Francisella tularensis regulates the expression of the amino acid transporter SLC1A5 in infected THP-1 human monocytes
Article first published online: 6 AUG 2012
© 2012 Blackwell Publishing Ltd
Volume 14, Issue 11, pages 1769–1783, November 2012
How to Cite
Barel, M., Meibom, K., Dubail, I., Botella, J. and Charbit, A. (2012), Francisella tularensis regulates the expression of the amino acid transporter SLC1A5 in infected THP-1 human monocytes. Cellular Microbiology, 14: 1769–1783. doi: 10.1111/j.1462-5822.2012.01837.x
- Issue published online: 16 OCT 2012
- Article first published online: 6 AUG 2012
- Accepted manuscript online: 13 JUL 2012 09:03AM EST
- Manuscript Accepted: 6 JUL 2012
- Manuscript Revised: 3 JUL 2012
- Manuscript Received: 25 APR 2012
- Université Paris Descartes
Fig. S1. Intracellular multiplication of the different bacteria used for cell infection. Thirty minutes before infection, LVS bacteria were opsonized at 37°C with human serum. 106 monocyte-like THP-1 cells rendered adherent by addition of PMA for 48 h, were infected with the specified bacteria for 1 h at 37°C, at a Multiplicity of Infection (MOI) of 100:1 for Francisella LVS or U112 (A), iglC or wbtA (C) or 10:1 for Listeria EGDe (B) (bacteria : cell). After washing cells with RPMI first without gentamicin for 1 h sample, then containing gentamicin, cells were further incubated for indicated times at 37°C. For counting bacterial load of cells, THP-1 cells were lysed with saponin. Serial dilutions were performed in 0.15 M NaCl and the number of intracellular bacteria was determined by plating 100 μl on chocolate agar plates. Experiments are representative of five independent experiments.
Fig. S2. Pretreatment of THP-1 cells with cytochalasin blocks entry of LVS and further intracellular multiplication. THP-1 cells infected for 24 h and pre-incubated (B) or not (A) for 15 min with cytochalasin were analysed by confocal analysis, as described in Experimental procedures. For counting bacterial load of cells (C), THP-1 cells were lysed with saponin. Serial dilutions were performed in 0.15 M NaCl and the number of intracellular bacteria was determined by plating 100 μl on chocolate agar plates. Experiments are representative of four independent experiments.
Fig. S3. Pretreatment of cells by the different chemicals does not affect cell viability. Tunicamycin, thapsigargin or monensin (A) and BCH or GPNA (B) were added to cells prior infection, as described in Experimental procedures. Cells were counted by trypan blue exclusion test after 1 h or 24 h infection. Experiments are representative of four independent experiments.
Fig. S4. Disc inhibition assay of the different chemicals used in cell assays. Thapsigargin, monensin and BCH at different concentrations and GPNA at the 3 mM concentration used in cell assays were tested for bactericidal effect, as described in Experimental procedures.
Fig. S5. SLC1A5 is localized on cell surface, after LVS infection. THP-1 cells uninfected (A) or infected for 1 h (B) or 24 h (C) with LVS-GFP (green) were incubated with anti-SLC1A5 (Cyan) and analysed by confocal microscopy, as described in Experimental procedures.
Fig. S6. A. SLC1A5 and CD 147 Abs do not cross-react with LVS protein band. 100 μg proteins solubilized from THP-1 cells either uninfected or infected for 24 h by LVS were loaded in parallel with 10 μg proteins (equivalent to 108 bacteria) obtained from lysed LVS bacteria. The membranes obtained after transfer were blotted either with anti-SLC1A5 or anti-CD147 (upper part) or with anti-actin or anti-EF-Tu (lower part).
B. Supplemented medium has no effect on uptake of bacteria on THP-1 cells. THP-1 cells rendered adherent by treatment with PMA were incubated for 48 h in RPMI-FCS. Cells were then incubated for 1 h in MEM before incubating for 1 h in the indicated medium. After infection by LVS for 1 h and washing the cells either with MEM or RPMI, cells were further incubated for 5 h or 24 h in the indicated medium containing gentamicin. At indicated times, cells were lysed with saponin and 100 μl were plated on chocolate plates for cfu counting. Experiments are representative of four independent experiments.
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