EXPRESSION AND PHOSPHORYLATION OF THE LISTERIA-MONOCYTOGENES ACTA PROTEIN IN MAMMALIAN-CELLS

Movement of Listeria monocytogenes within infected eukaryotic cells pr ovides a simple model system to study the mechanism of actin-based mot ility in nonmuscle cells. The actA gene of L. monocytogenes is require d to induce the polymerization of host actin filaments [Kocks, C., Gou in, E., Tabouret, M., Berche, P., Ohayon, H. & Cossart, P. (1990) Cell 68, 521-531; Domann, E., Wehland, J., Rohde, M., Pistor, S., Hartl, M ., Goebel, W., Leimeister-Wachter, M., Wuenscher, M. & Chakraborty, T. (1992) EMBO J. 11, 1981-1990]. In this study, an in-frame deletion mu tation within the actA gene was constructed and introduced into the L. monocytogenes chromosome by allelic exchange. This mutation resulted in a decrease (3 orders of magnitude) in virulence for mice. In tissue culture cells, the actA mutant was absolutely defective for the nucle ation of actin filaments and consequently was impaired in cell-to-cell spread. Antiserum raised to a synthetic peptide encompassing the prol ine-rich repeat (DFPPPPTDEEL) of ActA was used to characterize the exp ression of the ActA protein. The ActA protein derived from extracellul ar bacteria migrated as a 97-kDa polypeptide upon SDS/PAGE, whereas th e protein from infected cells migrated as three distinct polypeptides, one that comigrated with the 97-kDa extracellular form and two slight ly larger species. Treatment of infected cells with okadaic acid resul ted in decreased amounts of all forms of ActA and the appearance of a larger species of ActA. Phosphatase treatment of ActA immunoprecipitat ed from intracellular bacteria resulted in conversion of the larger tw o species to the 97-kDa form. Labeling of infected cells with P-32i fo llowed by immunoprecipitation showed that the largest molecular form o f ActA was phosphorylated. Taken together, these data indicate that Ac tA is phosphorylated during intracellular growth. The significance of the intracellular modification of ActA is not known, but we speculate that it may modulate the intracellular activity of ActA.