Quantitative Proteomics of Tissue-Infiltrating T Cells From CRC Patients Identified Lipocalin-2 Induces T-Cell Apoptosis and Promotes Tumor Cell Proliferation by Iron Efflux.

T cells play the most pivotal roles in antitumor immunity; the T-cell proteome and the differentially expressed proteins in the tumor immune microenvironment have rarely been identified directly from the clinical samples, especially for tumors that lack effective immunotherapy targets, such as colorectal cancer (CRC). In this study, we analyzed the protein expression pattern of the infiltrating T cells isolated from CRC patients using quantitative proteomics. CD4+ and CD8+ T cells were isolated from clinical samples and labeled by tandem mass tag reagents, and the differentially expressed proteins were quantified by mass spectrometry. The T-cell proteome profiling revealed dysfunctions in these tumor-infiltrating T cells. Specifically, antitumor immunity was suppressed because of differentially expressed metal ion transporters and immunity regulators. For the first time, lipocalin-2 (LCN2) was shown to be significantly upregulated in CD4+ T cells. Quantitative proteomic analysis of LCN2-overexpressed Jurkat cells showed that LCN2 damaged T cells by changes in iron transport. LCN2 induced T-cell apoptosis by reducing cellular iron concentration; moreover, the iron that was transported to the tumor microenvironment aided tumor cell proliferation, promoting tumor development. Meanwhile, LCN2 also influenced tumor progression through immune cytokines and cholesterol metabolism. Our results demonstrated that LCN2 has immunosuppressive functions that can promote tumor development; therefore, it is a potential immunotherapy target for CRC.

In vitro reconstitution of transition metal transporters.

Transition metal ions are critically important across all kingdoms of life. The chemical properties of iron, copper, zinc, manganese, cobalt, and nickel make them very attractive for use as cofactors in metalloenzymes and/or metalloproteins. Their versatile chemistry in aqueous solution enables them to function both as electron donors and acceptors, and thus participate in both reduction and oxidation reactions respectively. Transition metal ions can also function as nonredox multidentate coordination sites that play essential roles in macromolecular structure and function. Malfunction in transition metal transport and homeostasis has been linked to a wide number of human diseases including cancer, diabetes, and neurodegenerative disorders. Transition metal transporters are central players in the physiology of transition metals whereby they move transition metals in and out of cellular compartments. In this review, we provide a comprehensive overview of in vitro reconstitution of the activity of integral membrane transition metal transporters and discuss strategies that have been successfully implemented to overcome the challenges. We also discuss recent advances in our understanding of transition metal transport mechanisms and the techniques that are currently used to decipher the molecular basis of transport activities of these proteins. Deep mechanistic insights into transition metal transport systems will be essential to understand their malfunction in human diseases and target them for potential therapeutic strategies.

Siderophore-mediated iron acquisition by Klebsiella pneumoniae.

Microbes synthesize and secrete siderophores, that bind and solubilize precipitated or otherwise unavailable iron in their microenvironments. Gram (-) bacterial TonB-dependent outer membrane receptors capture the resulting ferric siderophores to begin the uptake process. From their similarity to fepA, the structural gene for the Escherichia coli ferric enterobactin (FeEnt) receptor, we identified four homologous genes in the human and animal ESKAPE pathogen Klebsiella pneumoniae (strain Kp52.145). One locus encodes IroN (locus 0027 on plasmid pII), and three other loci encode other FepA orthologs/paralogs (chromosomal loci 1658, 2380, and 4984). Based on the crystal structure of E. coli FepA (1FEP), we modeled the tertiary structures of the K. pneumoniae FepA homologs and genetically engineered individual Cys substitutions in their predicted surface loops. We subjected bacteria expressing the Cys mutant proteins to modification with extrinsic fluorescein maleimide (FM) and used the resulting fluorescently labeled cells to spectroscopically monitor the binding and transport of catecholate ferric siderophores by the four different receptors. The FM-modified FepA homologs were nanosensors that defined the ferric catecholate uptake pathways in pathogenic strains of K. pneumoniae. In Kp52.145, loci 1658 and 4984 encoded receptors that primarily recognized and transported FeEnt; locus 0027 produced a receptor that principally bound and transported FeEnt and glucosylated FeEnt (FeGEnt); locus 2380 encoded a protein that bound ferric catecholate compounds but did not detectably transport them. The sensors also characterized the uptake of iron complexes, including FeGEnt, by the hypervirulent, hypermucoviscous K. pneumoniae strain hvKp1. IMPORTANCE: Both commensal and pathogenic bacteria produce small organic chelators, called siderophores, that avidly bind iron and increase its bioavailability. Klebsiella pneumoniae variably produces four siderophores that antagonize host iron sequestration: enterobactin, glucosylated enterobactin (also termed salmochelin), aerobactin, and yersiniabactin, which promote colonization of different host tissues. Abundant evidence links bacterial iron acquisition to virulence and infectious diseases. The data we report explain the recognition and transport of ferric catecholates and other siderophores, which are crucial to iron acquisition by K. pneumoniae.

