Aliquots of conidial suspensions (200 conidia in 100 µl RPMI medium) were dispensed per well of a 96-well flat-bottom plate. RPMI medium (100 µl, control), purified PA metabolites pyoverdine (1–10 µM) or pyocyanin (50–500 µM) (both: Sigma-Aldrich), filtrates of WT PA strains and siderophore loss-of-function mutants, or viable PA cells were added. For live cell co-culture, PA/AF ratios of 0.01 to 100 were tested in 10-fold serial dilutions.
Pyoverdine
Manufactured by Merck Group
Sourced in United States
Pyoverdine is a fluorescent siderophore produced by certain bacteria, including Pseudomonas species. It functions as an iron-chelating compound, helping the bacteria acquire and utilize iron from their environment.
Automatically generated - may contain errors
Lab products found in correlation
Ferrichrome, by Merck Group (5 mentions)
Chrome azurol s, by Thermo Fisher Scientific (3 mentions)
Pyocyanin, by Merck Group (2 mentions)
Menadione, by Merck Group (2 mentions)
Pipes, by Merck Group (2 mentions)
Roswell park memorial institute (rpmi), by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Histopaque 1077 and 1119, by Merck Group (2 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Voriconazole, by Pfizer (1 mentions)
2 3 bis 2 methoxy 4 nitro 5 sulfophenyl 2h tetrazolium 5 carboxanilide inner salt xtt, by Merck Group (1 mentions)
Celastrol, by Merck Group (1 mentions)
L arabinose, by Thermo Fisher Scientific (1 mentions)
Fluorescein 5 maleimide, by Cayman Chemical (1 mentions)
Mannitol, by Merck Group (1 mentions)
Griess reagent kit, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh da, by Merck Group (1 mentions)
Sybr green mix, by Quanta Biosciences (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Qscript cdna synthesis kit, by Quanta Biosciences (1 mentions)
10 protocols using pyoverdine
1
Exploring PA Metabolites' Impact on Fungal Growth
2
Standardized A. fumigatus Biofilm Assay
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Pyocyanin, pyoverdine, pyochelin, ferric iron (FeCl3), 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT), menadione, and RPMI 1640 medium were purchased from Sigma-Aldrich (St. Louis, MO, USA). Iron contents in RPMI 1640 medium were below the detection limit (<1 µM, measured by inductively coupled plasma optical emission spectroscopy; Paolo Visca, Rome, Italy, personal communication). Voriconazole was obtained from Pfizer, New York City. Stock was prepared in DMSO and was further diluted to test conditions in RPMI. DMSO concentration in our combination experiments was 0.01%. DMSO concentrations below 1% do not affect A. fumigatus biofilm metabolism, and thus did not require dedicated DMSO controls. Large batches of the reagents were prepared in aliquots and frozen, and a fresh aliquot was used in each experiment.
3
Iron and Gallium Binding to Pyochelin and Pyoverdine
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Pyochelin I & II (Toronto Research Chemicals cat# P840365) and pyoverdine (Sigma cat# P8124) were purchased and used for the BMDM experiments. For pyochelin, 100 μM or 1 mM pyochelin was incubated with either 50 μM or 500 μM iron(III) chloride (Fe(III)) respectively, or 50 μM or 500 μM gallium(III) nitrate hydrate respectively, in 50mM Tris-HCl buffer pH 7.6 for 16h at room temperature. For pyoverdine, 100 μM or 1 mM pyoverdine was incubated with either 100 μM or 1 mM Fe(III) respectively, or 100 μM or 1 mM gallium(III) nitrate hydrate respectively, in 50mM Tris-HCl buffer pH 7.6 for 16h at room temperature. The solutions were then diluted 1:10 in macrophage growth medium, filtered sterilized and added to BMDM for 6 h.
4
Iron Assimilation Assay for Aspergillus
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All strains were grown on PDA plates containing 100 μM BPS and supplemented with ~25 ml lyophilized culture filtrates from the WT strain, the Δku70 parent strain, or the mutants. The ability of S. apiospermum to assimilate iron from xenosiderophores was also assessed by using 20 µM of iron-saturated ferrichrome, ferrioxamine, or pyoverdine (Sigma-Aldrich) as the sole source of iron. Plates were point-inoculated and then incubated for seven days at 37°C.
