Fig. 4

Electron microscopy analysis. Mtb lacking PDIM is much less frequently present in the cytosol. Human lymphatic endothelial cells were infected with Mtb WT, Mtb ΔPDIM or Mtb ΔPDIM::PDIM for 5 + 24 h and fixed. The samples were processed for resin embedding and then transmission electron microscopy was used to look at the subcellular localisation of Mtb. Three potential localisations were considered and classified. a–c Cytosolic (defined as no obvious host membrane surrounding bacteria outside the bacterial capsule-like material visible on the surface). d–f Phagosomal (defined as bacteria enclosed in a single membrane-bound organelle). Most of these are larger (membrane-bound) vacuoles, often with more than one Mtb cell as well as additional irregular membranes and contents in various stages of degradation, typical for phagolysosomes. Arrowheads indicate the phagosomal/phagolysosomal limiting membrane. g–i Autophagic. In this case, the bacteria and surrounding cytoplasm are enclosed by either single or multiple membranes that are mostly poorly preserved. In contrast to phagolysosomes, the inner contents of the autophagosomes do not appear to be degraded. The arrowheads here indicate the poorly visible membrane or remains of membranes on the periphery of the structure. In all images, black stars indicate bacteria. Scale bars are 500 nm. j Stereological analysis was performed to determine the proportion of Mtb in each compartment. Data are representative of two independent experiments with at least 32 infected cells analysed for each condition. Mtb Mycobacterium tuberculosis, PDIM phthiocerol dimycocerosate, WT wild type