Fig. 7: GBP1 is recruited to the bacterial surface and binds LPS through electrostatic interactions.

a Size exclusion chromatograms of recombinant His-tagged GBP1 incubated with E. coli LPS, or with E. coli LPS pre-treated with CaCl₂ (5 mM), Polymyxin B (10 µg/mL) or with alkaline phosphatase. Curves were corrected by subtracting the respective LPS-specific absorbance at 280 nm. Black curves representing control condition were overlayed. b Release of LDH from IFNγ-primed HeLa 5 h after transfection with E. coli LPS, or after transfection with LPS previously treated with alkaline phosphatase. c 3D structure of human GBP1 (PDB 1f5n), highlighting five different negatively charged patches (A to E). Residues comprising patch E are only visible in PDB 6k1z. For each patch, the indicated residues were all mutated to alanines and analyzed for GBP1-LPS interaction by size exclusion chromatography. Purple indicates GTPase domain, green indicates helical domain. d Size exclusion chromatograms of different His-tagged GBP1 mutants incubated with E. coli LPS. Curves were corrected by subtracting LPS-specific absorbance at 280 nm. e Fluorescence confocal microscopy of naive HeLa expressing eGFP-GBP1^wt, eGFP-GBP1^KKK61-63AAA or eGFP-GBP1^KK87-88AA and infected with Salmonella-dsRed for 1 h. DNA was stained with Hoechst (blue). Representative confocal images are shown and scale bar corresponds to 5 µm. f Percentage of eGFP-GBP1 positive Salmonella at 1 h p.i., as quantified by counting between 100–200 bacteria per coverslip. Graphs show the mean ± SD, and data are pooled from three independent experiments performed in duplicate (f), four independent experiments performed in triplicate (b), or are representative from three (a, d, e) independent experiments. ***P < 0.001; ns, not significant, two-tailed t-test.