RNAi screen of Salmonella invasion shows role of COPI in membrane targeting of cholesterol and Cdc42 - PubMed (original) (raw)

RNAi screen of Salmonella invasion shows role of COPI in membrane targeting of cholesterol and Cdc42

Benjamin Misselwitz et al. Mol Syst Biol. 2011.

Abstract

The pathogen Salmonella Typhimurium is a common cause of diarrhea and invades the gut tissue by injecting a cocktail of virulence factors into epithelial cells, triggering actin rearrangements, membrane ruffling and pathogen entry. One of these factors is SopE, a G-nucleotide exchange factor for the host cellular Rho GTPases Rac1 and Cdc42. How SopE mediates cellular invasion is incompletely understood. Using genome-scale RNAi screening we identified 72 known and novel host cell proteins affecting SopE-mediated entry. Follow-up assays assigned these 'hits' to particular steps of the invasion process; i.e., binding, effector injection, membrane ruffling, membrane closure and maturation of the Salmonella-containing vacuole. Depletion of the COPI complex revealed a unique effect on virulence factor injection and membrane ruffling. Both effects are attributable to mislocalization of cholesterol, sphingolipids, Rac1 and Cdc42 away from the plasma membrane into a large intracellular compartment. Equivalent results were obtained with the vesicular stomatitis virus. Therefore, COPI-facilitated maintenance of lipids may represent a novel, unifying mechanism essential for a wide range of pathogens, offering opportunities for designing new drugs.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Establishment of an automated assay to analyze S. Typhimurium invasion. (A) Overview showing the invasion process of S. Typhimurium divided into five major steps: (i) during the binding step, the bacteria attach to the cellular surface by reversible adhesion or irreversible docking; (ii) T1 is used as a molecular syringe to inject effectors (shown in red) into the eukaryotic cell; (iii) these effectors in turn induce membrane ruffling; (iv) subsequently the cellular membrane encloses a bacterium (membrane closure), thereby producing a _Salmonella_-containing vacuole (SCV, shown in blue); (v) after a maturation step, S. Tm genes important for intracellular survival are induced (green). (B) Fluorescence image showing GFP expression of S. Tmwt (pM975) only after invasion into HeLa cells (green=inside bacteria, red=actin, blue=DAPI, white=outside bacteria; scale bar=20 μm). (C) Automated image analysis strategy: S. Tmwt (pM975) infection of HeLa cells followed by the acquisition of nuclei (blue) and bacterial spots (green) using an automated microscope with a × 10 objective. Images were analyzed using CellProfiler as follows: recognition of nuclei, definition of cells, identification of bacterial spots and the allocation of these spots to cells (red outline=infected cell, blue outline=non-infected cell; scale bar whole image=100 μm, detailed image=50 μm). (D) Verification of the automated assay testing inhibitors of Salmonella invasion. HeLa cells were infected with S. Tmwt (pM975) and analyzed as described in (C). Pretreatment of HeLa cells with the inhibitors Cytochalasin D (Cyt. D), Latrunculin B (Lat. B), Toxin B (Tox. B) or the antibiotic gentamycin prevents invasion. (E) Invasion efficiencies of various Salmonella strains into HeLa cells analyzed by the automated assay showing S. TmSopE (pM975) invasion being as efficient as S. Tmwt (pM975). (F) Verification of the automated assay using siRNAs directed against different actin polymerization regulators. Depletion of ArpC3 and Cdc42 reduces S. TmSopE (pM975) invasion (red line=median of three siRNAs tested for each gene; log2 relative invasion=% infected cells with siRNA treatment divided by the median of % infected cells treated with control siRNA).

Figure 2

Genome-scale siRNA screen reveals host cell factors required for Salmonella invasion. (A) Overview of relative invasion of S. TmSopE (pM975) into HeLa cells transfected with siRNA of the druggable genome library. Values represent the median of three siRNAs per gene. Median values above and below 1.5 times of the interquartile range were defined as positive (green) and negative (red) hits, respectively. A confirmatory screen with 298 selected genes approved 72 hits from the genome-scale screen (inserted small graph). Positive (green) and negative (red) hits were determined with median values above 0.3 and below −0.5, respectively. (B) Example images of indicated hits from the screen demonstrating reduced invasion of S. TmSopE (pM975) for Actr3- and Cdc42-depleted cells and increased invasion in the absence of Itgb5 (blue=nuclei, green=invaded bacteria; scale bar=50 μm). (C) Relative invasion of S. TmSopE (pM975) for selected hits from both screens (*_P_-value <0.1, **_P_-value <0.05, NS=not significant, Mann–Whitney _U_-test).

