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. 2023 Oct 27;299(12):105390. doi: 10.1016/j.jbc.2023.105390

Figure 1.

Figure 1

ELMO1 and SifA interact directly and cooperate to promote SCV formation.A, immunoblot of control (shC) and ELMO1-depleted (shELMO1) J774 murine macrophages. B, schematic shows workflow for TEM on J774 macrophages infected with either Salmonella (SL) or an SifA-deleted variant strain (SifA) of the same. C and D, representative electron micrographs of control (shC) or ELMO1-depleted (shELMO1) J774 macrophages, infected with Salmonella typhimurium (SL or SifA; 30 MOI), at 6 h postinfection. Examples of bacteria within vacuoles (black arrowheads) and cytosolic bacteria (white arrowheads) are indicated. Bacteria that are either intact (black arrows) or partially degraded upon fusion with lytic compartments (white arrows) are also indicated. Scale bars are placed at the lower left corner of each micrograph. E–G, a montage (E) of pseudo-colored micrographs of SCVs at various stages of formation is shown. The presence (arrowheads) or absence (interrupted lines) of detectable vacuolar membrane are marked. The top panel (>75%) is derived from the “top-left side” image in the “shControl J774” column in 1C. The “50 to 75” pseudo-colored micrograph image (second from the top) is derived from the “lower-left side” image in the “shControl J774” column in (C). The “<25” pseudo-colored micrograph image in the lower-right side corner in (E) is derived from the “top-right side” image in the “shELMO J774” column in (D). Scale bars represent 1000 nm. Pie charts display the percentage of bacteria at each stage of SCV formation (F and G) encountered in (C and D). H, bar graph displays the percentage of complete (representing >75% in E) SCV formation in (C and D). I, a domain map of ELMO1 with major interacting partners. J, recombinant His-SifA (∼5 μg) was used in a pulldown assay with immobilized bacterially expressed recombinant GST-tagged full-length (FL) or a C-terminal domain (CT; amino acids 482–727) of ELMO1 or GST alone (control). Bound SifA was visualized by immunoblot using anti-His (SifA) antibody. GST proteins are visualized by Ponceau S staining. K, recombinant His-tagged CT (amino acids 482–727) or FL ELMO1 proteins (∼3 μg) were used in a pulldown assay with immobilized bacterially expressed recombinant full-length GST-SifA or GST alone (control). Bound ELMO1 proteins were visualized by immunoblot using anti-His (ELMO1) antibody. GST proteins are visualized by Ponceau S staining. BAI1, Brain-specific angiogenesis inhibitor 1 (ADGRB1); ELMO1, Engulfment and Cell Motility protein 1; ERM proteins, ezrin, radixin, and moesin; GST, glutathione-S-transferase; MOI, multiplicity of infection; SCV, Salmonella-containing vacuole; TEM, transmission electron microscopy.