The Actin Cytoskeleton and Intercellular Spread of Listeria monocytogenes

The ability to spread from cell to cell plays a critical role in infection by many intracellular pathogens. L. monocytogenes has evolved the ability to move directly from cell to cell without passing through the intercellular space, enabling it to evade the host immune response. The process of L. monocytogenes' spread was first observed by imaging infections in guinea pig corneal and intestinal epithelia using electron microscopy (Racz et al. 1970, 1972). These early studies documented that, after internalization into a vacuole and then escape into the cytoplasm, bacteria move into membrane protrusions that extend into invaginations of the membrane of a neighboring cell. Protrusions from an infected cell are then internalized by neighboring cells, completing the process of cell-to-cell transfer. Although these observations were remarkable, the process of L. monocytogenes cell-to-cell spread received little attention for many years.

The mechanism of spread was examined in greater detail nearly two decades later by Tilney and Portnoy (1989), who carefully imaged the process of infection in a macrophage-like cell line using electron microscopy. Their observations revealed the same basic phenomenon involving movement of Listeria into protrusions containing bacteria at their tips, protrusion internalization by neighboring cells to form a double membrane vacuole, and eventual escape from this vacuole (Figure 10.1.). Tilney and Portnoy also made the seminal observation that bacterial movement and protrusion formation involves an ability to associate with the host cell actin cytoskeleton (Figure 10.2.). This observation was also confirmed by other contemporary studies (Mounier et al. 1990). Interestingly, bacteria induce the formation of progressively more elaborate actin-containing structures as infection progresses. At short times (2 h) after infection, the majority of cytoplasmic bacteria are surrounded by an electron dense cloud of host actin filaments. At later times (4h) postinfection, many bacteria in the cytoplasm are associated with long comet tails of actin filaments, with the bacterium at the

Listeria Cell Cell Spread

Figure 10.1. Cartoon diagram of the process of L. monocytogenes actin-based motility and cell-to-cell spread. Internalization of bacteria into a vacuole is followed by escape into the cytoplasm. At short times after escape, bacteria are surrounded by clouds of actin filaments. At later times, bacteria initiate movement, and clouds are converted into comet tails that trail the moving bacteria. Movement propels bacteria into the plasma membrane, causing the formation of a protrusion that can be engulfed by a neighboring cell, where the cycle repeats itself.

Figure 10.1. Cartoon diagram of the process of L. monocytogenes actin-based motility and cell-to-cell spread. Internalization of bacteria into a vacuole is followed by escape into the cytoplasm. At short times after escape, bacteria are surrounded by clouds of actin filaments. At later times, bacteria initiate movement, and clouds are converted into comet tails that trail the moving bacteria. Movement propels bacteria into the plasma membrane, causing the formation of a protrusion that can be engulfed by a neighboring cell, where the cycle repeats itself.

Figure 10.2. Image of actin comet tails formed by moving L. monocytogenes in infected tissue culture cells. Bacteria (green) were visualized by immunofluorescence using an anti-L. monocytogenes primary antibody and a FITC conjugated secondary antibody. Actin (red) was visualized using rhodamine-phalloidin. Image courtesy of Justin Skoble, Daniel Portnoy, and Matthew D. Welch. Scale bar 10 |xm. (A color version of this figure appears between pages 196 and 197.)

apex of the tail. Bacteria located in membrane protrusions are also associated with actin comet tails that extend inward toward the cell body (Figure 10.1.). The ability to associate with actin is essential for spread as treatment of infected cells with cytochalasin D, an inhibitor of actin function, prevents bacterial movement to the plasma membrane and protrusion formation. Together these observations suggested a model for cell-to-cell spread in which the bacterium uses the host cell actin cytoskeleton to move through the cytoplasm of an infected cell and penetrate neighboring cells.

This basic model has been corroborated by more recent observations. Actin-based bacterial movement has been directly observed using video microscopy (Dabiri et al. 1990), and bacterial motility has been clocked at average speeds ranging from 1 to 36 ^m/ min, with the precise velocity differing between individual bacteria in a single cell and populations of bacteria in different cell types (Dabiri et al. 1990, Sanger et al. 1992, Theriot et al. 1992). Moreover, the coupling of actin-based movement and cell-to-cell spread has been directly observed in epithelial monolayers by time-lapse microscopy (Robbins et al. 1999). These live observations of spread show that moving bacteria often collide with the plasma membrane of the host cell, but collisions do not always result in the formation of protrusions that extend into neighboring cells. In many instances, bacteria ricochet off the membrane without forming a protrusion. Surprisingly, there is little correlation between the formation of productive protrusions and bacterial speed or angle of incidence. Instead, protrusion formation is correlated with the state of the host cell monolayer, suggesting that the nature or strength of cell-cell adhesions or the organization of the cortical cytoskeleton play critical roles. Nevertheless, if bacteria succeed in forming protrusions that extend into neighboring cells, the process of cell-to-cell transfer can occur.

It is now clear that this mechanism of cell-to-cell spread is not unique to L. monocytogenes. Numerous bacterial pathogens have been shown to use the host actin cytoskeleton to promote intracellular movement, including Shigella flexneri (Bernardini et al. 1989), L. ivanovii (Karunasagar et al. 1993), spotted fever group Rickettsia species (Heinzen et al. 1993), Burkholderia pseudomallei (Kespichayawattana et al. 2000), and Mycobacterium marinum (Stamm et al. 2003). Based on differences in the molecular mechanism by which they interact with actin (Gouin et al. 2005), it is likely that different pathogens have independently evolved the ability to manipulate the actin cytoskeleton of the host.

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