5
Siderophore-Mediated Iron Acquisition Assays
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Pyoverdine (PYOV), 3-hydroxy-1,2-dimethyl-4(1H)pyridine (deferiprone, DFP), celastrol, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT), and menadione were purchased from Sigma-Aldrich (St. Louis, MO). Amphotericin B (AmB) was derived from X-Gen Pharmaceuticals Inc. (Horseheads, NY). Chrome Azurol S (CAS) was purchased from MP Biomedicals (Solon, OH). Ferri- and desferri-triacetylfusarinine C (TAFC, DF-TAFC) were purified as described previously [32 (link)].
6
Bacterial Siderophore Characterization
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Ferrichrome and fluorescein-5-maleimide were purchased from Cayman Chemicals. Ferrioxamine B was purchased from Calbiochem. Pyoverdine and Pyoverdine-Fe3+ were purchased from Sigma. Arthrobactin was purchased from MolPort. Alexa Fluor 594 C5 maleimide was purchased from Fisher Scientific. Carbenicillin was purchased from AK Scientific. l -arabinose was purchased from BioShop. Glucose was purchased from Fisher Scientific. Stock solutions and powders were stored at −20°C. Lysogeny broth (LB) was purchased as a premixed powder from BioShop. 10:90 medium was prepared as previously described (13 (link), 14 (link)). l -arabinose was prepared as a 20% (wt/vol) stock solution in 10:90 and filter sterilized (0.2-μm pore size; Fisherbrand).
7
Siderophore-Mediated Iron Acquisition Assays
8
Enterobactin and Siderophore Assays
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Enterobactin (Ent; from Escherichia coli) procured from Sigma-Aldrich (St. Louis, MO) is supplied free of iron and endotoxin (≥98% purity; HPLC). Iron-free Pyoverdine (from Pseudomonas fluorescens), ferrichrome (from Ustilago sphaerogena), Deferiprone, deferoxamine mesylate salt (DFO; from Streptomyces pilosus), 2,3 dihydroxybenzoic acid (2,3-DHBA), lipopolysaccharide (LPS) from E. coli 0128:B12, dimethyl sulfoxide (DMSO), ferric chloride (FeCl3, Fe3+), 2,2ʹ-dipyridyl, and cyclosporin H were purchased from Sigma-Aldrich (St. Louis, MO). Flagellin (FliC) from Salmonella enterica subsp. enterica serovar Typhimurium (SL3201, fljB-), a kind gift from Dr. Andrew Gewirtz, Georgia State University, was purified through sequential cation and anion-exchange chromatography as previously described.52 (link) FITC Annexin-V apoptosis detection kit was purchased from Molecular Probes (Life Technologies, Columbus, OH). Chrome azurol S (CAS) was purchased from Acros Organics (Geel, Belgium). Recombinant mouse Lcn2 (alias neutrophil gelatinase-associated lipocalin) (rec-Lcn2; Cat# CM17) produced by a mammalian (human) expression system was procured from Novoprotein Scientific Inc. (Fremont, CA). Carrier-free rec-Lcn2 was supplied at a purity ≥95% as determined by reducing SDS-PAGE and free from endotoxin, siderophore, and iron.
9
Siderophore-Mediated Bacterial Interactions
10
Chemical Supplementation in C. elegans
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For chemical supplementation, each chemical was dissolved in water or DMSO to generate a stock. The stock solution was added to heat-killed OP50 and then spotted onto NGM plates. The chemical name, vendor, stock concentrations and volumes used for each chemical are listed as follows: 2,3-DHBA (Sigma 126209-5G, 300mM, 5ul), Enterobactin (Sigma E3910-1MG, 1mg/ml, 5 ml), Pyoverdine (Sigma P8124-1MG, 0.5 mg/ml, 5ml), Ferrichrome (Sigma F8014-1MG, 0.5mg/ml, 5ml), Hermin (Sigma 51280, 1mg/ml, 5ul), FeCl 3 (Sigma 236489, 175ug/ul, volumes indicated in assay).
For CaEDTA supplementation (Klang et al., 2014) , 50ul of CaEDTA (50ug/ul) was spread onto the center of the NGM plates seeded with OP50, then different volume of the FeCl 3 [175ug/ul] (0ul, 1ul, 5ul, 10ul, 50ul) or 20ul of the Ent (0.5mg/ml) was added onto the center of the bacterial lawn.
For CaEDTA supplementation (Klang et al., 2014) , 50ul of CaEDTA (50ug/ul) was spread onto the center of the NGM plates seeded with OP50, then different volume of the FeCl 3 [175ug/ul] (0ul, 1ul, 5ul, 10ul, 50ul) or 20ul of the Ent (0.5mg/ml) was added onto the center of the bacterial lawn.
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