Iron Siderophore Research Articles

Characterisation of cefiderocol-non-susceptible Acinetobacter baumannii isolates from Taiwan

Cefiderocol (CFDC), a siderophore cephalosporin, is active against Gram-negative bacteria including carbapenem-resistant Acinetobacter baumannii (CRAB). In this study, 100 CRAB isolates from patients with bacteraemia in Taiwan were characterised, among which 21 CFDC-non-susceptible isolates were identified with a minimum inhibitory concentration (MIC) of ≥8 mg/L. The effect of avibactam on CFDC activity was evaluated using broth microdilution methods according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Whole-genome sequencing (WGS) was performed on all CFDC-non-susceptible isolates (MIC ≥ 8 mg/L) for multilocus sequence typing (MLST) analysis, possession of β-lactamase genes and identification of possible variations in the PiuA iron transporter. Addition of avibactam, a diazabicyclooctane inhibitor for serine-type β-lactamases, resulted in a ≥8-fold decrease in the CFDC MIC for 15 of 21 CFDC-non-susceptible isolates compared with only 1 of 79 CFDC-susceptible isolates (MIC ≤ 4 mg/L). WGS analysis confirmed that all CFDC-non-susceptible isolates harboured multiple β-lactamases including ADC-30 homologues, OXA-23 and OXA-66. One isolate with a high MIC (>32 mg/L) had a PER-type extended-spectrum β-lactamase (ESBL) gene. Twenty other isolates belonged to ST455, ST473 and ST787. Among these, thirteen ST455 isolates were deficient in PiuA, a siderophore uptake receptor that may be required for optimal penetration of CFDC. MICs of CFDC-non-susceptible CRAB isolates from Taiwan could be significantly decreased to susceptible levels by the addition of avibactam, suggesting the involvement of β-lactamases in resistance. Among the 21 CFDC-non-susceptible isolates, 1 isolate had a PER-type ESBL gene and 13 isolates lacked a PiuA iron siderophore transporter.

Surface Plasmon Resonance Identifies High-Affinity Binding of l-DOPA to Siderocalin/Lipocalin-2 through Iron–Siderophore Action: Implications for Parkinson’s Disease Treatment

l-3,4-Dihydroxyphenylalanine (l-DOPA), the dopamine precursor, remains the frontline treatment for Parkinson's disease (PD). With the treatment progress, l-DOPA efficacy decreases, necessitating higher and more frequent doses, with higher risks of dyskinesia. l-DOPA chelates iron through its catechol group, forming the l-DOPA:Fe complex; however, the fate of this complex is unknown. Catechol siderophore-like compounds are known to bind siderocalin (Scn)/lipocalin-2 to form stable siderophore:Fe:Scn complexes. Scn is upregulated in PD patients' substantia nigra and may play a role in PD pathophysiology. Therefore, in this study, we used the surface plasmon resonance (SPR) technique to examine the binding properties of l-DOPA to Scn. We found that l-DOPA formed a stable complex with Scn in the presence of Fe3+. Our analysis of the binding properties of l-DOPA precursors and metabolites indicates that the catechol group is necessary but not sufficient to form a stable complex with Scn. Finally, the affinity constant (Kd) of DOPA:Fe3+ binding with Scn (0.8 μM) was lower than l-DOPA plasma peak concentrations in l-DOPA preparations in the past six decades. Our results speculate a significant role for the l-DOPA-Scn complex in the decreased bioavailability of l-DOPA with the progress of PD.

Toxicity of the iron siderophore mycobactin J in mouse macrophages: Evidence for a hypoxia response

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an obligate intracellular pathogen that lives within the phagosome of macrophages. Here we demonstrate that the siderophore mycobactin J, produced by the closely related intracellular pathogen Mycobacterium paratuberculosis, is toxic to murine macrophage cells. Its median lethal dose, 10 μM, is lower than that of the iron chelators desferrioxamine B and TrenCAM, an enterobactin analog. To determine the source of this toxicity, we conducted microarray, ELISA, and metabolite profiling experiments. The primary response is hypoxia-like, which implies iron starvation as the underlying cause of the toxicity. This observation is consistent with our recent finding that mycobactin J is a stronger iron chelator than had been inferred from previous studies. Mycobactin J is known to partition into cell membranes and hydrophobic organelles indicating that enhanced membrane penetration is also a likely factor. Thus, mycobactin J is shown to be toxic, eliciting a hypoxia-like response under physiological conditions.

