A candidate approach implicates the secreted Salmonella effector protein SpvB in P-body disassembly

Abstract

P-bodies are dynamic aggregates of RNA and proteins involved in several post-transcriptional regulation processes. P-bodies have been shown to play important roles in regulating viral infection, whereas their interplay with bacterial pathogens, specifically intracellular bacteria that extensively manipulate host cell pathways, remains unknown. Here, we report that Salmonella infection induces P-body disassembly in a cell type-specific manner, and independently of previously characterized pathways such as inhibition of host cell RNA synthesis or microRNA-mediated gene silencing. We show that the Salmonella-induced P-body disassembly depends on the activation of the SPI-2 encoded type 3 secretion system, and that the secreted effector protein SpvB plays a major role in this process. P-body disruption is also induced by the related pathogen, Shigella flexneri, arguing that this might be a new mechanism by which intracellular bacterial pathogens subvert host cell function.

Figure 1. Salmonella infection induces PB disassembly, but not SG formation.

(A) HeLa cells were mock-treated or infected for 20 hours with Salmonella expressing GFP, and either left untreated (control) or treated with puromycin for 1 hour. PBs were detected by staining the cells with anti-DDX6 antibody (red channel). Scale bar, 10 µm. The region highlighted by a white square is enlarged on the rightmost panel. (B) The average number of PBs per cell in mock-treated cells and Salmonella infected cells was calculated as described in Materials and Methods. Salmonella infected cells were divided into two populations: cells in which the bacteria were not internalized (Salmonella −) and cells in which the bacteria were internalized (Salmonella +). Mean values ± standard deviations from at least three independent experiments are shown. (C) SG formation was evaluated in HeLa cells mock-treated or infected for 20 hours with Salmonella, either left at 37°C (control) or after heat-shock for 1 hour at 46°C. Cells were stained with anti-TIAR antibody (red channel).

Figure 2. Salmonella infection does not interfere with host cell RNA synthesis and microRNA silencing pathways.

(A) Western-blot analysis of DDX6, AGO2 and GW182 protein levels in HeLa cells mock-treated or infected for 20 hours with Salmonella WT or ΔSpiC and ΔSpvB mutant strains. GFP signal is shown to confirm the presence of intracellular bacteria. (B) Analysis of host cell RNA synthesis in mock-treated and Salmonella infected cells (20 hours p.i.), through the incorporation of EU in newly transcribed RNA, followed by the detection using Alexa Fluor 594 azide (red channel), as described in Materials and Methods. Salmonella was stained with anti-LPS antibody (green channel). Scale bar, 10 µm. (C) HeLa cells were transfected with the psiCHECK2 empty plasmid or with psiCHECK2 plasmids carrying binding sites for the indicated microRNAs. Twenty-four hours after transfection, cells were mock-treated or infected with Salmonella. Luciferase activities were measured 20 hours after infection. Renilla luciferase (R-Luc) activity was normalized to that of the Firefly control (F-Luc) and normalized R-Luc activities of cells transfected with the empty vector were set to 100. Error bars represent standard deviations from three independent experiments.

Figure 3. Salmonella induced PB disruption is dependent on the activation of the SPI-2 T3SS.

(A) HeLa cells were mock-treated or treated with LPS (1 µg/ml) for 20 hours. Cells were stained with anti-DDX6 antibody. Scale bar, 10 µm. The region highlighted by a white square is enlarged on the rightmost panel. (B) HeLa cells were infected with Salmonella for 2, 4, 8 and 12 hours as indicated, and then processed for PB detection using anti-DDX6 antibody. (C) Average number of PBs per cell in the population of cells in which Salmonella was not internalized (Salmonella −) and in cells in which the bacteria were internalized (Salmonella +), at the indicated times post-infection. Mean values ± standard deviations from at least three independent experiments are shown. (D) HeLa cells were infected with wild-type Salmonella or with the indicated mutant strains and collected at 20 hours p.i. PBs were detected using anti-DDX6 antibody (red channel). (E) Ratio of the average number of PBs per cell between the cell population in which bacteria where not internalized (Salmonella −) and the infected cells (Salmonella +) for wild-type and mutant Salmonella strains. Mean values ± standard deviations from at least three independent experiments are shown.

Figure 4. SpvB effector protein is involved in PB disassembly induced by Salmonella infection.

(A) HeLa cells were infected with wild-type or single mutants of all Salmonella effector proteins. The ratio of the average number of PBs per cell, between the cell population in which bacteria where not internalized (Salmonella −) and the infected cells (Salmonella +), was calculated. Mean values ± standard deviations from at least three independent experiments are shown. Representative images of cells infected with the different mutants are shown in Figure S4. (B) HeLa cells were infected with wild-type Salmonella or with the indicated mutant strains. Twenty-hours post-infection cells were processed for immunofluorescence and PBs were detected using anti-DDX6 antibody (red channel). Scale bar, 10 µm. The region highlighted by a white square is enlarged on the rightmost panel. (C) Ratio of the average number of PBs per cell between the cell population in which bacteria where not internalized (Salmonella −) and the infected cells (Salmonella +) for wild-type and mutant Salmonella strains. (D) Intracellular replication assays were performed in HeLa cells with the indicated strains. Values indicate the fold increase, calculated as the ratio between the intracellular bacteria at 20 hours p.i. and the input bacteria used for infection. Error bars represent standard deviations from three independent experiments.

Figure 5. Shigella infection also induces PB disassembly.

HeLa cells were mock-treated or infected with Shigella for 12 and 20 hours. PBs and Shigella were detected using anti-GW182 (green channel) and anti-Shigella (red channel) antibodies, respectively. Scale bar, 10 µm. The region highlighted by a white square is enlarged on the rightmost panel.