Shigella flexneri Regulation of ARF6 Activation during Bacterial Entry via an IpgD-Mediated Positive Feedback Loop

Virulent S. flexneri activates ARF6 early during infection. (A) ARF6 activation determined by pulldown of GTP-bound ARF6 from HeLa cells 20 or 40 min after initial contact of WT or noninvasive S. flexneri (top panel) with cells, versus total ARF6 in each lysate (bottom panel). GTPγS or GDP added to lysates of uninfected cells and uninfected cells alone (uninfect) are positive and negative controls. Ratio of ARF6-GTP to total ARF6 for blots shown, by densitometry. Blots are representative. (B and C) ARF6-HA recruitment to AFA-I-expressing WT or noninvasive S. flexneri in HeLa cells infected (40 min), fixed, and stained with antibody to HA (green), DAPI (blue), and phalloidin (red). ND, not detectable. Mean ± standard error of the mean for 3 independent experiments. Images are representative. Arrows, ARF6 recruitment to entering bacteria. Arrowheads, bacteria without ARF6 recruitment. Bar, 10 µm.

The phosphoinositide phosphatase activity of Shigella T3SS effector IpgD is required for efficient ARF6 activation at entry sites. (A) ARF6 activation determined by pulldown of GTP-bound ARF6 from HeLa cells infected with indicated S. flexneri strains for 40 min after initial bacterial contact with cells (top panel) versus total ARF6 in each lysate (bottom panel). Ratio of ARF6-GTP to total ARF6 for blots shown, by densitometry. Uninfect, uninfected; Non-invas, noninvasive strain; ΔipgB1 ΔB2, ΔipgB1 ΔipgB2 mutant. Blots are representative. (B to E) Infection of ARF6-HA-transfected HeLa cells (40 min) with WT, ΔipgD, ΔipgD pIpgD, or ΔipgD pIpgD C438S S. flexneri, fixed and labeled with antibody to HA (green), DAPI (blue), and phalloidin (red). (B) Percentage of cells with bacteria associated that show ARF6 recruitment to bacteria. (C) Representative images. Arrows, bacteria with ARF6 recruitment. Arrowheads, bacteria without ARF6 recruitment. Bar, 10 µM. (D and E) Percentage of cells infected or with ruffles. Mean ± standard error of the mean of at least 3 independent experiments.

Efficient ARF6 recruitment to entering S. flexneri requires cytohesin GEFs. Recruitment of ARF6-HA to HeLa cells infected with S. flexneri for 40 min in the presence of SecinH3 or dimethyl sulfoxide (DMSO). (A) Percentage of cells infected. (B) Percentage of cells with ruffles. (C) Percentage of cells with bacteria associated showing ARF6 recruitment to bacteria. Mean ± standard error of the mean of at least 3 independent experiments. (D) Representative images with labeling with antibody to HA (green), DAPI (blue), and phalloidin (red). Arrows, bacteria with ARF6 recruitment. Arrowheads, bacteria without ARF6 recruitment. Bar, 10 µm.

ARNO is recruited to entering S. flexneri independent of ARF6. (A and B) Recruitment of Myc-ARNO 3G to AFA-I-expressing WT or noninvasive S. flexneri in HeLa cells, infected for 30 min. Representative images labeled with antibody to Myc (green), DAPI (blue), and phalloidin (red). (C and D) Recruitment of Myc-ARNO 2G or Myc-ARNO 3G to S. flexneri entering ARF6^K/D or ARF6⁺ MEFs, infected for 25 min. Labeling as in panel B. Arrows, entering bacteria with ARNO recruitment. Arrowheads, bacteria without ARNO recruitment. ND, not detectable. NS, not significant. Mean ± standard error of the mean of at least 3 independent experiments. Bars, 10 µm.

IpgD phosphatase activity is required for ARNO recruitment to entry sites, and PI 3-kinase contributes to ARNO 2G but not ARNO 3G recruitment. (A and B) WT, ΔipgD, ΔipgD pIpgD, and ΔipgD pIpgD C438S S. flexneri infection for 30 min of HeLa cells transfected with Myc-ARNO 3G. (A) Percentage of cells with ARNO recruitment to entering bacteria. (B) Representative images, labeled with antibody to Myc (green), DAPI (blue), and phalloidin (red). (C and D) Recruitment of PI(3,4,5)P₃ to bacterial entry sites upon WT or ΔipgD infection for 40 min of HeLa cells transfected with BTK-PH-GFP, which specifically binds PI(3,4,5)P₃. (C) Percentage of cells with BTK-PH-GFP recruitment to entering bacteria. (D) Representative images, labeled with DAPI (blue), and phalloidin (red), and with GFP signal. (E and F) Inhibition of recruitment of ARNO 2G, but not ARNO 3G, upon treatment with LY294002 upon WT infection of ARF6^K/D MEFs for 25 min. (E) Percentage of cells with ARNO recruitment to entering bacteria. (F) Representative images, labeled as in panel B. Arrows, bacteria with ARNO recruitment. Arrowheads, bacteria without ARNO recruitment. Bars, 10 µm. NS, not significant. Mean ± standard error of the mean of at least 3 independent experiments. DMSO, dimethyl sulfoxide; LY, LY294002.