How Zinc-Binding Systems, Expressed by Human Pathogens, Acquire Zinc from the Colonized Host Environment: A Critical Review on Zincophores.

Some transition metals, like manganese, iron, cobalt, nickel, copper and zinc, required for the biosynthesis of metalloenzymes and metalloproteins, are essential micronutrients for the growth and development of pathogenic microorganisms. Among the defenses put in place by the host organism, the so-called “nutritional immunity” consists of reducing the availability of micronutrients and thus “starving” the pathogen. In the case of metals, microorganisms can fight the nutritional immunity in different ways, i.e. by directly recruiting the metal ion or capturing an extracellular metalloprotein or also through the synthesis of specific metallophores which allow importing the metal in the form of a chelate complex. The best known and most studied metallophores are those directed to iron (siderophores), but analogous chelators are also expressed by microorganisms to capture other metals, such as zinc. An efficient zinc recruitment can also be achieved by means of specialized zinc-binding proteins. A deep knowledge of the properties, structure and action mechanisms of extracytoplasmic zinc chelators can be a powerful tool to find out new therapeutic strategies against the antibiotic and/or antifungal resistance. This review aims to collect the knowledge concerning zincophores (small molecules and proteins in charge of zinc acquisition) expressed by bacterial or fungal microorganisms that are pathogenic for the human body.

Characterization of siderophore-producing microorganisms associated to plants from high-Andean heavy metal polluted soil from Callejón de Huaylas (Ancash, Perú)

Endophytic and rhizospheric microorganisms associated with six native plants adapted to heavy metal polluted soil from Punta Olímpica and Chahuapampa, located in Callejón de Huaylas mountains, were evaluated as potential candidates for technologies to clean polluted ecosystems. It was selected 14 bacteria and 9 fungi strains by their iron and/or aluminum siderophore production trait, where BEP17-Dm showed higher production. According to the 16S rDNA analysis, bacteria belong to Pseudomonas, Bacillus, and Achromobacter genera, whereas by ITS analysis fungi belong to Talaromyces, Hypoxylon, Tolypocladium, and Penicillium. All bacteria strains tolerated lead (2−8 mM) and eigth tolerated cadmium (1−6 mM); also all fungi tolerated lead (9−70 mM) and cadmium (3−10 mM). Two bacteria and six fungi solubilized cadmium carbonate, while eleven bacteria and two fungi solubilized tricalcium phosphate, where P. japonica BEP18-Dm and B. subtilis BRU16-Sr exhibited higher solubilization index. None strains solubilized lead carbonate. BEP18-Dm produced higher concentration of IAA (53.42 μgml−1); while six bacteria and all fungi strains produced a low concentration of auxins. Medicago sativa seedlings inoculated with BEP17-Dm, BEP18-Dm, or BRU16-Sr showed more surviving percentage under in vitro culture in presence of Cd, Pb (0.5–1.0 mM), or Al (2.5–5.0 mM). Finally, it is the first report of siderophore-producing microorganisms from polluted soil of Callejón de Huaylas highlands, interestedly they displayed metabolic properties useful to enhance phytoremediation and biotechnology application.

Genome analysis of a salinity adapted Achromobacter xylosoxidans rhizobacteria from the date palm

