ActA Mediates Actin Polymerization

A bacterial gene that is required for actin polymerization, called actA, was identified in a screen for transposon mutants that failed to spread between cells in a plaque assay (Kocks et al. 1992) and also by directed insertional mutagenesis (Domann et al. 1992). Mutations in actA cause a complete failure to assemble actin at the bacterial surface and to undergo intracellular motility. Entry and intracellular growth are not affected, but the motility defect results in the failure to spread from cell to cell. In frame deletion mutants in actA have also been generated and cause similar phenotypes (Brundage et al. 1993). Importantly, these actA deletion mutants have an LD50 (lethal dose, 50%) in mice of -107, compared to ~104 for the wild-type strain, suggesting that the ability to polymerize actin is critical for virulence.

As expected for a protein that is required to manipulate the host actin cytoskeleton, ActA is present on the bacterial surface (Kocks et al.1992) (Figure 10.3.). ActA has a canonical signal sequence that mediates secretion and

Listeria Acta Domain

Figure 10.3. Cartoon diagram depicting ActA localization at the bacterial surface, ActA domain organization, and the sequences of different regions within ActA aligned with the sequences of homologous host proteins. ActA domains are indicated as follows: SS signal sequence, A acidic, AB actin-binding, C connector, P proline rich, TM transmembrane. Sequence alignments are for the listed proteins from the following species: Lm, Listeria monocytogenes; Hs, Homo sapiens.

Figure 10.3. Cartoon diagram depicting ActA localization at the bacterial surface, ActA domain organization, and the sequences of different regions within ActA aligned with the sequences of homologous host proteins. ActA domains are indicated as follows: SS signal sequence, A acidic, AB actin-binding, C connector, P proline rich, TM transmembrane. Sequence alignments are for the listed proteins from the following species: Lm, Listeria monocytogenes; Hs, Homo sapiens.

a transmembrane domain that anchors it to the bacterial cytoplasmic membrane (Vazquez-Boland et al. 1992). It is observed only on the bacterial surface and is not found within actin comet tails (Kocks et al. 1993, Niebuhr et al. 1993), suggesting that surface-bound protein is the functional form. Interestingly, ActA exhibits a polarized distribution, with the highest concentration at the older pole and the lowest concentration at the newer pole or the septum (Kocks et al. 1993). The pole with the highest concentration corresponds to the location from which actin polymerizes and the comet tail emanates. Polarization of ActA is correlated with efficient comet tail formation and rapid motility (Rafelski and Theriot 2005), and therefore may play a key role in spread. How the polarized distribution of ActA is generated is not well understood, although polarization is known to occur after initial surface targeting (Rafelski and Theriot 2006).

In addition to being necessary for actin polymerization, ActA is also sufficient to promote actin polymerization in the absence of other bacterial proteins. This was first demonstrated by expressing ActA in uninfected eukaryotic cells (Pistor et al. 1994), where it targets to mitochondria and induces actin to polymerize around these organelles. However, mitochondria decorated with ActA do not undergo actin-based motility, perhaps because the surface distribution of ActA is not polarized. ActA can also be targeted to the plasma membrane rather than mitochondria by replacing its transmembrane domain with a CAAX box, which causes actin polymerization at the membrane and changes in cell shape (Friederich et al. 1995).

A direct demonstration of the sufficiency of ActA in actin-based motility was achieved by expressing or tethering ActA on the surface of bacteria that are not normally able to polymerize actin, such as L. innocua (Kocks et al. 1995) or Streptococcus pneumoniae (Smith et al. 1995), or by coating ActA on the surface of inert plastic beads (Cameron et al. 1999). The direct demonstration of sufficiency also relied on the ability to reconstitute L. monocytogenes actin-based motility in cell-free cytoplasmic extracts made from Xenopus laevis eggs (Theriot et al. 1994), which enables addition of ActA-coated bacteria or beads to cytoplasm without the complications of the infection process. When ActA-expressing L. innocua (Kocks et al. 1995), ActA-coated S. pneumoniae (Smith et al. 1995), or ActA-coated plastic beads (Cameron et al. 1999) are added to Xenopus egg extracts in place of L. monocytogenes, they undergo actin-based motility. Moreover, killed L. monocytogenes also undergo motility in cell extracts (Theriot et al. 1994). Thus, ActA is the only bacterial protein that is required for actin-based motility, and once ActA is displayed on their surface, bacteria can be passive participants while the host cell expends energy to move them within and between cells.

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