Fig. 4.

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).