Electron microscopy of ejecting bacteria and schematic representation of an ejection event. Scanning electron microscopy (A–B, A’ is the magnified inset of A) showed bulges of the plasma membrane (small arrows), some ruptured at the tip of the ejecting bacterium (B, asterisk). Scale bars 0.5 µm. (C) Serial sections through an ejectosome revealed the organization of the Factin (white arrowheads) and the plasma membrane. The posterior of the ejecting bacterium (black arrowhead) was in the cytosol. The bacterium was separated from the F-actin by the invaginated plasma membrane, which was tightly apposed to its surface. Membrane fragments were scattered along the extracellular part of the bacterium (small arrows). (D) Schematic representation of an ejection. During outward movement of a bacterium (green), the F-actin barrel exerts contraction (red arrows), and the invaginating plasma membrane (PM) reseals at the posterior of the bacterium (black arrows), maintaining a tight septum despite partial membrane rupture. (E) Micrograph of a phagocytic cup. The actin-filled lamellipodia (white arrowheads) extend and engulf a bacterium. Scale bars 1µm.

Biogenesis, structure and topology of ejectosomes. Paraformaldehyde-fixed cells infected with GFP-expressing M. marinum (green), stained for F-actin (red). (A) A phagocytic cup lined with F-actin (arrows). A single (B) or multiple bacteria (C, D) spanning the host cell plasma membrane (asterisks) through ejectosomes (white arrowheads) with the intracellular part of the bacterium devoid of actin-labeling (black arrowhead). (E) Cell-to-cell transmission of bacteria from a donor (do) to an acceptor (ac) cell through an ejectosome into a phagocytic cup (arrows). (F) Movement of cytosolic bacteria (blue) through ejectosomes (F-actin, green) induces a plasma membrane bulge (small arrows) that ruptures at the tip (F, asterisk). The intracellular part of the ejecting bacterium is devoid of p80, a plasma membrane marker (F), and vacuolin, a niche marker (asterisk, G). Some extracellular bacteria were positive for p80 (small arrows, H). (I–K) Live infected cells (M. marinum, blue, actin, green) were incubated in the presence of an anti-M. marinum serum. The extracellular parts of ejecting (small arrows, I, K) or outside (small arrow, J) bacteria were accessible to the antibody and labeled red, in contrast to intracellular bacteria (black arrowhead, J), confirming partial loss of plasma membrane integrity. Scale bars 1 µm.

Ejection is a process involving both host and pathogen factors and is a strategy shared by tubercular mycobacteria. (A) In wild type cells (Dd wt) both, M. marinum (mar) and M. tuberculosis (tuber) were able to escape their vacuolin–coated (green) niche (upper row, black arrowheads) and form ejectosomes (lower row, green, white arrowheads). Plasma membrane fragments on ejecting bacteria were positive for p80 (red, small arrows), but the intracellular part of the bacteria was devoid of p80 labeling (black arrowhead). In contrast M. avium remained in a vacuolin-positive compartment (upper row) and did not form ejectosomes. (B) In racH-cells GFP-expressing M. marinum (red) translocated into the cytosol (upper row), but no ejectosome was detected (lower row; actin, green; p80, red). M. marinum ΔRD1 escaped its niche in wild type and ESAT-6 (E6) expressing cells; however, ejectosomes (white arrowhead) were only detected in ESAT-6 expressing cells. (C–E) Trans-complementation by expression of M. marinum ESAT-6 in the Dictyostelium cytosol. Removal of tetracycline-induced ESAT-6 expression (C). Wild type and ESAT-6 expressing cells were infected with GFP-expressing M. marinum ΔRD1 and the infection monitored by FACS. Quantification of total fluorescence of infected cells indicated increased replication of M. marinum ΔRD1 in ESAT-6 expressers (D, red curve, normalized to T=0.5 hpi). Infected ESAT-6 expressers with high fluorescence (FL1) persisted longer (E, red curve), compared to infected wild type Dictyostelium (D, E, blue curve).

Direct cell-to-cell transmission of M. marinum occurs via ejectosomes and is RacH dependent. (A) Quantitative dissemination assay. A donor strain (wild type or racH-cells) was infected with DsRed-expressing M. marinum and, at 12 hpi, mixed with a green fluorescent acceptor strain. Presence of bacteria in donor and acceptor cells was scored. Dissemination efficiency for wild type (B) and racH-(C) donor cells. Number of bacteria per cell (classified into groups of 1–3, 4–10 and >10 bacteria/cell) are indicated. (D–F) Live GFP-ABD expressing Dictyostelium cells (green) infected with DsRed-expressing M. marinum (red) were imaged at 35 hpi for the indicated times (upper left corner). (D) Nonlytic ejection of mycobacteria occurs through actin-dense ejectosomes (white arrowhead). (E) Cells with mycobacteria spanning the plasma membrane retain normal motility. (F) Cell-to-cell transmission of a cytosolic bacterium (black arrowhead) from a donor cell (do) to an acceptor cell (ac) through an ejectosome (white arrowhead) into a phagocytic cup (white arrows). (G) A bundle of bacteria are ejected from a donor cell (do). The plasma membrane bulge (small arrows) is ruptured at the tip (asterisk), where it contacts an acceptor cell (ac). Actin tails stained by phalloidin (green, arrowheads), were polarized at the posterior of bacteria (blue). Vertical distance from the first section is indicated in µm. Scale bars 1 µm.