Rhizospheric bacteria play an indispensable role in plant growth, development, and stress response in plants. This project aims to isolate and functionally characterize plant-growth-promoting rhizobacteria (PGPR) from date palm (Phoenix dactylifera L.) rhizosphere, to investigate its potential uses as a biofertilizer in date palm. Here, we report an Achromobacter xylosoxidans SQU-1 (SQU-1), as a rhizobacteria strain isolated from date palms grown under salinity. The strain was visualized using electron microscopy, identified using protein profiling and 16 S rRNA gene sequencing, functionally characterized, and its genome was sequenced, assembled, and annotated. The results showed that SQU-1 is a Gram-negative flagellated strain and possesses several plant-growth-promoting traits, including the ability to solubilize micronutrients such as zinc, potassium, phosphorus and to produce ammonia, iron siderophores, indole acetic acid (IAA) phytohormone, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. SQU-1 was also able to significantly enhance date palm seed germination and Arabidopsis growth under normal and saline conditions. Whole-genome sequencing of SQU-1 revealed a total of 6,441,875 bp reads that were assembled into 15 scaffolds. Genome sequence analysis indicated the presence of 5968 open reading frames (ORFs), including 5848 coding genes, 56 pseudogenes, and 64 rRNA DNA sequences. Sequence and cluster of orthologous groups (COG) analysis of SQU-1 enabled the identification of potential genes involved in plant growth-promoting characteristics such as ACC DEAMINASE (acdS), D-CYSTEINE DESULFHYDRASE, and various siderophore production and transporter genes. In conclusion, SQU-1 can be a good candidate for a sustainable agricultural system that aims at producing date fruits under saline conditions with minimal usage of chemical fertilizers.

Bioremediation of petroleum contaminated soil through biosurfactant and Pseudomonas sp. SA3 amended design treatments.

Toxicity of agricultural soil due to petroleum contamination has become a serious issue in recent times. Petrol oil exhibits toxic effects in agricultural crops due to the presence of various hazardous hydrocarbons. The degradation of petroleum hydrocarbon has been widely studied by the researchers that signify the requirement of effective treatments for the detoxification of petroleum contaminated soil and their reuse for growing crops. Hence, with this intention in the present study secondary metabolites "biosurfactant" (natural surfactant) along with the potent plant growth promoting (PGP) bacterial strain Pseudomonas sp. SA3 was used in the designed treatments for growing agricultural crop. The biosurfactant produced by the strain has the emulsification capacity of 43% and surface tension reduction ability to 34.5mN/m whereas the plant growth promoting traits demonstrates 93.46µg/mL phosphate solubilisation ability, siderophores (iron chelating compound) production upto 69.41% units and 81.41µg/mL indole acetic acid (IAA) production ability. Further, the results of the design treatments signifies that treatments amended with the strain SA3 and biosurfactant is effective in the management of petroleum contaminated soil indicating treatment EX 5 (1kg soil+1L water+Pseudomonas sp. SA3+300mL crude biosurfactant), as an efficient treatment in increment of phytochemical constituents and 10-15% enhancement in growth parameters as compared to negative control. Hence, the developed treatments can be efficaciously used for the management of petroleum contaminated soil for agronomy.

A novel NRPS cluster, acquired by horizontal gene transfer from algae, regulates siderophore iron metabolism in Burkholderia seminalis R456

In an environment with limited iron levels, sufficiently high intracellular iron concentrations are critical for bacterial survival. When iron levels are low, many bacteria including those of the Burkholderia cepacia group secrete chemically diverse siderophores to capture Fe3+. The synthesis of the two main siderophores, ornibactin and pyochelin, is regulated in an iron concentration dependent manner via the regulator protein Fur. In this study, we identified a novel Nonribosomal Peptide Synthetase (NRPS) cluster in strain R456 of Burkholderia seminalis, a member of the B. cepacia group. We show that the NRPS cluster not only allows the production of a so-far undescribed siderophore, but is also required for ornibactin and pyochelin production as it is a crucial component in the signaling pathway targeting the global iron regulating effector Fur which regulates siderophore production. Furthermore, the NRPS cluster is also involved in cell motility and biofilm formation, both of which are directly dependent on iron concentration in various bacteria. Interestingly, our data suggests that this newly discovered NRPS cluster which regulates siderophore iron metabolism in bacteria was obtained by horizontal gene transfer from algae.

Understanding the fundamental role of virulence determinants to combat Aspergillus fumigatus infections: exploring beyond cell wall

Aspergillus fumigatus is a ubiquitous fungal pathogen associated with several life-threatening infections in patients suffering from chronic lung inflammatory diseases and immune deficiencies. Multiple factors contribute towards the survival and adaptation of A. fumigatus in the host and determine its pathogenicity. A limited spectrum of antifungal drugs is available for treating A. fumigatus infections which mainly target either cell wall or cell membrane biosynthesis. The clinical usage of these drugs is becoming progressively restricted due of the emergence of resistant A. fumigatus strains. Thus, there is an urgent unmet need to analyse other virulence determinants of A. fumigatus, in addition to screening suitable and effective targets for the prospective antifungal drugs. The present review elaborates A. fumigatus virulence determinants associated with cell surface organisation such as hydrophobins (rodlet layer formation), chitin, glucans and galactosaminogalactan. Besides, secondary metabolites such as gliotoxin, melanin and siderophores (iron homeostasis mechanism) have also been discussed. The exhaustive understanding of these virulence determinants could potentially lead to the discovery of novel targets that could be exploited to either discover new molecules or enhance the efficacy of known antifungal drugs.

Conjugation to Enterobactin and Salmochelin S4 Enhances the Antimicrobial Activity and Selectivity of β-Lactam Antibiotics against Nontyphoidal Salmonella.

The pathogen Salmonella enterica is a leading cause of infection worldwide. Nontyphoidal Salmonella (NTS) serovars typically cause inflammatory diarrhea in healthy individuals, and can cause bacteremia in immunocompromised patients, children, and the elderly. Management of NTS infection poses a challenge because antibiotic treatment prolongs fecal shedding of the pathogen and is thus not recommended for most patients. In recent years, the emergence of antibiotic resistance in NTS has also become a major issue. Thus, new therapeutic strategies to target NTS are needed. Here, we evaluated whether six siderophore-β-lactam conjugates based on enterobactin (Ent) and salmochelin S4 (digulcosylated Ent, DGE) provide antimicrobial activity against the two highly prevalent NTS serovars Typhimurium and Enteritidis by targeting the siderophore receptors FepA and/or IroN. The conjugates showed 10- to 1000-fold lower minimum inhibitory concentrations against both serovars Typhimurium and Enteritidis compared to the parent antibiotics under iron limitation and were recognized and transported by FepA and/or IroN. NTS treated with the Ent/DGE-β-lactam conjugates exhibited aberrant cellular morphologies suggesting inhibition of penicillin-binding proteins, and the conjugates selectively killed NTS in coculture with Staphylococcus aureus. Lastly, the DGE-based conjugates proved to be effective at inhibiting growth of NTS in the presence of the Ent-sequestering protein lipocalin-2. This work describes the successful use of siderophore-antibiotic conjugates against NTS and highlights the opportunity for narrowing the activity spectrum of antibiotics by using Ent and DGE to target enteric bacterial pathogens.

Virulence genotypes and phylogenetic distribution of the community-onset Escherichia coli

Background: The concept that not all E. coli are equally able to cause UTI, and that the uropathogenic E. coli (UPEC) strains possess distinctive traits that confer enhanced virulence potential compared to other source E. coli, is supported by abundant epidemiologic and experimental evidence. Virulence factors (VFs) are the specific genes encoding a diverse array of specialized properties that are postulated to contribute to virulence. The aim of this study was to find the prevalence difference of virulence genes and phylogenetic lineages between E. coli from community-onset urinary tract infection (CO-UPEC) and hepatobiliary infection (CO-HBEC) compared to rectal commensal E. coli (RCEC) in a healthy population. Methods and materials: We conducted a case-control study of patients with positive UPEC & HBEC between 2006 and 2008 at a teaching hospital in South Korea. Community-onset (CO) was the isolated from the patients from whom the specimen was obtained within 2 calendar days of hospitalization or at outpatient department. We also collected the rectal commensal E. coli (RCEC) of healthy people. All isolates underwent PCR-based microarray for phylotyping and detection of 11 VFs, including adhesins (prf, sfa, drb, prsGj96, papGad), toxins (hly, cnf1), siderophores (iroN, aer), capsule (kpsMT), and other (ompT). The virulence score was defined as the number of virulence genes identified. Results: A total of 548 CO-UPEC and 140 HBEC with 486 CREC isolates were collected. Compared with CREC, CO-UPEC showed higher VFs (3.92 ± 2.22 vs. 2.28 ± 1.90, p < 0.001) and CO-HBEC averaged fewer VFs (1.56 ± 1.64 vs. 2.28 ± 1.90, p < 0.001). Overall distribution of phylogenic group were B2(52.5%) and D(33%) in CO-UPEC, A(37.9%) and D(31.4%) in CO-HBEC and A(38.3%) and B2(29%) in RCEC. Conclusion: In conclusion, we observed the differences of VFs between clones from community- onset UPECs and HBECs compared to RCEC, and this result explain that UPECs would be more virulent than HBECs which could be less virulent than RCECs considering only VFs distribution. In phylogenetic analysis, each group of E. coli showed different phylogenic distribution, which imply group B E. coli might be more specifically associated with uropathogenecity.

