gentamicin protection assays  (ATCC)

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: Where indicated, cells were stimulated during the gentamicin-protected infection phase (directly after washing and addition of gentamicin-containing medium) with either 50 μg/mL iron-loaded transferrin (TF, Sigma-Aldrich), 1 μM STAT-6 inhibitor AS1517499 (MedChem Express), 40 μg/mL anti-human IL-10 blocking antibody (Biolegend) or 40 ng/mL recombinant human IL-10 (Peprotech).

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 infects human macrophages and induces TFR1. (A) Quantification of intracellular bacteria during the 24 h course of infection. PMA-differentiated THP-1 cells were infected with KP for 1 h at a MOI of 10 and were lysed directly after incubation phase (0 h time point) or at noted time intervals of gentamicin-protected, intracellular infection. Bacteria-containing lysates were serially diluted and plated onto LB-agar plates for CFU quantification. Data are shown as mean CFU/well lysate ±95% CI of three separate experiments. (B) Western blot of the iron uptake protein TFR1 and the iron storage protein FT in KP infected cells. Representative blot after 24 h of infection of three separate experiments. (C) Immune fluorescence imaging revealing intracellular bacteria and induction of TFR1 in infected cells after 24 h of infection. Representative images, showing Ypet expressing bacteria (green rods) in cells stained for TFR1 (red) and nuclei (blue) at 400x magnification with a 20 μM scale bar. *** denotes p < 0.001 for post-hoc statistical testing. KP, Klebsiella pneumoniae ; ctrl, control; CFU, colony forming units; TFR1, transferrin-receptor-1; FT, ferritin; ACTB, β-actin.

Article Snippet: Where indicated, cells were stimulated during the gentamicin-protected infection phase (directly after washing and addition of gentamicin-containing medium) with either 50 μg/mL iron-loaded transferrin (TF, Sigma-Aldrich), 1 μM STAT-6 inhibitor AS1517499 (MedChem Express), 40 μg/mL anti-human IL-10 blocking antibody (Biolegend) or 40 ng/mL recombinant human IL-10 (Peprotech).

Techniques: Bacteria, Infection, Incubation, Western Blot, Fluorescence, Imaging, Expressing, Staining, Control

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: Where indicated, cells were stimulated during the gentamicin-protected infection phase (directly after washing and addition of gentamicin-containing medium) with either 50 μg/mL iron-loaded transferrin (TF, Sigma-Aldrich), 1 μM STAT-6 inhibitor AS1517499 (MedChem Express), 40 μg/mL anti-human IL-10 blocking antibody (Biolegend) or 40 ng/mL recombinant human IL-10 (Peprotech).

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: TFR1 induction is mediated by the STAT6-IL-10 axis. (A) TFR1 induction in infected cells is STAT-6 dependent. PMA-differentiated THP-1 cells were infected with KP for 1 h at MOI of 10. During the 24 h gentamicin protected, intracellular infection phase, cells were treated with 1 μM of the STAT-6-inhibitor (AS1517499) and subsequently harvested for Western blotting. Representative blot of three separate experiments. (B) Differential IL-10 mRNA expression of KP infected cells treated with STAT-6 inhibitor. Infected cells and uninfected controls were harvested at indicated time intervals of intracellular, gentamicin protected infection. Data shown as mean ± SEM of two separate experiments. (C) IL-10 levels in supernatants of infected cells treated with STAT-6 inhibitor, as determined by ELISA. Supernatants of infected cells and uninfected controls were collected at indicated time intervals of intracellular, gentamicin protected infection. Data shown as mean ± SD of two separate experiments. (D) TFR1 induction in infected cells is IL-10 dependent. Infected cells were treated with 40 μg/mL anti-human IL-10 blocking antibody during the 24 h intracellular, gentamicin protected infection phase und subject to Western blotting. Representative blot of three separate experiments. (E) Western blot of the iron uptake protein TFR1 and the iron storage protein FT in cells treated with inactivated bacteria or 40 ng/mL recombinant human IL-10. Representative blot after 24 h of infection of two separate experiments. (F) Intracellular bacterial numbers are decreased in cells treated with STAT-6 inhibitor. PMA-differentiated THP-1 cells were infected with KP for 1 h at a MOI of 10 and treated with 1 μM STAT-6 inhibitor (AS1517499) 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. (G) Intracellular bacterial numbers are decreased in cells treated with anti IL-10. Infected cells were treated with 40 μg/mL anti-human IL-10 blocking antibody 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 isotype-treated control condition. (H) MFI of TFR1 in infected cells treated with 40 μg/mL anti-human IL-10 blocking antibody during the 24 h gentamicin-protected, intracellular infection phase. MFI was measured from at least 100 cells per condition using the automated CellSense software. Data shown as mean ± SD. (I) Representative immune fluorescence imaging revealing the effect of IL-10 blockade on TFR1 expression in infected cells after 24 h of infection. Representative images, showing Ypet expressing bacteria (green rods) in cells stained for TFR1 (red) and nuclei (blue) at 400x magnification with a 20 μM scale bar. # denotes p < 0.05 compared to untreated controls, ** denotes p < 0.01, *** denotes p < 0.001 for post-hoc statistical testing. KP, Klebsiella pneumoniae ; ctrl, control; stat6i, STAT-6 inhibitor; IL-10, interleukin 10; CFU, colony forming units; HI, heat-inactivated; TFR1, transferrin-receptor-1; FT, ferritin; ACTB, β-actin.

Article Snippet: Where indicated, cells were stimulated during the gentamicin-protected infection phase (directly after washing and addition of gentamicin-containing medium) with either 50 μg/mL iron-loaded transferrin (TF, Sigma-Aldrich), 1 μM STAT-6 inhibitor AS1517499 (MedChem Express), 40 μg/mL anti-human IL-10 blocking antibody (Biolegend) or 40 ng/mL recombinant human IL-10 (Peprotech).

Techniques: Infection, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Blocking Assay, Bacteria, Recombinant, Control, Software, Fluorescence, Imaging, Staining

Journal: bioRxiv

Article Title: Generation of an optimised Cumate toolkit for tuneable protein expression during in vitro and in vivo studies of Burkholderia cenocepacia

doi: 10.1101/2025.11.26.690688

Figure Lengend Snippet: Cumate induction allows inducible control of protein expression from B. cenocepacia within THP-1 cells . A) Gentamicin protection assays demonstrate comparable bacterial burden, assessed as CFU/ml, within THP-1 cells under induced and non-induced conditions as compared to the MH1K strain. B) Immunoblot of sfGFP expression after 2, 4, 6, and 24 hours of cumate induction within THP-1 cells reveals detectible levels of sfGFP after 2 hours of induction. C) Immunofluorescence microscopy of internalised MH1K and MH1K containing CymR GV -sfGFP strains within THP-1 cells under induced and uninduced conditions reveal co-localisation of sfGFP and B. cenocepacia in an induction dependent manner. DNA has been stained with Hoechst 33342, B. cenocepacia with Alexa Fluor 568, sfGFP visualised through intrinsic fluorescence, and actin filaments labelled with SiR-actin 647.

Article Snippet: Gentamicin protection assays were undertaken using the approach of Hamad et al [ ].

Techniques: Control, Expressing, Western Blot, Immunofluorescence, Microscopy, Staining, Fluorescence