Intracellular Motility and Cellto Cell Spread The Surface Protein ActA

L. monocytogenes moves rapidly through the cytoplasm by the polymerization of host cell actin which is induced by the listerial surface protein ActA [39, 40]. The actA gene is located downstream of mpl in the lecithinase operon [34] and codes for a proline-rich protein of 639 amino acids, which is sufficient to stimulate F-actin assembly and to promote intracellular movement. ActA consists of three domains: the N-terminal domain with the transport signal sequence, the central proline-rich repeat region, and the C-terminal part, which includes a membrane anchor [39, 40].

The expression of mutated forms of ActA allowed the definition of regions of the ActA protein with specific functions in the recruitment of cellular proteins and hence in actin polymerization and movement. Deletion of the whole N-termi-nal domain of ActA resulted in a total abolishment of actin polymerization and intracellular movement, showing the absolute necessity of this domain in ActA function [41]. Within the N-terminal part of ActA, two smaller regions were identified which are either required for filament elongation or for the continuity of the actin tail since their deletion led to discontinuous actin tail formation [42]. In contrast, deletion of the C-terminal domain did not inhibit actin assembly [41]. The actin tails produced by L. monocytogenes strains expressing ActA lacking the central proline-rich repeats were significantly shorter and the speed of the movement was drastically reduced [43].

Several actin binding proteins and proteins regulating actin dynamics colocalize with the F-actin tails. From these proteins only VASP, Mena, and the Arp2/3 complex were shown to directly bind to ActA [44, 45, 46]. The Arp2/3 complex initiates

F-actin polymerization due to its nucleation activity, which is significantly activated by the presence of ActA [46], thus mimicking the activity of proteins of the cellular WASP family which are natural ligands and activators of Arp2/3.

The phosphoprotein VASP binds directly to the proline-rich repeats of ActA [44]. On the other hand VASP is a natural ligand of profilin and could stimulate actin assembly by binding to ActA and enhancing the profilin concentration in the vicinity of the bacterium. ActA itself can bind G-actin with a region in its N-terminal part, but deletion of this region does not interfere with actin tail formation in infected cells [47]. It is believed that inside cells, the VASP-mediated profi-lin/G-actin recruitment can bypass defects in actin binding of ActA.

ActA expression is maximally induced when the bacteria have reached the host cell cytoplasm, and the expression level is increased more than 200-fold in cyto-solic bacteria in comparison to broth-grown cultures [48] as shown either by reporter gene fusions to actA [30, 48] or by direct quantification of actA-specific transcripts [30].

L. monocytogenes can spread from cell to cell without leaving the cytoplasm by forming microvilli-like protrusions on the host cell surface which are phagocy-tosed by neighboring cells [49, 50]. The mechanisms of microvilli formation and of induction of phagocytosis by the neighboring cell are largely unknown. Listeria moves randomly through the cytoplasm and the bacteria are finally propelled into the host's plasma membrane, which is distended and long protrusions are formed. These protrusions enter neighboring cells, are taken up subsequently, and a secondary vacuole with a double membrane is formed [51]. This secondary vacuole is disrupted, requiring the action of LLO and the listerial phospholipase PC-PLC [34, 52].

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