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. Tm^wt (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. Tm^wt (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. Tm^wt (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. Tm^SopE (pM975) invasion being as efficient as S. Tm^wt (pM975). (F) Verification of the automated assay using siRNAs directed against different actin polymerization regulators. Depletion of ArpC3 and Cdc42 reduces S. Tm^SopE (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).