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Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization, Pathogen Vacuole Motility and Infection

Figure 5

Microbial microinjection of LegG1 promotes microtubule polymerization.

(A) Yersinia enterocolitica strain WA (pT3SS), encoding the Ysc T3SS but lacking type III-secreted effectors, produced and secreted fusion proteins of YopE1–53 or YopE1–138 with the Legionella Ran activator LegG1 or the Rab1 GEF SidM. The proteins in the pellet (P) or supernatant (S) were precipitated by chloroform/methanol treatment and visualized by Western blot using an antibody against YopE. (B) HeLa cells were infected (MOI 10, 2 h) with Y. enterocolitica WA (pT3SS) producing YopE1–53-LegG1, washed several times and lysed with 1% digitonin. After centrifugation, proteins in the pellet (intact bacteria, debris) and in the supernatant (translocated bacterial/soluble host proteins) were precipitated, and the fusion protein was visualized by Western blot using an anti-YopE antibody. Controls: HeLa cells alone, bacteria treated with 1% digitonin, 1% SDS or with the T3SS inhibitor CCCP (50 µM). (C) Fluorescence microscopy of YopE1–138-LegG1 translocation into HeLa cells infected (MOI 10, 2 h) with Y. enterocolitica WA (pT3SS) producing YopE1–138-LegG1, YopE1–138-SidM or YopE. Controls: uninfected cells or cells treated with 30 µM taxol or nocodazole. The cells were immuno-stained for α-tubulin and YopE, and nuclei were labeled with DAPI. Bars, 10 µm. (D) Fluorescence microscopy of YopE1–53-LegG1 translocation into HeLa cells treated with nocodazole (1 µM, 1 h) and infected (MOI 10, 2 h) with Y. enterocolitica WA (pT3SS) producing YopE1–53 or YopE1–53-LegG1. The cells were immuno-stained for α-tubulin (green) and YopE (red), nuclei were labeled with DAPI (grey). Bars, 40 µm or 10 µm (insets). (E) Western blot of YopE1–53-LegG1 translocation into HeLa cells treated with nocodazole (1 µM, 1 h) and infected (MOI 10, 2 h) with Y. enterocolitica WA (pT3SS) producing YopE1–53 or YopE1–53-LegG1. The soluble microtubule fraction in the cell supernatant (S) and the pellet (P) was analyzed with an anti-α-tubulin antibody; ratio of polymerized to soluble α-tubulin (R). (F) Fluorescence microscopy of A549 cells treated or not with siRNA against Ran and infected (MOI 10, 2 h) with Y. enterocolitica WA (pT3SS) producing YopE1–53 or YopE1–53-LegG1. The cells were immuno-stained for α-tubulin (green) and YopE (red), and nuclei were labeled with DAPI (grey). Bars, 10 µm or 5 µm (insets). The data shown is representative of 3 independent experiments (A–F).

Figure 5

doi: https://doi.org/10.1371/journal.ppat.1003598.g005