Shigella T3SS establishes direct contact with the vacuole membrane during infection.

(A–C) HeLa eGFP-Lysenin mOrange-Galectin-3 infected with Tag-BFP Shigella were processed through a correlative cryo-ET workflow to capture intact (A), damaged (B), or ruptured (C) vacuoles. The first panel displays cryo-fluorescence images of the bacteria imaged by cryo-ET. Scale bars 2 µm. The second panel shows tomographic slices, with T3SSs regions marked by dashed boxes. Scale bars 500 nm. Insets show enlarged views of the T3SSs. Scale bars 50 nm. The right side panel shows membrane segmentation with vacuole coloured according to the infection stage identified by correlative cryo-ET. Yellow arrowheads indicate the T3SSs shown in the insets. White arrowheads point to bacterial membranes deformations while outlined arrowheads point at unknown densities. Vac., Vacuole; OM, Outer membrane; IM, Inner membrane. See also S3A–S3C Fig for full correlation strategy and S4–S6 Videos for tomogram and 3D rendering of vacuole and bacteria membranes. (D) Quantification of T3SSs surface distribution (pole/body) across all infection stages. n = 147 individual T3SS analyzed. (E) Violin plot of the point-based calculation of distances between T3SSs across all infection stages and corresponding summary table. Dashed lines correspond to 25% and 75% percentiles, solid line indicates the median. n = 147. (F) Contingency graph of the percentage of T3SSs contacting or not the vacuole membrane according to the infection stage. Only the T3SSs for which a basal body and a needle were identified are plotted. Intact n = 49, Damaged n = 15, Ruptured n = 36. The data underlying Fig 2 can be found in S2 Data.