A Haemophilus ducreyi strain lacking the yfeABCD iron transport system is virulent in human volunteers.

Haemophilus ducreyi causes the genital ulcer disease chancroid and painful cutaneous ulcers in children who live in the tropics. To acquire heme from the host, H. ducreyi expresses a TonB-dependent hemoglobin receptor, HgbA, which is necessary and sufficient for H. ducreyi to progress to the pustular stage of disease in a controlled human infection model. HgbA transports hemoglobin across the outer membrane; how heme is transported across the cytoplasmic membrane is unclear. In previous studies, transcripts encoding the YfeABCD heme transporter were upregulated in experimental lesions caused by H. ducreyi in human volunteers, suggesting the latter may have a role in virulence. Here we constructed a double deletion mutant, 35000HPΔyfeABΔyfeCD, which exhibited growth defects relative to its parent 35000HP in media containing human hemoglobin as an iron source. Five human volunteers were inoculated at three sites on the skin overlying the deltoid with each strain. The results of the trial showed that papules formed at 100% (95% CI, 71.5, 100) at both 35000HP and 35000HPΔyfeABΔyfeCD-inoculated sites (P = 1.0). Pustules formed at 60% (95% CI, 25.9, 94.1) at parent-inoculated sites and 53% (95% CI, 18.3, 88.4) at mutant-inoculated sites (P = 0.79). Thus, the ABC transporter encoded by yfeAB and yfeCD was dispensable for H. ducreyi virulence in humans. In the absence of YfeABCD, H. ducreyi likely utilizes other periplasmic binding proteins and ABC-transporters such as HbpA, SapABCDF, and DppBCDF to shuttle heme from the periplasm into the cytoplasm, underscoring the importance of redundancy of such systems in gram-negative pathogens.

Emergence of cefiderocol resistance during ceftazidime/avibactam treatment caused by a large genomic deletion, including ampD and piuCD genes, in Pseudomonas aeruginosa.

We report the emergence of cefiderocol resistance during the treatment of a ST312 Pseudomonas aeruginosa respiratory infection with ceftazidime/avibactam. whole genome sequencing (WGS) revealed that resistance was caused by a large genomic deletion, including PiuDC (iron transport system) and AmpD (ampC negative regulator), driven by the integration of phage DNA. Thus, our findings alert that this type of deletion could be an efficient (two mechanisms in one step) specific cefiderocol resistance mechanism that might occur nonspecifically upon treatment with ß-lactams that select for AmpC overexpression.

Identification of a system for hydroxamate xenosiderophore-mediated iron transport in Burkholderia cenocepacia.

One of the mechanisms employed by the opportunistic pathogen Burkholderia cenocepacia to acquire the essential element iron is the production and release of two ferric iron chelating compounds (siderophores), ornibactin and pyochelin. Here we show that B. cenocepacia is also able to take advantage of a range of siderophores produced by other bacteria and fungi ('xenosiderophores') that chelate iron exclusively by means of hydroxamate groups. These include the tris-hydroxamate siderophores ferrioxamine B, ferrichrome, ferricrocin and triacetylfusarinine C, the bis-hydroxamates alcaligin and rhodotorulic acid, and the monohydroxamate siderophore cepabactin. We also show that of the 24 TonB-dependent transporters encoded by the B. cenocepacia genome, two (FhuA and FeuA) are involved in the uptake of hydroxamate xenosiderophores, with FhuA serving as the exclusive transporter of iron-loaded ferrioxamine B, triacetylfusarinine C, alcaligin and rhodotorulic acid, while both FhuA and FeuA are able to translocate ferrichrome-type siderophores across the outer membrane. Finally, we identified FhuB, a putative cytoplasmic membrane-anchored ferric-siderophore reductase, as being obligatory for utilization of all of the tested bis- and tris-hydroxamate xenosiderophores apart from alcaligin.