Figure 3

Host cell factors affect Salmonella binding and effector injection. (A) Establishment of the binding assay for indicated siRNA-transfected HeLa cells (left) and corresponding image analysis (right). To analyze binding independent of cell cycle state, mitotic cells and their neighbors (showing an increased binding phenotype; indicated by yellow/white border) were excluded from the analysis using Enhanced CellClassifier. The remaining nuclei either had bound bacteria or not (nuclei with red/blue border; gray=nuclei, green=S. TmΔ4, scale bar=50 μm). (B) Differential quantification of S. TmΔ4 and S. TmΔT1 binding for mitotic cells, neighbors of mitotic cells and other cells. Each bar shows the median and standard deviation from 72 wells of two independent experiments. (C) Binding efficiency of S. TmΔ4 onto HeLa cells transfected with the confirmatory siRNA library. Data are displayed as log2 relative binding corresponding to the percentage of cells with bound bacteria of siRNA-treated cells and control siRNA-treated cells. (D) Binding efficiency of S. TmΔ4 for selected genes. The depletion of Atp1A1 and Rbx1 strongly inhibits binding of S. TmΔ4, whereas the depletion of Itgav and Itgb5 stimulates adherence to HeLa cells. (E) Scheme of the effector injection assay. HeLa cells were loaded with CCF2-AM (green) and infected with Salmonella carrying a fusion protein of SipA with β-lactamase (SipA–TEM). Translocated β-lactamase mediates cleavage of CCF2-AM inducing a shift from green to blue fluorescence. Effector translocation is defined as the ratio between blue (460 nm) and green (435 nm) fluorescence. (F) Validation of the effector injection assay: HeLa cells were infected with S. TmSipA or S. TmSipA-TEM, an increase of effector injection signal (arbitrary units) was observed with increasing m.o.i. of the bacteria. (G) Effector injection analysis of selected genes. HeLa cells transfected with siRNAs directed against copG and copB1 and infected with S. TmSipA-TEM showed a twofold reduction in effector injection efficiency. The ratio between blue and green fluorescence was normalized to control siRNAs and is displayed as log2 of the relative translocation. Two siRNAs per gene have been analyzed (*P<0.1, NS=not significant).

Figure 4

Depletion of Atp1a1 and Rbx1 results in strong inhibitory effects on ruffling and membrane closure. (A) Functionality of the image analysis of ruffling cells. Upper panels: details of the original automated microscopy images (blue=nuclei, gray=actin). Lower panels: results of the automated image analysis (red outlines=ruffling cells, blue outlines=non-ruffling cells; scale bar=20 μm). (B) Examples of the hits with a positive or a negative effect on ruffling induced upon S. TmSopE invasion. Multiple test correction for 298 genes was performed to obtain the _P_-value (*_P_-value <0.1, **_P_-value <0.05, NS=not significant). (C) Fluorescence images showing cells treated either with control siRNAs or siRNAs directed against the indicated genes and infected with S. TmSopE (pM965) (red/green double stain=extracellular bacteria; blue=nuclei; gray=actin; green=intracellular bacteria; scale bar=20 μm). (D) Relative invasion of S. TmSopE into cells transfected with siRNAs directed against the indicated genes. Multiple test correction for 60 genes was performed on the results (**_P_-value <0.05; NS=not significant).

Figure 5

Classification of hits according to their profile in the different assays and cluster analysis of the results of the whole screen. (A) Overview showing the results of the different assays describing Salmonella invasion steps for selected genes. While Odc1 shows a pure binding phenotype, Cdc42 and Rab7a are examples for pure ruffling or maturation phenotypes. Several proteins show combined phenotypes: Actr3 depletion has effects on binding and ruffling, whereas depletion of CopB1 and CopG have inhibitory effects on effector translocation and ruffling. For consistency only the results of the two strongest siRNAs are shown. (B) Scatter plot of relative binding versus relative effector injection. A strong correlation between binding and effector injection is observed for most of the hits. The outliers CopB1 and CopG are stained in purple. Each point indicates the median of the two strongest siRNAs. The results for 90 genes are shown. (C) Heatmap and clustering dendrogram based on the 300 ‘candidate hits’ obtained in the initial screen. (D, E) Functional interactions within the clusters were annotated using the STRING database (confidence cutoff=0.4, additional white nodes: five in cluster c and one in cluster d).

