The pH of the phagolysosome does not affect the cytosolic translocation of M. marinum. (A) Electron micrograph of a THP-1 cell infected with M. marinum for 24 h in the presence of ConB showing M. marinum in the cytosol without membrane and CD63 labeling. CD63 immunolabeling indicated by 10-nm gold particles is present on the multivesicular lysosome in the top left corner. (A′) Schematic representation of the micrograph in panel A, with blue lines representing host membranes, black dots representing CD63 labeling indicated by 10-nm gold particles, and orange representing bacteria. (B) Quantification of the percentage of M. marinum bacteria in the cytosol 24 and 48 h after infection using immunogold labeling for CD63 (see also Fig. S2A in the supplemental material). Cells were treated with ConB to raise the lysosomal pH, which did not affect the percentage of cytosolic M. marinum bacteria. Error bars represent the standard deviations from 3 experiments.

The cytosolic localization of M. marinum in zebrafish is abundant when the adaptive immune system is not yet developed. Embryo and adult zebrafish infected with M. marinum were analyzed using TEM. (A) Cytosolic M. marinum in zebrafish embryo tissue. (B) Phagosomal M. marinum ?ESX-5 bacteria in adult zebrafish tissue. (A? and B?) Schematic representations of panels A and B, with black dots indicating actin immunogold labeling, orange lines indicating M. marinum, blue lines indicating phagosomal and host membranes, and green lines indicating mitochondria. (C) Quantification of the percentage of cytosolic M. marinum bacteria at embryo day 9 and in the spleen of adult zebrafish at day 11 (see also Fig. S2B in the supplemental material). Error bars indicate standard deviations between 3 different zebrafish embryos and 3 adult fish, and n represents the total number of bacteria counted.

Restrained cytosolic M. leprae localization in armadillo skin biopsy specimens. (A and B) Live M. leprae bacteria were injected into the skin of the armadillo at the red circle indicated by the white arrow text box. The skin was observed 3 days after infection (A) and 21 days after infection (B). At infection sites, loss of pigmentation was detected. (C) TEM image of an infected armadillo skin biopsy specimen 3 days after infection with viable M. leprae. Immunogold labeling using anti-CWP was used to indicate M. leprae. (C′) Enlargement of the upper boxed area in panel C. M. leprae is enclosed by host membranes and is thus phagosomal. (C″) Enlargement of the lower boxed area in panel C. M. leprae was not enclosed by host membranes and was thus cytosolic. (C‴ and C⁗) Schematic representations of panels C′ and C″, with orange indicating M. leprae, the blue lines indicating host membranes, and green indicating mitochondrial membranes. The total numbers of bacteria detected were 47 on day 3 and 45 on day 21.

Restrained cytosolic localization of M. leprae in human skin biopsy specimens. Leprosy patient skin biopsy specimens were analyzed using TEM. Immunogold labeling for cathepsin D was used to label lysosomes and phagolysosomes. (A) TEM image of a patient skin biopsy specimen with cytosolic M. leprae. The mycobacteria were not enclosed with host membranes. (A′) Schematic representation of panel A. Orange indicates M. leprae, blue lines indicate host membranes, and green indicates mitochondria. (B) M. leprae present in the phagolysosome. The mycobacterium is enclosed by host membranes and immunogold labeled for the lysosomal marker cathepsin D. (B′) Schematic representation of panel B. Black dots indicate cathepsin D labeling, orange indicates M. leprae, and the blue lines indicate phagolysosomal membranes. (C) Quantification of the average percentage of M. leprae bacteria present in the cytosol. The error bar indicates the standard deviation from 4 different patients, and n represents the total number of intracellular bacteria (see also Fig. S2C in the supplemental material).

Early cytosolic localization of M. tuberculosis in SCID mouse lungs. (A and B) Sections of SCID mouse lung tissue infected with M. tuberculosis H37Rv for 21 days were labeled for LAMP1 using immunogold labeling to indicate lysosomes. (A′ and B′) Schematic representations, with orange indicating M. tuberculosis, blue lines indicating host membranes, black dots representing gold particles indicating LAMP1-decorated lysosomes, and green indicating mitochondria. Panel A shows a cross section of M. tuberculosis surrounded by cellular membranes, which is thus phagosomal. Panel B shows M. tuberculosis present in the cytosol, without cellular membranes surrounding the bacteria. For information on the number of imaged bacteria and mice, see Table S1 in the supplemental material.

M. tuberculosis preferentially localizes to the cytosol in Il1r1^?/? mice. (A and B) Fluorescence microscopy of 200-nm sections stained with DAPI (4?,6-diamidino-2-phenylindole) for nuclei (white) and anti-cell wall protein to detect M. tuberculosis (green) in granulomas in lung tissues of WT B6 mice (A) and Il1r1^?/? mice (B) infected with M. tuberculosis for 28?days. (C) Immunogold labeling using LAMP1 to indicate lysosomal membranes in Il1r1^?/? mice imaged using TEM. (C?) Schematic representation of panel C. M. tuberculosis is depicted in orange, host membranes are in blue, and the host nucleus is in pink. (D) Quantification of the localization of M. tuberculosis in B6 and Il1r1^?/? lungs, here presenting cytosolic localization (see also Fig. S2D in the supplemental material). The analysis included 897 (WT) or 618 (Il1r1^?/?) bacteria, and error bars indicate standard deviations based on in multiple granulomas of 2 WT B6 and 2 Il1r1^?/? mice. KO, knockout.