Proteome analysis of virulent Aeromonas hydrophila reveals the upregulation of iron acquisition systems in the presence of a xenosiderophore.

The Gram-negative bacterium, Aeromonas hydrophila, has been responsible for extensive losses in the catfish industry for over a decade. Due to this impact, there are ongoing efforts to understand the basic mechanisms that contribute to virulent A. hydrophila (vAh) outbreaks. Recent challenge models demonstrated that vAh cultured in the presence of the iron chelating agent deferoxamine mesylate (DFO) were more virulent to channel catfish (Ictalurus punctatus). Interestingly, differential gene expression of select iron acquisition genes was unremarkable between DFO and non-DFO cultures, posing the question: why the increased virulence? The current work sought to evaluate growth characteristics and protein expression of vAh after the addition of DFO. A comparative proteome analysis revealed differentially expressed proteins among tryptic soy broth (TSB) and TSB+DFO treatments. Upregulated proteins identified among the TSB+DFO treatment were enriched for gene ontology groups including iron ion transport, siderophore transport and siderophore uptake transport, all iron acquisition pathways. Protein-protein interactions were also evaluated among the differentially expressed proteins and predicted that many of the upregulated iron acquisition proteins likely form functional physiological networks. The proteome analysis of the vAh reveals valuable information about the basic biological processes likely leading to increased virulence during iron restriction in this organism.

Beauveria bassiana Multifunction as an Endophyte: Growth Promotion and Biologic Control of Trialeurodes vaporariorum, (Westwood) (Hemiptera: Aleyrodidae) in Tomato.

Simple SummaryThe tomato, Solanum lycopersicum L. has great importance worldwide for its nutritional characteristics and its antioxidant content. It is cultivated in different geographical areas, under field and greenhouse conditions, and it can be subjected to abiotic and abiotic factors that negatively affect production and quality. In this study, we evaluated the effect of five native endophytic strains of Beauveria bassiana on the reproduction of greenhouse whiteflies and the growth of tomatoes. The endophyte was inoculated in the substrate, and plants were afterward exposed to adult populations of the insect. For plant-growth promoter activity, the effect of endophytic strains on phosphate solubilization, iron siderophores production, plant height, and biomass was determined. The RGM-557 strain reduced the number of eggs and nymphs per cm2 on leaflet by 66 and 65%, respectively, compared with the control (water); while in comparison with the chemical insecticide the reduction was 32 and 55%, respectively. Most strains showed some degree of phosphate solubilization and siderophores production. Plants inoculated with strains RGM-557 and RGM-731 produced the greatest plant heights; RGM-731 obtained the greatest plant biomass. Endophytic B. bassiana provide important protection levels against whiteflies in tomato—in addition to promoting their growth.The tomato, Solanum lycopersicum L. is one of the most consumed vegetables in the world; nevertheless, it is affected by biotic and abiotic factors that reduce its productivity. The whitefly is globally considered as the main pest under protected crop conditions, where biologic control using endophytic fungi emerges as a sustainable alternative. We evaluated the indirect effects of five native endophytic strains of Beauveria bassiana on the reproduction of greenhouse whiteflies and the growth of tomatoes. The plant growth substrate was inoculated with five strains of this endophyte and the resulting plants were then exposed to whiteflies afterwards. The effect that endophytic strains had on phosphate solubilization, iron siderophore production, plant height, and plant biomass were evaluated. The evaluated endophytes reduced the number of eggs per cm2 on leaflets compared to the control and behaved similarly to the commercial synthetic insecticide. Leaflets inoculated with strains RGM-557, RGM-644 and RGM-731 showed fewer nymphs than the control and those treated with insecticide. RGM-557 and RGM-731 produced the greatest plant heights; RGM-731 obtained the greatest plant biomass. Our study provides evidence that native endophytic strains of B. bassiana have a biocontrol effect on whiteflies and could be used to promote tomato growth.

