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. 2017 Jul 28;13(7):e1006534. doi: 10.1371/journal.ppat.1006534

Fig 7. NleL promotes the ability of EHEC O157:H7 to cause A/E lesions on mammalian cells by inhibiting JNK activation.

Fig 7

(A and B) NleL promoted the formation of actin pedestals induced by EHEC O157:H7. HeLa cells stably expressing control shRNA (shCtrl) or shRNA for Jnk1/2 (shJNK1/2) were co-cultured with indicated EHEC strains for 2 h, then washed with PBS and further cultured for 2.5 h in the fresh medium. After infection, cells were subjected to immunofluorescence microscopy analysis. Shown are representative cell images where anti-E. coli LPS staining indicates bacteria (red), DAPI staining marks the nucleus (blue) and F-actin denotes the filamentous actin stained by CytoPainter Phalloidin-iFluor 488 Reagent (green) (A). Shown in (B) are numbers of actin pedestals per 100 cells. More than 100 infected cells were examined for each infection experiment and data are represented as mean ± s.d. from three independent experiments. Statistical significance was determined by Student’s t-test. *P < 0.05, **P < 0.01, n.s., not significant. (C) NleL enhances the ability of EHEC O157:H7 to infect Caco-2 monolayer (grown for 6 days) by targeting host JNKs. Caco-2 monolayers (grown for 6 days) treated with DMSO or JNK inhibitor SP600125 (10 μM) were infected with EHEC strains for 2.5 h, then washed with PBS and further cultured for 4 h in fresh medium. After infection, cells were subjected to scanning electron microscopy (SEM) analyses. The white arrow represents an actin pedestal; the orange arrows represent bacteria. (D) NleL promotes the ability of EHEC O157:H7 to form A/E lesions on Caco-2 monolayers (grown for 21 days) by inhibiting JNK proteins. Caco-2 monolayers (grown for 21 days) treated with DMSO or JNK inhibitor SP600125 (10 μM) were infected with EHEC strains for 2.5 h, then washed with PBS and further cultured for 4 h in fresh medium. After infection, cells were subjected to SEM analyses. (E) A schematic diagram showing how NleL-mediated JNK ubiquitylation promotes A/E lesion and EHEC colonization through suppressing JNK phosphorylation and impairing MKK7/JNK/AP-1 signaling.