Inhibition of Slc39a14/Slc39a8 reduce vascular calcification via alleviating iron overload induced ferroptosis in vascular smooth muscle cells.

BACKGROUND: Vascular calcification (VC) is an independent risk factor for cardiovascular diseases. Recently, ferroptosis has been recognised as a novel therapeutic target for cardiovascular diseases. Although an association between ferroptosis and vascular calcification has been reported, the role and mechanism of iron overload in vascular calcification are still poorly understood. Specifically, further in-depth research is required on whether metalloproteins SLC39a14 and SLC39a8 are involved in ferroptosis induced by iron overload. METHODS: R language was employed for the differential analysis of the dataset, revealing the correlation between ferroptosis and calcification. The experimental approaches encompassed both in vitro and in vivo studies, incorporating the use of iron chelators and models of iron overload. Additionally, gain- and loss-of-function experiments were conducted to investigate iron's effects on vascular calcification comprehensively. Electron microscopy, immunofluorescence, western blotting, and real-time polymerase chain reaction were used to elucidate how Slc39a14 and Slc39a8 mediate iron overload and promote calcification. RESULTS: Ferroptosis was observed in conjunction with vascular calcification (VC); the association was consistently confirmed by in vitro and in vivo studies. Our results showed a positive correlation between iron overload in VSMCs and calcification. Iron chelators are effective in reversing VC and iron overload exacerbates this process. The expression levels of the metal transport proteins Slc39a14 and Slc39a8 were significantly upregulated during calcification; the inhibition of their expression alleviated VC. Conversely, Slc39a14 overexpression exacerbates calcification and promotes intracellular iron accumulation in VSMCs. CONCLUSIONS: Our research demonstrates that iron overload occurs during VC, and that inhibition of Slc39a14 and Slc39a8 significantly relieves VC by intercepting iron overload-induced ferroptosis in VSMCs, providing new insights into the VC treatment.

Gene expression of iron transporters, markers of vascularization and structural integrity in placenta of mothers with and without anemia.

OBJECTIVE: To compare the mRNA expression of placental iron transporters (TfR-1 and FPN), markers of placental vascularization (VEGF and sFLT1) and marker of structural integrity (LMN-A) in term women with and without iron deficiency anemia. MATERIALS AND METHODS: A total of 30 pregnant women were enrolled; 15 cases of iron deficiency anemia (Hb 7-10.9 gm/dL) and 15 gestational age matched healthy controls (Hb ≥ 11 gm/dL). Peripheral venous blood was collected for assessment of hemoglobin levels and serum iron profile. Placental tissue was used for assessing the mRNA expression of TfR-1, FPN, VEGF, sFLT-1 and LMN-A via real time PCR. RESULTS: Placental expression of TfR-1, VEGF and LMN-A was increased in pregnant women with anemia compared to healthy pregnant controls. Placental expression of sFLT-1 was decreased in pregnant women with anemia compared to healthy pregnant controls. There was no change in the placental expression of FPN. CONCLUSION: The increased expression of TfR-1, VEGF and LMN-A in cases of iron deficiency anemia are most likely to be compensatory in nature to help maintain adequate fetal iron delivery. WHAT DOES THIS STUDY ADDS TO THE CLINICAL WORK: Compensatory changes in the placenta aimed at buffering transport of iron to the fetus are seen in pregnant women with anemia compared to healthy pregnant controls.

Dynamic Expressions of Yellow Stripe-Like (YSL) Genes During Pod Development Shed Light on Associations with Iron Distribution in Phaseolus vulgaris.