A High-Denticity Chelator Based on Desferrioxamine for Enhanced Coordination of Zirconium-89.

Herein we report a new high-denticity chelator based on the iron siderophore desferrioxamine (DFO). Our new chelator-DFO2-is acyclic and was designed and synthesized with the purpose of improving the coordination chemistry and radiolabeling performance with radioactive zirconium-89. The radionuclide zirconium-89 ([89Zr]Zr4+) has found wide use for positron emission tomography (PET) imaging when it is coupled with proteins, antibodies, and nanoparticles. DFO2 has a potential coordination number of 12, which uniquely positions this chelator for binding large, high-valent, and oxophilic metal ions. Following synthesis of the DFO2 chelator and the [natZr]Zr-(DFO2) complex we performed density functional theory calculations to study its coordination sphere, followed by zirconium-89 radiolabeling experiments for comparisons with the "gold standard" chelator DFO. DFO (CN 6) can coordinate with zirconium in a hexadentate fashion, leaving two open coordination sites where water is thought to coordinate (total CN 8). DFO2 (potential CN 12, dodecadentate) can saturate the coordination sphere of zirconium with four hydroxamate groups (CN 8), with no room left for water to directly coordinate, and only binds a single atom of zirconium per chelate. Following quantitative radiolabeling with zirconium-89, the preformed [89Zr]Zr-(DFO) and [89Zr]Zr-(DFO2) radiometal-chelate complexes were subjected to a battery of in vitro stability challenges, including human blood serum, apo-transferrin, serum albumin, iron, hydroxyapatite, and EDTA. One objective of these stability challenges was to determine if the increased denticity of DFO2 over that of DFO imparted improved complex stability, and another was to determine which of these assays is most relevant to perform with future chelators. In all of the assays DFO2 showed superior stability with zirconium-89, except for the iron challenge, where both DFO2 and DFO were identical. Substantial differences in stability were observed for human blood serum using a precipitation method of analysis, apo-transferrin, hydroxyapatite, and EDTA challenges. These results suggest that DFO2 is a promising next-generation scaffold for zirconium-89 chelators and holds promise for radiochemistry with even larger radionuclides, which we anticipate will expand the utility of DFO2 into theranostic applications.

Mathematical modelling of fungi-initiated siderophore-iron interactions.

Nearly all life forms require iron to survive and function. Microorganisms utilize a number of mechanisms to acquire iron including the production of siderophores, which are organic compounds that combine with ferric iron into forms that are easily absorbed by the microorganism. There has been significant experimental investigation into the role, distribution and function of siderophores in fungi but until now no predictive tools have been developed to qualify or quantify fungi-initiated siderophore-iron interactions. In this investigation, we construct the first mathematical models of siderophore function related to fungi. Initially, a set of partial differential equations are calibrated and integrated numerically to generate quantitative predictions on the spatio-temporal distributions of siderophores and related populations. This model is then reduced to a simpler set of equations that are solved algebraically giving rise to solutions that predict the distributions of siderophores and resultant compounds. These algebraic results require the calculation of zeros of cross products of Bessel functions and thus new algebraic expansions are derived for a variety of different cases that are in agreement with numerically computed values. The results of the modelling are consistent with experimental data while the analysis provides new quantitative predictions on the time scales involved between siderophore production and iron uptake along with how the total amount of iron acquired by the fungus depends on its environment. The implications to bio-technological applications are briefly discussed.

Neutrophil Gelatinase-Associated Lipocalin Protects from ANCA-Induced GN by Inhibiting TH17 Immunity.

