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. 2003 Nov 3;198(9):1361-8.
doi: 10.1084/jem.20031072.

Mycobacterium marinum escapes from phagosomes and is propelled by actin-based motility

Luisa M Stamm  1 , J Hiroshi Morisaki, Lian-Yong Gao, Robert L Jeng, Kent L McDonald, Robyn Roth, Sunao Takeshita, John Heuser, Matthew D Welch, Eric J Brown
Affiliations

Mycobacterium marinum escapes from phagosomes and is propelled by actin-based motility

Luisa M Stamm et al. J Exp Med. .

Abstract

Mycobacteria are responsible for a number of human and animal diseases and are classical intracellular pathogens, living inside macrophages rather than as free-living organisms during infection. Numerous intracellular pathogens, including Listeria monocytogenes, Shigella flexneri, and Rickettsia rickettsii, exploit the host cytoskeleton by using actin-based motility for cell to cell spread during infection. Here we show that Mycobacterium marinum, a natural pathogen of fish and frogs and an occasional pathogen of humans, is capable of actively inducing actin polymerization within macrophages. M. marinum that polymerized actin were free in the cytoplasm and propelled by actin-based motility into adjacent cells. Immunofluorescence demonstrated the presence of host cytoskeletal proteins, including the Arp2/3 complex and vasodilator-stimulated phosphoprotein, throughout the actin tails. In contrast, Wiskott-Aldrich syndrome protein localized exclusively at the actin-polymerizing pole of M. marinum. These findings show that M. marinum can escape into the cytoplasm of infected macrophages, where it can recruit host cell cytoskeletal factors to induce actin polymerization leading to direct cell to cell spread.

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Figures

Figure 1.
Figure 1.
M. marinum is propelled by actin-based motility in primary macrophages. (A) Time lapse images show movement of M. marinum within macrophages. Select motile bacteria are followed with colored arrowheads (also see corresponding Video 1, available at http://www.jem.org/cgi/content/full/jem. 20031072/DC1). (B) A macrophage (shown in phase contrast in the top left) infected with M. marinum–expressing GFP (bottom left) was stained for F-actin (top right). A merged image (bottom right) demonstrates the association of actin tails with the bacterial pole. (C) The appearance of actin tails behind M. marinum as a function of time after infection of primary macrophages is shown. The y axis is the fraction of the total intracellular bacteria that have actin tails. Data shown are from one of two detailed experiments with similar results.
Figure 2.
Figure 2.
M. marinum induces actin polymerization in a fish macrophage cell line and is capable of polymerizing actin in vitro. (A) CLC cells (shown in phase contrast in the top left) were infected with M. marinum–expressing GFP (bottom left). 48 h after infection, the cells with fixed and stained for F-actin (top right). A merged image is shown in the bottom right. (B) M. marinum–expressing GFP (green) grown in macrophages for 48 h were isolated and added to mouse brain extracts. Within 30 min, M. marinum polymerized actin (red) in diffuse clouds surrounding the bacteria (top) and by 1 h, M. marinum polymerized actin into tails at its pole (bottom).
Figure 3.
Figure 3.
M. marinum with actin tails are found free in the host cell cytoplasm. (A) Macrophages were infected with M. marinum (shown in phase contrast in the top left) and stained with DiI (bottom left), a membrane marker, and for F-actin (top right). A merged image is shown in the bottom right. Arrowheads indicate three bacteria with actin tails that are in the plane of focus and not surrounded by DiI. (B and C) Macrophages were infected with M. marinum and observed by TEM. (B) Many M. marinum are found in membrane-bound compartments and show no evidence of actin polymerization. Detail of boxed area is shown at right to highlight the bacterial cell wall and the host membrane lipid bi-layer. (C) An example of M. marinum that polymerizes actin. The detail at right demonstrates the close apposition of actin filaments to the bacterial cell wall. Bars: left, 1.0 μm; right, 0.2 μm. (D) An FFEM image shows the intimate association of M. marinum with its actin tail. Bar, 0.5 μm. Although more often found at a pole, actin polymerization can occur at the side of a bacterium. Inset, a phase contrast image of another M. marinum with actin polymerized at the side and fluorescently labeled F-actin superimposed in red.
Figure 4.
Figure 4.
Arp2/3, WASP, and VASP localize in the actin tails of M. marinum. Macrophages were infected with M. marinum and stained with antibodies for actin (red) and for the host cell proteins. (A) p34-Arc, subunit of the Arp2/3 complex, (B) WASP, and (C) VASP (all shown in green). For orientation, the entire macrophage is shown at the left and details of the boxed area are shown to the right. Images reveal that the Arp2/3 complex and VASP are located throughout the actin tail of M. marinum, whereas WASP is located exclusively at the pole at which the actin tail is formed.
Figure 5.
Figure 5.
Focal growth of intracellular M. marinum in the presence of antibiotics is evidence of direct cell to cell spread. A confluent cell monolayer was infected with GFP-expressing M. marinum and growth was assessed in the (A) presence or (B) absence of amikacin to kill extracellular bacteria. (A) The top row depicts the cell monolayer in phase contrast, and directly below the corresponding fluorescence image demonstrates the focal pattern of GFP-expressing M. marinum. Three representative fields are shown. (B) In parallel experiments where the media did not contain antibiotics, the pattern of M. marinum growth is diffuse.
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