The global prevalence of iron deficiency-induced "hidden hunger" highlights a critical health concern, underscoring the pressing need to improve iron nutrition through safe and efficient means, such as increasing iron intake from plant-based foods. Yellow Stripe-Like (YSL) genes play a crucial role in long-distance iron transport between source and sink tissues in plants. Here, we report on the analysis of YSL family genes in the common bean (Phaseolus vulgaris L.), an iron-rich legume crop. We identified 9 YSL genes in the common bean genome using BLAST and HMM methods. Gene duplication analysis revealed that PvYSL7a and PvYSL7b originated through tandem duplication events. Structural analysis noted an absence of conservative motifs in PvYSL3b and PvYSL7a, which led to distinct predicted 3D protein structures. Leveraging publicly available RNA-seq data from developing bean pods, the expression patterns of PvYSL genes alongside pod and seed development were analyzed. Notably, PvYSL7a and PvYSL7b, as well as PvYSL1a and PvYSL1b, exhibited diverged expression patterns in seeds, signifying their functional divergence in this tissue. Moreover, PvYSL3a and PvYSL3b exhibited divergent expression patterns in both pod walls and seeds during pod development, underscoring their distinct roles in facilitating iron transportation between pods and seeds. This study provides valuable insights into the gene regulatory basis of iron accumulation in bean pods and seeds.

First detection of a cefiderocol-resistant and extensively drug-resistant Acinetobacter baumannii clinical isolate in Bulgaria.

Cefiderocol (CFDC) is a first-in-class siderophore cephalosporin with potent activity against multidrug-resistant Gram-negative bacteria including carbapenem-resistant Acinetobacter baumannii. The present study aimed to explore the CFDC resistance mechanisms of an extensively drug-resistant A. baumannii isolate from Bulgaria. The A. baumannii Aba52 strain (designated Aba52) was obtained in 2018 from a blood sample of a critically ill patient. The methodology included antimicrobial susceptibility testing, whole-genome sequencing (WGS), reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), multilocus sequence typing, and phylogenomic analysis. The isolate demonstrated high-level resistance to CFDC (MIC = 64 mg L-1), resistance to carbapenems, aminoglycosides, fluoroquinolones, sulfamethoxazole-trimethoprim, and tigecycline, as well as susceptibility only to colistin. WGS-based resistome analysis revealed the existence of blaOXA-23, blaOXA-66 and blaADC-73. Seven non-conservative missense mutations affecting iron transport-related genes were detected: exbD4 (p.Ser61Pro), tonB2 (p.Ala268Val), bauA (p.Thr61Ala), ftsI (p.Ala515Val), piuA (p.Gly216Val), and feoB (p.Ser429Pro and p.Thr595Ala). A variety of virulence factors associated with adherence, biofilm formation, enzyme production, immune invasion, iron uptake, quorum sensing, and two-component regulatory systems were identified, suggesting a significant pathogenic potential of Aba52. The performed RT-qPCR analysis showed diminished (0.17) and absent expression of the pirA and piuA genes, respectively, encoding TonB-dependent siderophore receptors. Aba52 belonged to the widespread high-risk sequence type ST2 (Pasteur scheme). To the best of our knowledge, this is the first documented case of CFDC-resistant A. baumannii in Bulgaria even though, CFDC has never been applied in our country. The emerging resistance highlights the crucial need for nationwide surveillance targeting the implementation of novel antibiotics.

Molecular insights into placental iron transfer mechanisms and maternofetal regulation.

PURPOSE: Adequate iron transportation from the mother across the placenta is crucial for fetal growth and establishing sufficient iron stores in neonates at birth. The past decade has marked significant discoveries in iron metabolism with the identification of new players and mechanisms. Immunohistochemical studies rendered valuable data on the localization of substantial iron transporters on placental syncytiotrophoblasts. However, the function and regulation of maternal-placentofetal iron transporters and iron handling is still elusive and requires more attention. METHODS: A thorough literature review was conducted to gather information about placental iron transfer, the role of regulators and maintenance of iron homeostasis. RESULTS: The role of classical and new players in maternal-fetal iron transport and the regulation in the placenta has been addressed in this review. Animal and human studies have been discussed. The role of placental iron regulation in thalassemia and hemochromatosis pregnancies has been reviewed. CONCLUSIONS: The current advances that highlight the mechanisms of placental iron regulation and transport in response to maternal and fetal signals have been presented.

Location, Location, Location: Establishing Design Principles for New Antibacterials from Ferric Siderophore Transport Systems.

