Efficient L. monocytogenes actin polymerization and motility also require host proteins other than the Arp2/3 complex. These include the Ena/VASP proteins, which operate in conjunction with the actin monomer-binding protein profilin. In mammalian cells the Ena/VASP family is comprised of three members: VASP, Mena (mouse Ena), and EVL (Ena VASP like) (Krause et al. 2003). Each has a similar modular structure (Haffner et al. 1995) that includes an N-terminal Ena/VASP homology 1 (EVH1) domain that binds directly to the proline-rich motifs in ActA (Niebuhr et al. 1997), a central proline-rich region that binds to profilin (Reinhard et al. 1995, Kang et al. 1997), and a C-terminal Ena/VASP homology 2 region (EVH2) that binds to filamentous actin and also mediates multimerization (Bachmann et al. 1999). In uninfected host cells, Ena/VASP proteins and profilin are concentrated at the cortex and in cell-matrix adhesion complexes (Krause et al. 2003). During infection by L. monocytogenes, both are specifically recruited to and concentrated at the surface of intracellular bacteria (Theriot et al. 1994, Chakraborty et al. 1995) (Figure 10.4.), unlike the Arp2/3 complex, which is distributed throughout comet tails.
Several lines of evidence indicate that Ena/VASP proteins are functionally important, but not essential, for actin-based motility. Deletion or mutation of the proline-rich Ena/VASP binding sites in ActA prevents Ena/VASP surface recruitment (Smith et al. 1996, Niebuhr et al. 1997) and causes a reduction in the rate of movement (Auerbuch et al. 2003), the percentage of bacteria that initiate movement (Smith et al. 1996, Auerbuch et al. 2003), and the directional persistence of movement (Auerbuch et al. 2003). Moreover, bacteria exhibit reduced motility rates in MVD7 cells (Bear et al. 2000), which are deficient in the expression of all three Ena/VASP proteins (Geese et al. 2002). The similarity between the phenotypes caused by mutation of Ena/VASP binding sites in ActA and global depletion of these proteins from host cells suggests that Ena/VASP proteins function primarily via recruitment to the bacterial surface, and that the general cytoplasmic pool makes only a residual contribution to actin-based bacterial movement.
One key function of Ena/VASP proteins is to modulate the organization of actin filaments within comet tails. Ena/VASP proteins decrease the frequency of actin y-branching by the Arp2/3 complex in the context of purified proteins (Skoble et al. 2001), as well as in the comet tails formed by beads coated with ActA (Samarin et al. 2003) or host nucleation-promoting factors (Plastino et al. 2004). This is similar to the effect of Ena/VASP proteins on actin architecture at the cortex of host cells (Bear et al. 2002). The ability to inhibit y-branching is correlated with an increase in the rates of actin-based motility (Samarin et al. 2003, Plastino et al. 2004). It has been proposed that this increased bacterial speed is caused by an ability of Ena/VASP proteins to promote the dissociation of y-branched filaments from the bacterial surface, which would stimulate the rate of polymerization and retard the drag force associated with filament-surface attachments (Samarin et al. 2003). It is still unclear how Ena/VASP exerts these activities. One hypothesis is that the effects on actin organization and motility may be mediated by F-actin-binding activity. However, deletion of the F-actin-binding region does not affect the rates of bacterial movement (Geese et al. 2002, Auerbuch et al. 2003), suggesting that other activities must be more relevant. The ability to bind F-actin does play a role in promoting straighter movement trajectories, however, which contributes to the ability of the bacteria to spread from cell to cell in a plaque assay (Auerbuch et al. 2003).
A second key activity of Ena/VASP proteins is their ability to recruit profilin to the bacterial surface. Mutations in ActA that prevent Ena/VASP binding also prevent profilin accumulation (Smith et al. 1996), highlighting the functional connection between these molecules. The ability to recruit profilin is critical for bacterial motility, as mutant Ena/VASP proteins lacking the proline-rich profilin binding site are unable to promote rapid motility (Geese et al. 2002, Auerbuch et al. 2003). The importance of profilin is also highlighted by the observation that depletion of the protein from cell extracts (Theriot et al. 1994, Marchand et al. 1995) and interference with its function in cells (Southwick and Purich 1995, Grenklo et al. 2003) either halts motility or reduces motility rates. Moreover, there is a correlation between the presence of profilin at the bacterial surface and rapid bacterial movement—profilin is not recruited to the surface of stationary bacteria, accumulates at the bacterial surface as motility initiates, and disappears as motility slows (Geese et al. 2000). At the biochemical level, profilin is thought to promote actin polymerization by binding actin monomers, displacing them from the sequestering protein thymosin P4, and ushering them onto the barbed ends of actin filaments (Pantaloni and Carlier 1993, Kang et al. 1999). Concentrating profilin at the bacterial surface may in turn concentrate polymerization competent actin, enabling rapid actin polymerization and bacterial movement.
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