Neutrophil gelatinase-associated lipocalin (NGAL) is a diagnostic marker of intrinsic kidney injury produced by damaged renal cells and by neutrophils. ANCA-associated vasculitis features necrotizing crescentic GN (NCGN), and ANCA-activated neutrophils contribute to NCGN. Whether NGAL plays a mechanistic role in ANCA-associated vasculitis is unknown. We measured NGAL in patients with ANCA-associated vasculitis and mice with anti-myeloperoxidase (anti-MPO) antibody-induced NCGN. We compared kidney histology, neutrophil functions, T cell proliferation and polarization, renal infiltrating cells, and cytokines in wild-type and NGAL-deficient chimeric mice with anti-MPO antibody-induced NCGN. To assess the role of TH17 immunity, we transplanted irradiated MPO-immunized MPO-deficient mice with bone marrow from either wild-type or NGAL-deficient mice; we also transplanted irradiated MPO-immunized MPO/IL-17A double-deficient mice with bone marrow from either IL-17A-deficient or NGAL/IL-17A double-deficient mice. Mice and patients with active ANCA-associated vasculitis demonstrated strongly increased serum and urinary NGAL levels. ANCA-stimulated neutrophils released NGAL. Mice with NGAL-deficient bone marrow developed worsened MPO-ANCA-induced NCGN. Intrinsic neutrophil functions were similar in NGAL-deficient and wild-type neutrophils, whereas T cell immunity was increased in chimeric mice with NGAL-deficient neutrophils with more renal infiltrating TH17 cells. NGAL-expressing neutrophils and CD3+ T cells were in close proximity in kidney and spleen. CD4+ T cells showed no intrinsic difference in proliferation and polarization in vitro, whereas iron siderophore-loaded NGAL suppressed TH17 polarization. We found significantly attenuated NCGN in IL-17A-deficient chimeras compared with MPO-deficient mice receiving wild-type bone marrow, as well as in NGAL/IL-17A-deficient chimeras compared with NGAL-deficient chimeras. Our findings support that bone marrow-derived, presumably neutrophil, NGAL protects from ANCA-induced NCGN by downregulating TH17 immunity.

Antibiotic resistant Pseudomonas aeruginosa upregulate iron piracy genes during macrophage infection and evade killing

Abstract Antibiotic resistant bacterial lung infections are the primary cause of morbidity and mortality in people with cystic fibrosis (CF). Recently, we showed that aztreonam resistant P. aeruginosa with a mutated nalD gene were more virulent than wild type (WT), even in the absence of antibiotic treatment. Yet, the mechanisms underlying the increased virulence were unknown. Because nalD− mutants increased antibiotic efflux pump expression, we hypothesized that their hypervirulence was dependent on the efflux pump. Surprisingly, deletion of the efflux pump did not affect nalD− virulence in an acute lung infection. To determine other potential mechanisms underlying the enhanced virulence, we performed RNA-seq on WT and nalD− grown in vitro and found that nalD− increased efflux pump expression relative to WT, as expected, but also differentially regulated hundreds of genes. Exopolysaccharide genes involved in biofilm formation were among the most highly upregulated genes. Consistent with this differential gene expression, the nalD− mutant exhibited increased biofilm formation, reduced C3 complement opsonization, and also replicated better than WT in the presence of macrophages. Subsequent RNA-seq analyses of infected macrophages revealed that nalD− upregulated iron siderophore systems relative to WT. We predict that this increased iron piracy facilitates subversion of nutritional immunity during infection. Because individuals with CF frequently experience anemia, antibiotic resistance mutations that increase iron piracy could promote bacterial growth during these periods of iron limitation.

The ABC exporter IrtAB imports and reduces mycobacterial siderophores.