Figure 6

Cdc42, sphingolipid GM1 and cholesterol are mislocalized after depletion of the COPI complex. (A) Confocal images showing the localization of Cdc42–GFP in water-transfected cells (left), cells transfected with siRNA directed against copB1 (middle) or copG (right). The intensity plots along the lines indicated in yellow are shown below the pictures; the position of the membrane is indicated by an asterisk; scale bar=10 μm. (B) Quantification of the Cdc42–GFP signal on the membrane relative to the cytosol (***P<0.005). (C) Confocal images showing the distribution of sphingolipid GM1 (left two panels) and cholesterol (right two panels) in the cells. For each staining, water-transfected cells are shown on the left side and cells transfected with siRNA against copG on the right side; scale bar=10 μm. (D) Quantification of the relative cholera toxin β/filipin staining signal at the membrane compared with the cytosol (***P<0.005).

Figure 7

CopG depletion abolishes actin recruitment to the site of Salmonella binding and ruffling. (A) Confocal images showing the distribution of actin and Rho GTPases upon S. TmSopE invasion for 10 min in stable Rac1- or Cdc42-GFP-expressing cell lines. The cells were transfected either with water (mock) or with siRNA directed against copG; scale bar=10 μm. (B) Size of membrane ruffles after infection with S. TmSopE for 10 min of CopG-depleted cells with or without cholesterol replenishing. The ruffle size was measured in the focal plane showing the largest diameter of the ruffle. (C) Infection of cells either transfected with water (mock) or with siRNA directed against copG by VSV for 5 h. The relative invasion was quantified using a virus construct, which leads to a green fluorescent staining of the cytoplasm upon invasion. Quantification of the amount of infected cells in untransfected cells (mock), or cells depleted of CopG, which were either complemented with cholesterol, with GM1 or with both prior to infection are shown (***P<0.005; *P<0.05; NS=not significant).

Figure 8

Model for S. Typhimurium entry into HeLa cells. In order to invade, S. Typhimurium binds onto cells and injects a cocktail of effectors. This study investigates the action of the key effector SopE. SopE mediates the GTP nucleotide exchange and thereby the activation of the Rho GTPases Rac1 and Cdc42. This leads to the activation of the Arp2/3 complex, actin polymerization cellular ruffling and bacterial entry. The screen identified key regulators of this process, including Nap1, Cdc42, Profilin 1 and the Arp2/3 complex, as well as proteins mediating actin depolymerization, a process probably counteracting the activity of Rac1 and Cdc42. The correct localization seems to be as important as the activation of the Rho GTPases. Upon depletion of the COPI complex, cholesterol and Rho GTPases are mislocalized away from the cell membrane, leading to a much less efficient S. Typhimurium entry. Proteins newly identified to be implicated in S. Typhimurium entry are indicated in red. Proteins that have already been described to have a role in the infection and were found in the screen are indicated in black.

References

1. Agaisse H, Burrack LS, Philips JA, Rubin EJ, Perrimon N, Higgins DE (2005) Genome-wide RNAi screen for host factors required for intracellular bacterial infection. Science 309: 1248–1251 - PubMed
1. Balcer HI, Goodman AL, Rodal AA, Smith E, Kugler J, Heuser JE, Goode BL (2003) Coordinated regulation of actin filament turnover by a high-molecular-weight Srv2/CAP complex, cofilin, profilin, and Aip1. Curr Biol 13: 2159–2169 - PubMed
1. Barwe SP, Anilkumar G, Moon SY, Zheng Y, Whitelegge JP, Rajasekaran SA, Rajasekaran AK (2005) Novel role for Na,K-ATPase in phosphatidylinositol 3-kinase signaling and suppression of cell motility. Mol Biol Cell 16: 1082–1094 - PMC - PubMed
1. Beck R, Rawet M, Wieland FT, Cassel D (2009) The COPI system: molecular mechanisms and function. FEBS Lett 583: 2701–2709 - PubMed
1. Benjamini Y, Hochberg J (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B (Methodological) 57: 289–300

RNAi screen of Salmonella invasion shows role of COPI in membrane targeting of cholesterol and Cdc42 - PubMed (original) (raw)