LaXp180, a mammalian ActA-binding protein, identified with the yeast two-hybrid system, co-localizes with intracellular Listeria monocytogenes

The Listeria monocytogenes surface protein ActA is an important virulence f actor required for listerial intracellular movement by inducing actin polym erization. The only host cell protein known that directly interacts with Ac tA is the phosphoprotein VASP, which binds to the central proline-rich repe at region of ActA. To identify additional ActA-binding proteins, we applied the yeast two-hybrid system to search for mouse proteins that interact wit h ActA. A mouse cDNA library was screened for ActA-interacting proteins (AI Ps) using ActA from strain L. monocytogenes EGD as bait. Three different AI Ps were identified, one of which was identical to the human protein LaXp180 (also called CC1). Binding of LaXp180 to ActA was also demonstrated in vit ro using recombinant histidine-tagged LaXp180 and recombinant ActA. Using a n anti-LaXp180 antibody and fluorescence microscopy, we showed that LaXp180 co-localizes with a subset of intracellular, ActA-expressing L. monocytoge nes but was never detected on intracellularly growing but ActA-deficient mu tants. Furthermore, LaXp180 binding to intracellular L. monocytogenes was a symmetrical and mutually exclusive with F-actin polymerization on the bacte rial surface. LaXp180 is a putative binding partner of stathmin, a protein involved in signal transduction pathways and in the regulation of microtubu le dynamics. Using immunofluorescence, we showed that stathmin co-localizes with intracellular ActA-expressing L. monocytogenes.