Extended Data Fig. 6. Generation of PtdIns(3,4)P₂ by the Shigella flexneri effector IpgD does not require class I or class II PI3Ks.

(a) Sequence alignment of the phosphate-binding (P-loop) sequence from SopB (Salmonella enterica) and IpgD (Shigella flexneri). Residue 439 of IpgD encodes the cysteine of the C(X)₅R motif.

(b) IpgD reduces PM PtdIns(4,5)P₂ levels in a C(X)₅R-dependent manner. Heterologous expression of EGFP-IpgD (WT or C439S) and PH-PLCδ1 (inverted gray, main panels). The PM was stained with CellMask prior to imaging live.

(c) Quantification of PH-PLCδ1 intensity in the PM from (b) across n=3 independent experiments quantifying 82 cells (IpgD^WT) and 74 cells (IpgD^C439S. Data are trial means ± SEM (foreground) overlaid on cell measurements (gray, background). ****P < 0.0001.

(d) Live cell imaging of heterologously-expressed EGFP-IpgD (WT or C439S) and NES-mCherry-cPHx3 following PM staining with CellMask. Cells were treated with the indicated inhibitors (PI-103, 500 nM; wortmannin, 100 nM) for 20 min prior to and throughout imaging.

(e) PM cPHx3 intensity was quantified from (d) across 3 independent trials (IpgD^WT, 70 cells; IpgD^C439S, 62 cells; IpgD^WT(PI-103), 68 cells; IpgD^{WT(Wortmannin)}, 76 cells). Data are trial means ± SEM (foreground) overlaid on cell measurements (gray, background). ****P < 0.0001.

(f) PM PtdIns(3,4)P₂ synthesis in S. cerevisiae. Galactose-inducible empty vector (control), IpgD^WT, or IpgD^C439S were induced for 2 hours in yeast that expressed cPHx3. Trypan Blue staining demarcates the cell wall and non-viable yeast.

(g) Yeast from (f) were scored for plasmalemmal cPHx3 localization across n=4 independent experiments analyzing the following number of yeast: control, 881 cells; IpgD^WT, 1165 cells; IpgD^C439S, 938 cells. Data are mean ± SEM. ****P < 0.0001. Source numerical data are available in source data.