This review strives to assemble a set of molecular design principles that enables the delivery of antibiotic warheads to Gram-negative bacterial targets (ESKAPE pathogens) using iron-chelating siderophores, known as the Trojan Horse strategy for antibiotic development. Principles are derived along two main lines. First, archetypical siderophores and their conjugates are used as case studies for native iron transport. They enable the consideration of the correspondence of iron transport and antibacterial target location. The second line of study charts the rationale behind the clinical antibiotic cefiderocol. It illustrates the potential versatility for the design of new Trojan Horse-based antibiotics. Themes such as matching the warhead to a location where the siderophore delivers its cargo (i.e., periplasm vs. cytoplasm), whether or not a cleavable linker is required, and the relevance of cheaters to the effectiveness and selectivity of new conjugates will be explored. The effort to articulate rules has identified gaps in the current understanding of iron transport pathways and suggests directions for new investigations.

Exploring the Biological Pathways of Siderophores and Their Multidisciplinary Applications: A Comprehensive Review.

Siderophores are a class of small molecules renowned for their high iron binding capacity, essential for all life forms requiring iron. This article provides a detailed review of the diverse classifications, and biosynthetic pathways of siderophores, with a particular emphasis on siderophores synthesized via nonribosomal peptide synthetase (NRPS) and non-NRPS pathways. We further explore the secretion mechanisms of siderophores in microbes and plants, and their role in regulating bioavailable iron levels. Beyond biological functions, the applications of siderophores in medicine, agriculture, and environmental sciences are extensively discussed. These applications include biological pest control, disease treatment, ecological pollution remediation, and heavy metal ion removal. Through a comprehensive analysis of the chemical properties and biological activities of siderophores, this paper demonstrates their wide prospects in scientific research and practical applications, while also highlighting current research gaps and potential future directions.

A fusion of the Bacteroides fragilis ferrous iron import proteins reveals a role for FeoA in stabilizing GTP-bound FeoB.

Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe2+ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system is the primary prokaryotic Fe2+ import machinery, and two constituent proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remains enigmatic. In this work, we explored the distribution of feoAB operons encoding a fusion of FeoA tethered to the N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion poises FeoA to interact with FeoB to affect function. To test this hypothesis, we characterized the soluble NFeoAB fusion protein from Bacteroides fragilis, a commensal organism implicated in drug-resistant infections. Using X-ray crystallography, we determined the 1.50-Å resolution structure of BfFeoA, which adopts an SH3-like fold implicated in protein-protein interactions. Using a combination of structural modeling, small-angle X-ray scattering, and hydrogen-deuterium exchange mass spectrometry, we show that FeoA and NFeoB interact in a nucleotide-dependent manner, and we mapped the protein-protein interaction interface. Finally, using guanosine triphosphate (GTP) hydrolysis assays, we demonstrate that BfNFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis that is not potassium-stimulated. Importantly, truncation of FeoA from this fusion demonstrates that FeoA-NFeoB interactions function to stabilize the GTP-bound form of FeoB. Taken together, our work reveals a role for FeoA function in the fused FeoAB system and suggests a function for FeoA among prokaryotes.

Fluorescent sensors of siderophores produced by bacterial pathogens.

Siderophores are iron-chelating molecules that solubilize Fe3+ for microbial utilization and facilitate colonization or infection of eukaryotes by liberating host iron for bacterial uptake. By fluorescently labeling membrane receptors and binding proteins, we created 20 sensors that detect, discriminate, and quantify apo- and ferric siderophores. The sensor proteins originated from TonB-dependent ligand-gated porins (LGPs) of Escherichia coli (Fiu, FepA, Cir, FhuA, IutA, BtuB), Klebsiella pneumoniae (IroN, FepA, FyuA), Acinetobacter baumannii (PiuA, FepA, PirA, BauA), Pseudomonas aeruginosa (FepA, FpvA), and Caulobacter crescentus (HutA) from a periplasmic E. coli binding protein (FepB) and from a human serum binding protein (siderocalin). They detected ferric catecholates (enterobactin, degraded enterobactin, glucosylated enterobactin, dihydroxybenzoate, dihydroxybenzoyl serine, cefidericol, MB-1), ferric hydroxamates (ferrichromes, aerobactin), mixed iron complexes (yersiniabactin, acinetobactin, pyoverdine), and porphyrins (hemin, vitamin B12). The sensors defined the specificities and corresponding affinities of the LGPs and binding proteins and monitored ferric siderophore and porphyrin transport by microbial pathogens. We also quantified, for the first time, broad recognition of diverse ferric complexes by some LGPs, as well as monospecificity for a single metal chelate by others. In addition to their primary ferric siderophore ligands, most LGPs bound the corresponding aposiderophore with ∼100-fold lower affinity. These sensors provide insights into ferric siderophore biosynthesis and uptake pathways in free-living, commensal, and pathogenic Gram-negative bacteria.

Identification of Multiple Iron Uptake Mechanisms in Enterococcus faecalis and Their Relationship to Virulence.

Among the unfavorable conditions bacteria encounter within the host is restricted access to essential trace metals such as iron. To overcome iron deficiency, bacteria deploy multiple strategies to scavenge iron from host tissues, with abundant examples of iron acquisition systems being implicated in bacterial pathogenesis. Yet the mechanisms utilized by the major nosocomial pathogen Enterococcus faecalis to maintain intracellular iron balance are poorly understood. In this study, we conducted a systematic investigation to identify and characterize the iron acquisition mechanisms of E. faecalis and to determine their contribution to virulence. Bioinformatic analysis and literature surveys revealed that E. faecalis possesses three conserved iron uptake systems. Through transcriptomics, we discovered two novel ABC-type transporters that mediate iron uptake. While inactivation of a single transporter had minimal impact on the ability of E. faecalis to maintain iron homeostasis, inactivation of all five systems (Δ5Fe strain) disrupted intracellular iron homeostasis and considerably impaired cell growth under iron deficiency. Virulence of the Δ5Fe strain was generally impaired in different animal models but showed niche-specific variations in mouse models, leading us to suspect that heme can serve as an iron source to E. faecalis during mammalian infections. Indeed, heme supplementation restored growth of Δ5Fe under iron depletion and virulence in an invertebrate infection model. This study revealed that the collective contribution of five iron transporters promotes E. faecalis virulence and that the ability to acquire and utilize heme as an iron source is critical to the systemic dissemination of E. faecalis.

Functional Characterization of the Co2+ Transporter AitP in Sinorhizobium meliloti: A New Player in Fe2+ Homeostasis.

Co2+ induces the increase of the labile-Fe pool (LIP) by Fe-S cluster damage, heme synthesis inhibition, and "free" iron import, which affects cell viability. The N2-fixing bacteria, Sinorhizobium meliloti, is a suitable model to determine the roles of Co2+-transporting cation diffusion facilitator exporters (Co-eCDF) in Fe2+ homeostasis because it has a putative member of this subfamily, AitP, and two specific Fe2+-export systems. An insertional mutant of AitP showed Co2+ sensitivity and accumulation, Fe accumulation and hydrogen peroxide sensitivity, but not Fe2+ sensitivity, despite AitP being a bona fide low affinity Fe2+ exporter as demonstrated by the kinetic analyses of Fe2+ uptake into everted membrane vesicles. Suggesting concomitant Fe2+-dependent induced stress, Co2+ sensitivity was increased in strains carrying mutations in AitP and Fe2+ exporters which did not correlate with the Co2+ accumulation. Growth in the presence of sublethal Fe2+ and Co2+ concentrations suggested that free Fe-import might contribute to Co2+ toxicity. Supporting this, Co2+ induced transcription of Fe-import system and genes associated with Fe homeostasis. Analyses of total protoporphyrin content indicates Fe-S cluster attack as the major source for LIP. AitP-mediated Fe2+-export is likely counterbalanced via a nonfutile Fe2+-import pathway. Two lines of evidence support this: (i) an increased hemin uptake in the presence of Co2+ was observed in wild-type (WT) versus AitP mutant, and (ii) hemin reversed the Co2+ sensitivity in the AitP mutant. Thus, the simultaneous detoxification mediated by AitP aids cells to orchestrate an Fe-S cluster salvage response, avoiding the increase in the LIP caused by the disassembly of Fe-S clusters or free iron uptake. IMPORTANCE Cross-talk between iron and cobalt has been long recognized in biological systems. This is due to the capacity of cobalt to interfere with proper iron utilization. Cells can detoxify cobalt by exporting mechanisms involving membrane proteins known as exporters. Highlighting the cross-talk, the capacity of several cobalt exporters to also export iron is emerging. Although biologically less important than Fe2+, Co2+ induces toxicity by promoting intracellular Fe release, which ultimately causes additional toxic effects. In this work, we describe how the rhizobia cells solve this perturbation by clearing Fe through a Co2+ exporter, in order to reestablish intracellular Fe levels by importing nonfree Fe, heme. This piggyback-ride type of transport may aid bacterial cells to survive in free-living conditions where high anthropogenic Co2+ content may be encountered.