SummaryIntracellular replication of the deadly pathogen Mycobacterium tuberculosis relies on the production of small organic molecules called siderophores to scavenge iron from host proteins1. M. tuberculosis produces two classes of siderophores, lipid-bound mycobactin and soluble carboxymycobactin2, 3. Functional studies revealed that iron-loaded carboxymycobactin is imported into the cytoplasm by the ABC transporter IrtAB4, which features an additional cytoplasmic siderophore interaction domain (SID)5. However, IrtAB’s predicted ABC exporter fold seemingly contradicts its import function. Here, we show that membrane-reconstituted IrtAB is sufficient to import mycobactins, which are then reduced by the SID to facilitate iron release. Structure determination by X-ray crystallography and cryo-EM confirms IrtAB’s ABC exporter fold, but also reveals structural peculiarities at the transmembrane region of IrtAB resulting in a partially collapsed inward-facing substrate binding cavity. The SID is positioned in close proximity to the inner membrane leaflet, which allows the reduction of membrane-inserted mycobactin. Enzymatic ATPase activity and in vivo growth assays show that IrtAB prefers mycobactin over carboxymycobactin as its substrate. Our study provides insights into an unusual ABC exporter that evolved as highly specialized siderophore import machinery in mycobacteria.

Hydroxamate Titanium-Organic Frameworks and the Effect of Siderophore-Type Linkers over Their Photocatalytic Activity.

The chemistry of metal-organic frameworks (MOFs) relies on the controlled linking of organic molecules and inorganic secondary building units to assemble an unlimited number of reticular frameworks. However, the design of porous solids with chemical stability still remains limited to carboxylate or azolate groups. There is a timely opportunity to develop new synthetic platforms that make use of unexplored metal binding groups to produce metal-linker joints with hydrolytic stability. Living organisms use siderophores (iron carriers in Greek) to effectively assimilate iron in soluble form. These compounds make use of hard oxo donors as hydroxamate or catecholate groups to coordinate metal Lewis acids such as iron, aluminum, or titanium to form metal complexes very stable in water. Inspired by the chemistry of these microorganisms, we report the first hydroxamate MOF prepared by direct synthesis. MUV-11 (MUV = materials of Universidad de Valencia) is a crystalline, porous material (close to 800 m2·g-1) that combines photoactivity with good chemical stability in acid conditions. By using a high-throughput approach, we also demonstrate that this new chemistry is compatible with the formation of single-crystalline phases for multiple titanium salts, thus expanding the scope of accessible precursors. Titanium frameworks are regarded as promising materials for photocatalytic applications. Our photoelectrochemical and catalytic tests suggest important differences for MUV-11. Compared to other Ti-MOFs, changes in the photoelectrochemical and photocatalytic activity have been rationalized with computational modeling, revealing how the chemistry of siderophores can introduce changes to the electronic structure of the frontier orbitals, relevant to the photocatalytic activity of these solids.

Components of rhizospheric bacterial communities of barley and their potential for plant growth promotion and biocontrol of Fusarium wilt of watermelon.

This work aimed to characterize antagonistic bacteria from the field-grown barley rhizosphere, and evaluate their potential for growth promotion and biocontrol of Fusarium wilt on watermelon caused by Fusarium oxysporum f. sp. Niveum (FON). Seven bacteria were isolated and screened for plant growth promoting and antagonistic traits. Based on the results of phenotypic characterization and 16S rRNA gene sequencing, the isolates were identified to be related to Bacillus methylotrophicus (DMK-1), Bacillus amyloliquefaciens subsp. plantarum (DMK-7-2), Bacillus cereus (DMK-12), Pseudomonas brassicacearum subsp. brassicacearum (DMK-2), Pseudomonas veronii (DMK-3), Paenibacillus polymyxa (DMK-8), and Ensifer adhaerens (DMK-17). All the isolates were positive for the production of indole-3-acetic acid (IAA) and ammonia (NH3), while negative for the production of hydrogen cyanide (HCN). Six bacteria strains (except DMK-17) were able to phosphate solubilization. All the bacteria strains, except DMK-8, were able to produce iron siderophore complexes, and possessed the proteolytic activity. Greenhouse experiment indicated six strains can decrease diseased percentage caused by FON. All the isolates enhanced plant biomass, six strains increased root volume, six strains increased root system activity in greenhouse test. Inoculation of mixtures of seven plant growth promoting rhizobacteria could be more effective in plant growth promotion and biocontrol of Fusarium wilt in watermelon.