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. 2021 Mar 28;296:100607. doi: 10.1016/j.jbc.2021.100607

Figure 6.

Figure 6

Plasma membrane targeting of BteA effector does not contribute to its cytotoxicity.A, protein-lipid overlay assay. Recombinant GST-tagged full-length B. pertussis BteA (BteA/BtcA) and its mutated variant with charge reversal substitutions within the loop L1 (BteA-L1/BtcA; R50E + H52E + H53E) were incubated with home-made lipid arrays at 5 μg/ml. The BteA binding was detected using an anti-GST antibody, followed by chemiluminescence detection. Recombinant GST was used as a control. See the legend of Figure 1B for the description of spotted lipids. Results are representative of two independent experiments. B and C, localization of the GFP-tagged BteA protein variants. B, S. cerevisiae BY4741 cells harboring plasmids encoding GFP-tagged full-length BteA of B. pertussis and its variant BteA-GFP-L1 (L1; R50E + H52E + H53E) were cultivated for 20 h in the medium supplemented with galactose to induce protein expression. Scale bar, 5 μm. C, HeLa cells were transiently transfected with plasmids expressing GFP-tagged variants of B. pertussis BteA (BteA-1-642-GFP, BteA-1-642-L1-GFP) or B. bronchiseptica BteA (BbBteA-1-644-GFP, BbBteA-1-644-L1-GFP). After 18 h, the HeLa cells were fixed and examined by fluorescence microscopy. Scale bar, 20 μm. Representative images from two independent experiments with the same outcome are presented. D and E, HeLa cells were infected with WT, ΔbteA, and bteA-L1 mutant derivatives of B. bronchiseptica RB50 at the indicated multiplicity of infection (MOI). Cytotoxicity was measured as lactate dehydrogenase (LDH) release 3 h postinfection (D) and as real-time kinetics of membrane permeabilization determined by fluorescent DNA binding dye CellTox Green (E). Values represent the means ± SD from at least two independent experiments performed in duplicate (n = 4). ∗p < 0.05, bteA-L1 versus WT-infected cells, unpaired two-tailed t test using the corresponding infection time and MOI. GST, glutathione-S-transferase; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine.