Journal: Frontiers in Microbiology

Article Title: Klebsiella pneumoniae manipulates human macrophages to acquire iron

doi: 10.3389/fmicb.2023.1223113

Figure Lengend Snippet: Inhibition of STAT6-IL-10 axis leads to bacterial iron starvation. (A) Illustrative image of bacteria containing the pAH05 iron-sensing plasmid. Bacterial expression of BFP is dependent on iron availability (RhyB2-promotor). Created with Biorender.com . (B) FI of BFP in bacterial cultures under iron starved and iron sufficient conditions. Bacteria containing the pAH05 plasmid were grown in LB to OD 600 0.5 and afterwards diluted to OD 600 of 0.005 in iron free media (IMDM) or media supplemented with 50 μM iron (III) nitrate nonahydrate and immediately incubated in an automated microplate reader where FI of BFP was acquired every 15 min. Data shown as mean with ± SD as colored error bands. (C) Gating strategy of cells infected with bacteria containing the pAH05 iron-sensing plasmid. PMA-differentiated THP-1 cells were infected with KP (pAH05) for 1 h at MOI of 10 and followed by a 24 h gentamicin-protected, intracellular infection phase, and afterwards subject to flow cytometry analysis. Infected cells were identified as mCherry+. (D) MFI of BFP, as marker for iron starvation in intracellular bacteria, measured in infected (mCherry+) cells. Data shown as mean ± SD of three separate experiments. (E) Measurement of cellular iron levels via the fluorescent probe FerroOrange. Cells underwent 24 h gentamicin-protected KP infection and treatment with 50 μg/mL iron loaded TF or 40 μg/mL anti-human IL-10 blocking antibody. Data from three separate experiments shown as mean ± SD, normalized to KP infected cells. * denotes p < 0.05, *** denotes p < 0.001 for post-hoc statistical testing. KP, Klebsiella pneumoniae ; BFP, blue fluorescent protein; TF, transferrin; MFI, median fluorescent intensity; FI, fluorescent intensity; IL-10, interleukin 10.

Article Snippet: For evaluation of free cellular iron, cells were washed with PBS and stained with 250 μM FerroOrange (FO, Dojindo) for 30 min. FO is a fluorescent probe that detects intracellular Fe 2+ ions with high specificity and does not react with chelated iron or other bivalent metals ( ).

Techniques: Inhibition, Bacteria, Plasmid Preparation, Expressing, Incubation, Infection, Flow Cytometry, Marker, Blocking Assay

Journal: Frontiers in Microbiology

Article Title: Klebsiella pneumoniae manipulates human macrophages to acquire iron

doi: 10.3389/fmicb.2023.1223113

Figure Lengend Snippet: Klebsiella induces iron uptake to establish infection. (A) Intracellular bacteria benefit from iron loaded TF stimulation. PMA-differentiated THP-1 cells were infected with KP for 1 h at a MOI of 10 and treated with 50 μg/mL iron loaded TF during the 24 h gentamicin-protected, intracellular infection phase. Afterwards, bacteria-containing lysates were serially diluted and plated onto LB-agar plates for CFU quantification. Data shown as mean ± 95% CI of three separate experiments, normalized to control condition. (B) Western blot of the iron uptake protein TFR1 and the iron storage protein FT in cells treated with inactivated bacteria or infected with viable KP. Representative blot after 24 h of infection of two separate experiments. (C) Differential TFR1 mRNA expression in KP infected cells. Infected cells and uninfected controls were harvested at indicated time intervals of intracellular, gentamicin protected infection. Data shown as mean ± SEM of three separate experiments. (D) Measurement of cellular iron levels via the fluorescent probe FerroOrange. Cells underwent 24 h gentamicin-protected KP infection or treatment with iron loaded TF, and subsequently FI of FerroOrange was measured. Data from two separate experiments shown as mean ± SD, normalized to untreated controls. * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001 for post-hoc statistical testing. KP, Klebsiella pneumoniae ; TF, transferrin; ctrl, control; CFU, colony forming units; HI, heat-inactivated; GM, gentamicin-killed; TFR1, transferrin-receptor-1; FT, ferritin; ACTB, β-actin.

Article Snippet: For evaluation of free cellular iron, cells were washed with PBS and stained with 250 μM FerroOrange (FO, Dojindo) for 30 min. FO is a fluorescent probe that detects intracellular Fe 2+ ions with high specificity and does not react with chelated iron or other bivalent metals ( ).

Techniques: Infection, Bacteria, Control, Western Blot, Expressing

Journal: Frontiers in Microbiology

Article Title: Klebsiella pneumoniae manipulates human macrophages to acquire iron

doi: 10.3389/fmicb.2023.1223113

Figure Lengend Snippet:

Article Snippet: For evaluation of free cellular iron, cells were washed with PBS and stained with 250 μM FerroOrange (FO, Dojindo) for 30 min. FO is a fluorescent probe that detects intracellular Fe 2+ ions with high specificity and does not react with chelated iron or other bivalent metals ( ).

Techniques: Fluorescence, Infection, Real-time Polymerase Chain Reaction