosolic iron accumulation only in the brain and not in other tissues.

Molecular insights into the unusually promiscuous and catalytically versatile Fe(II)/α-ketoglutarate-dependent oxygenase SptF

Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis revealed that SptF catalyzes several continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced unique cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF revealed the molecular basis of the enzyme reactions, and suggested that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions.

A proteomics informed by transcriptomics insight into the proteome of Ornithodoros erraticus adult tick saliva

Background The argasid tick Ornithodoros erraticus is the main vector of tick-borne human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin. The prevention and control of these diseases would greatly benefit from the elimination of O. erraticus populations, and anti-tick vaccines are envisaged as an effective and sustainable alternative to chemical acaricide usage for tick control. Ornithodoros erraticus saliva contains bioactive proteins that play essential functions in tick feeding and host defence modulation, which may contribute to host infection by tick-borne pathogens. Hence, these proteins could be candidate antigen targets for the development of vaccines aimed at the control and prevention of O. erraticus infestations and the diseases this tick transmits. The objective of the present work was to obtain and characterise the proteome of the saliva of O. erraticus adult ticks as a means to identify and select novel salivary antigen targets. Methods A proteomics informed by transcriptomics (PIT) approach was applied to analyse samples of female and male saliva separately using the previously obtained O. erraticus sialotranscriptome as a reference database and two different mass spectrometry techniques, namely liquid chromatography–tandem mass spectrometry (LC–MS/MS) in data-dependent acquisition mode and sequential window acquisition of all theoretical fragment ion spectra MS (SWATH-MS). Results Up to 264 and 263 proteins were identified by LC–MS/MS in the saliva of O. erraticus female and male ticks, respectively, totalling 387 non-redundant proteins. Of these, 224 were further quantified by SWATH-MS in the saliva of both male and female ticks. Quantified proteins were classified into 23 functional categories and their abundance compared between sexes. Heme/iron-binding proteins, protease inhibitors, proteases, lipocalins and immune-related proteins were the categories most abundantly expressed in females, while glycolytic enzymes, protease inhibitors and lipocalins were the most abundantly expressed in males. Ninety-seven proteins were differentially expressed between the sexes, of which 37 and 60 were overexpressed in females and males, respectively. Conclusions The PIT approach demonstrated its usefulness for proteomics studies of O. erraticus, a non-model organism without genomic sequences available, allowing the publication of the first comprehensive proteome of the saliva of O. erraticus reported to date. These findings confirm important quantitative differences between sexes in the O. erraticus saliva proteome, unveil novel salivary proteins and functions at the tick–host feeding interface and improve our understanding of the physiology of feeding in O. erraticus ticks. The integration of O. erraticus sialoproteomic and sialotranscriptomic data will drive a more rational selection of salivary candidates as antigen targets for the development of vaccines aimed at the control of O. erraticus infestations and the diseases it transmits. Graphical Abstract

Catfish Oil (Pangasius hypophthalmus) effect to ferritin and sTfR in iron deficiency anemia

Background: Iron deficiency anemia is a micronutrient problem and the prevalence is still high. Catfish oil (Pangasius hypophthalmus) is a natural source of heme iron which can improve body iron levels.Objectives: This study was aimed to examine and analyze the effect of catfish oil on ferritin and sTfR levels in male wistar rats with iron deficiency anemia models.Materials and Methods: This study was conducted on male wistar rats which were divided into groups C- (standard feed), C+ (standard feed but had the iron removed), X1 (standard feed without iron but was supplemented with catfish oil), X2 (standard feed without iron but was supplemented with ferrous sulfate) for 14 days. Ferritin and sTfR levels were measured before and after intervention using ELISA.Results: The study showed an increase ferritin levels in X1 (21.87 ng/ml ±0.76), X2 (24.47 ng/ml ±0.54) and there was no significant difference between the two (p=0.069; p>0.05); a decrease in C- (0.25 ng/ml ±0.43), C+ (0.32 ng/ml ±0.059) (p=0.00; p<0.05). The sTfR levels decreased before and after intervention (p=0.00; p<0.05) in C+ (0.24 μ/mL ±0.99), X1 (60.66 μ/mL ±0.29), X2 (62.10 μ/mL ±0.90) and increased in C- (0.40 μ/mL ±0.97).Conclusions: The study indicates ferritin levels increased in the rats receiving catfish oil is not different from the rats that received ferrous sulfate and sTfR levels decreased significantly in wistar rats with iron deficiency anemia receiving catfish oil although the results were not as good as ferrous sulfate supplementation