Biochemical interactions between the Atm1-like transporter from Novosphingobium aromaticivorans and heavy metals.

Novosphingobium aromaticivorans has the ability to survive in harsh environments by virtue of its suite of iron-containing oxygenases that biodegrade an astonishing array of aromatic compounds. It is also resistant to heavy metals through Atm1, an ATP-binding cassette protein that mediates active efflux of heavy metals conjugated to glutathione. However, Atm1 orthologues in higher organisms have been implicated in the intracellular transport of organic iron complexes. Our hypothesis suggests that the ability of Atm1 to remove heavy metals is related to the need for regulated iron handling in N. aromaticivorans to support high oxygenase activity. Here we provide the first data demonstrating a direct interaction between an iron-porphyrin compound (hemin) and NaAtm1. Hemin displayed considerably higher binding affinity and lower EC50 to stimulate ATP hydrolysis by Atm1 than Ag-GSH, GSSG or GSH, established substrates of the transporter. Co-incubation of NaAtm1 and hemin with Ag-GSH in ATPase assays revealed a non-competitive interaction, indicating distinct binding sites on NaAtm1 and this property was reinforced using molecular docking analysis. Our data suggests that NaAtm1 has considerable versatility in transporting organic conjugates of metals and that this versatility enables it to play roles in detoxification processes for toxic metals and in homeostasis of iron. The ability to play these distinct roles is enabled by the plasticity of the substrate binding site within the central cavity of NaAtm1.

Siderophores: Chemical tools for precise antibiotic delivery.

The success of precision medicine coupled with the disappointing impact of broad-spectrum antibiotic use on microbiome stability and bacterial resistance, has triggered a shift in antibiotic design strategies toward precision antibiotics. This also includes the implementation of novel vectorization approaches directed to improve the internalization of antibacterial agents into deadly gram-negative pathogens through precise and well-defined mechanisms. The conjugation of antibiotics to siderophores (iron scavengers), which are compounds that are able to afford stable iron-complexes that facilitate the internalization into the cell by using bacterial iron uptake pathways as gateways, is a strategy that has begun to show excellent results with the commercialization of the first antibiotic based on this principle, cefiderocol. This digests review provides an overview of the molecular basis for this antibiotic-siderophore conjugation approach, along with recent successful examples and highlights future challenges facing this booming research area.

Structural and functional insights into iron acquisition from lactoferrin and transferrin in Gram-negative bacterial pathogens.

Iron is an essential element for various lifeforms but is largely insoluble due to the oxygenation of Earth's atmosphere and oceans during the Proterozoic era. Metazoans evolved iron transport glycoproteins, like transferrin (Tf) and lactoferrin (Lf), to keep iron in a non-toxic, usable form, while maintaining a low free iron concentration in the body that is unable to sustain bacterial growth. To survive on the mucosal surfaces of the human respiratory tract where it exclusively resides, the Gram-negative bacterial pathogen Moraxella catarrhalis utilizes surface receptors for acquiring iron directly from human Tf and Lf. The receptors are comprised of a surface lipoprotein to capture iron-loaded Tf or Lf and deliver it to a TonB-dependent transporter (TBDT) for removal of iron and transport across the outer membrane. The subsequent transport of iron into the cell is normally mediated by a periplasmic iron-binding protein and inner membrane transport complex, which has yet to be determined for Moraxella catarrhalis. We identified two potential periplasm to cytoplasm transport systems and performed structural and functional studies with the periplasmic binding proteins (FbpA and AfeA) to evaluate their role. Growth studies with strains deleted in the fbpA or afeA gene demonstrated that FbpA, but not AfeA, was required for growth on human Tf or Lf. The crystal structure of FbpA with bound iron in the open conformation was obtained, identifying three tyrosine ligands that were required for growth on Tf or Lf. Computational modeling of the YfeA homologue, AfeA, revealed conserved residues involved in metal binding.