Deferoxamine

CB17/Icr-Prkdc^scid/IcrIcoCrl female mice were grafted with human skin as described by Join-Lambert et al. [14 (link)]. Nm strains were grown overnight at 37°C on GCB agar plates prepared without iron and supplemented with deferoxamine (Desferal, Novartis) at a final concentration of 15 μM. Bacterial colonies were harvested and cultured in RPMI with 1% bovine serum albumin medium and 0.06 μM deferoxamine with gentle agitation to reach the exponential phase of growth. Bacteria were then resuspended in physiological saline. All mice received 10 mg of human holotransferrin (R&D Systems) administered intraperitoneally just before infection.
For the single infection model, six grafted mice were infected IV with 10⁷ CFU of WT strain or the ΔMDA isogenic strain. The injected dose is just below the LD50. The numbers of CFU in blood and in the graft were determined at 1 and 18 hours.
To obtain a competitive index, three grafted mice were infected intravenously with a mix of 5.10⁶ CFU of each strain. The numbers of CFU in the blood and in the graft were determined. The number of CFU corresponding to the ΔMDA strain was obtained by determining the number of spectinomycin resistant colonies. The competitive index was calculated by the ratio of [log (UFC_Z5463ΔMDA)/log (UFC_WT) in the blood or in the graft] on [log(UFC_Z5463ΔMDA)/log(UFC_WT) of the inoculum].

The catecholamines epinephrine (EPI, Figure 1(a)) and ISO (Figure 1(a)), copper chelator D-PA (Figure 1(c)), ferric chloride hexahydrate (FeCl₃·6H₂O), ferrous sulphate heptahydrate (FeSO₄·7H₂O), 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4′′-disulphonic acid sodium salt (ferrozine), sodium acetate, acetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), HEPES sodium salt, hydroxylamine, salicylic acid, and 2,3-dihydroxybenzoic and 2,5-dihydroxybenzoic acids were purchased from Sigma-Aldrich (USA). Deferoxamine was purchased from Novartis (Switzerland), dimethyl sulfoxide (DMSO) was purchased from Avantor Performance Materials (USA), and methanol for HPLC was purchased from JT Baker (USA). The chemicals and solutions used for cellular cultivation (cultivation media, sera, etc.) were purchased from Sigma-Aldrich or Lonza Group (Switzerland).

Drugs for precomplex experiment were first mixed with FeCl3 in 1:1 molar ratio and incubated at room temperature for 5 mins. Cells were treated at 70% confluence with the drugs or precomplexed drugs at their specified concentrations for 48 h in a 5% CO₂, 37 °C incubator. Chemical compounds used: deferoxamine (Novartis NDC 0078-0467-91, Basel, Switzerland), deferiprone (ApoPharma, Toronto, ON, Canada), deferasirox (Novartis), Dp44mT (gift from Prof. Des Richardson, University of Sydney, Australia), C7, C7-1, C7-2, C7-3, C7-4, C7-5, C7-6 (ChemBridge, ID#5632947, ID#5636413, ID#6120380, ID#5631431, ID#5335854, ID#5630707, and ID#5636784, respectively), PD98059 (Merck 513000, Kenilworth, NJ, USA), romidepsin (Selleck S3020, Houston, TX, USA), 3-MA (Sigma Aldrich M9281, St. Louis, MO, USA), and chloroquine (Sigma Aldrich C6628).

Berberine Cl, isocorydine HCl, cupric sulfate pentahydrate (CuSO₄ ∙ 5H₂O), cuprous chloride (CuCl), DMSO, ferric chloride hexahydrate (FeCl₃ ∙ 6H₂O), ferrous sulfate heptahydrate (FeSO₄ ∙ 7H₂O), 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4′′-disulfonic acid sodium salt (ferrozine), hydroxylamine hydrochloride (hydroxylamine), hematoxylin, hydrochloric acid (HCl), BCS, sodium chloride (NaCl), sodium acetate, acetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), HEPES sodium salt and trientine were purchased from Sigma-Aldrich (Czech Republic), while deferoxamine was from Novartis (Switzerland), boldine was from Carl Roth (Germany), glaucine was from Cayman Chemical (Ann Arbor, MI, USA) and galanthamine hydrobromide was from Changsha Organic Herb (Changsha, China). Chlidanthine and galanthamine were isolated from Chlidanthus fragrans herb [30 (link)], haemanthamine, lycorine and galanthine from Zephyranthes robusta (Herb. ex Sweet) [31 (link)], bulbocapnine, corydine, sinoacutine, canadine, corydaline, scoulerine, tetrahydropalmatine, allocryptopine and corycavamine from Corydalis cava (L.) [32 (link)], californidine iodide, eschscholtzine and protopine from Eschscholia californica [33 (link)], platycerine from Argemona platyceras [34 (link)] and sinactine, stylopine, cryptopine, fumaricine and parfumine from Fumaria officinalis L. [35 (link)].