Sedimentary Cobalt Protoporphyrin as a Potential Precursor of Prosthetic Heme Group for Bacteria Inhabiting Fossil Organic Matter-Rich Shale Rock

This study hypothesizes that bacteria inhabiting shale rock affect the content of the sedimentary cobalt protoporphyrin present in it and can use it as a precursor for heme synthesis. To verify this hypothesis, we conducted qualitative and quantitative comparative analyses of cobalt protoporphyrin as well as heme, and heme iron in shale rock that were (i) inhabited by bacteria in the field, (ii) treated with bacteria in the laboratory, and with (iii) bacterial culture on synthetic cobalt protoporphyrin. Additionally, we examined the above-mentioned samples for the presence of enzymes involved in the heme biosynthesis and uptake as well as hemoproteins. We found depletion of cobalt protoporphyrin and a much higher heme concentration in the shale rock inhabited by bacteria in the field as well as the shale rock treated with bacteria in the laboratory. Similarly, we observed the accumulation of protoporphyrin in bacterial cells grown on synthetic cobalt protoporphyrin. We detected numerous hemoproteins in metaproteome of bacteria inhabited shale rock in the field and in proteomes of bacteria inhabited shale rock and synthetic cobalt protoporhyrin in the laboratory, but none of them had all the enzymes involved in the heme biosynthesis. However, proteins responsible for heme uptake, ferrochelatase and sirohydrochlorin cobaltochelatase/sirohydrochlorin cobalt-lyase were detected in all studied samples.

15-Lipoxygenase Worsens Renal Fibrosis, Inflammation, and Metabolism in a Murine Model of Ureteral Obstruction

INTRODUCTION: 15-Lipoxygenase (15-LO) is a non-heme iron-containing dioxygenase that has both pro- and anti-inflammatory roles in many tissues and disease states. 15-LO is thought to influence macrophage phenotype; and silencing 15-LO reduces fibrosis after acute inflammatory triggers. The goal of this study was to determine if altering 15-LO expression influences inflammation and fibrogenesis in a murine model of unilateral ureteral obstruction (UUO). METHODS: C57BL/6J mice, 15-lipoxygenase knockout (Alox15-/-) mice, and 15-lipoxygenase transgenic overexpressing mice (15LOTG) were subjected UUO and kidneys were analyzed at 3, 10, and 14-days post injury. Histology for fibrosis, cytokine quantification, flow cytometry, and metabolomics were performed on injured tissues and controls. PD146176, a specific 15-LO inhibitor, was used to complement studies involving knockout animals. RESULTS: Compared to WT animals undergoing UUO, Alox15-/- mouse kidneys had less pro-inflammatory, pro-fibrotic message along with less fibrosis. PD146176 inhibited 15-LO, and resulted in reduced fibrosis similar to Alox15-/- mice. Flow cytometry revealed that Alox15-/- UUO-injured kidneys had a dynamic change in macrophage phenotype, with an early blunting of CD11bHiLy6CHi "M1" macrophages and increase in anti-inflammatory CD11bHiLy6CInt "M2c" macrophages and reduced expression of the fractalkine receptor, CX3CR1. Many of these findings were reversed when UUO was performed on 15LOTG mice. Metabolomics analysis revealed that WT kidneys developed a glycolytic shift post-injury, while Alox15-/- kidneys exhibited increased oxidative phosphorylation. CONCLUSIONS: 15-LO manipulation by genetic or pharmacologic means induces dynamic changes in the inflammatory microenvironment in the UUO-model and appears to be critical in the progression of UUO-induced fibrosis.