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. 2019 Jan 24;15(5):796–812. doi: 10.1080/15548627.2019.1569297

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© 2019 Leiden University. Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

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Figure 2. — Types of Salmonella-containing vesicles in professional phagocytes. (a) Regions of interest (blue squares) for confocal images of infected phagocytes in the blood circulation in b. (b) Representative confocal micrographs for 6 distinctive patterns of GFP-Lc3 association with mCherry-expressing S. Typhimurium (b_i-b_vi) and 3 types of Lc3-negative cases (b_vii-b_ix) observed in motile phagocytes in the tail and yolk sac regions at 4 hpi following systemic infection by caudal vein injection. Under each image the symbolic representation of the pattern is shown. Dotted lines outline the cellular boundaries of the phagocytes and yellow arrows point towards GFP-Lc3 association with bacterial cells. (c) Quantification of types of Lc3-positive and Lc3-negative Salmonella associations observed in b. Percentages of the 9 types of Lc3-positive and Lc3-negative vesicles were determined from images of Salmonella-infected phagocytes and averaged from 5 embryos (n > 30 Lc3⁺/Lc3⁻ Salmonella associations per embryo, 196 in total). Error bars represent the SD. (d) Region of interest (blue square) in subcutaneously infected embryos for image acquisition through TEM in e to j. (e) TEM micrograph of a heavily infected macrophage with phagocytosed S. Typhimurium at 4 hpi, located in the lumen underneath the epidermis. Cytoplasm and nucleus (n) of the cell are pushed towards the cellular periphery and a large number of phagocytosed S. Typhimurium cells (S.T) are centrally contained in a large phagosomal compartment. The long white arrow indicates a dividing bacterial cell. The short thick white arrows indicate examples of dissociating Salmonella cells recognized by the retraction of the cytoplasm from the inner membrane. The black arrow indicates phagocytosing activity of the macrophage which is enlarged in e’. The yellow arrows in e’ point to membrane ruffles of the macrophage formed to enclose extracellular S. Typhimurium (1 and 2). Figure S1 shows a broader view of this macrophage in the tissue context. (f) TEM micrograph showing two S. Typhimurium-containing compartments. The larger compartment (long arrow) contains cellular debris in addition to bacterial cells (S.T1 and S.T2). The lumen of the smaller vesicle (short thick arrow), which holds a single bacterium (S.T3), is clean. The nucleus (n) and a mitochondrion (mt) in close vicinity of the bacterial compartments are indicated. (g) TEM micrograph of S. Typhimurium (S.T1) inside a multi-vesicular body (MVB, long arrow). The short thick arrow indicates a cytoplasmic Salmonella cell (S.T2) with intact outer and inner bacterial membrane. (h) TEM of S. Typhimurium (S.T1) inside a vesicle that forms tubular extensions into the cytoplasm (arrows). (i) TEM showing S. Typhimurium cells (S.T1 and S.T2) inside two single membrane-bound compartments. The presence of ribosomes (arrow) in the internal environment suggests membrane damage. (j) TEM of three S. Typhimurium (S.T) cells in the cytoplasm, one of which (S.T1) is replicating. Scale bars: b = 5 μm, e-j = 2 μm, e’ = 1 μm.