Listeria monocytogenes

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11890-11894, December 1993
Microbiology

Expression and phosphorylation of the Listeria monocytogenes ActA protein in mammalian cells
(microbial pathogeness/actwn/okadaic acid)

RODNEY A. BRUNDAGE*, GREGORY A. SMITHt, ANDREW CAMILLIt, JULIE A. THERIOT§1, AND DANIEL A. PORTNOYtII tDepartment of Microbiology, University of Pennsylvania, School ofMedicine, Philadelphia, PA 19104-6076; and 1Departments of Biochemistry and
Biophysics and Pharmacology, University of California, San Francisco, CA 94143 Communicated by Emil C. Gotschlich, September 3, 1993

Movement of Listeria monocytogenes within ABSTRACT infected eukaryotic cells provides a simple model system to study the mechanism of actin-based motilit in nonmuscle cells. The act4 gene of L.monocytogenes is requhred to induce the polymerization of host actin filaments [Kocks, C., Gouin, E., Tabouret, M., Berche, P., Ohayon, H. & Cossart, P. (1990) Cel 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 mutation within the actAgene 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 nucleation of actin fiaments and consequently was impaired in cell-to-cell spread. Antiserum raised to a synthetic peptide encompassing theproline-rich repeat (DFPPPPTDEEL) of ActA was used to characterize the expression of the ActA protein. The ActA protein derived from extracellular bacteria migrated as a 97-kDa polypeptide upon SDS/ PAGE, whereas the protein from infected cells migrated as three distinct polypeptides, one that comigrated with the 97-kDa extracellular form and two slightly larger species. Treatment of infected cells withokadaic acid resulted in decreased mounts of all forms of ActA and the appearance of a larger species of ActA. Phosphatase treatment of ActA immunoprecipitated from intracellular bacteria resulted in conversion of the larger two species to the 97-kDa form. Labeling of infected cells with 32P1 followed by immunoprecipitation showed that the largest molecular form of ActA was phosphorylated. Takentogether, these data indicate that ActA 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.
Listeria monocytogenes is a facultative intracellular Grampositive bacterium that has been studied for decades as a model for the study of host-pathogen interactions.There is an excellent mouse model of infection in which immunity is exclusively cell mediated (1, 2). Also, tissue culture models of infection have been developed that facilitate quantitation of intracellular growth and cell-to-cell spread (3-7). The intracellular phase of the L. monocytogenes life cycle can be divided broadly into four stages: (i) internalization (8), (ii) escape from the hostvacuole (7, 9, 10), (iii) multiplication in the cytosol (11), and (iv) cell-to-cell spread without an extracellular phase (9, 10). Cell-to-cell spread is initiated by using components of the host cell's actin cytoskeleton that
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propel the bacteria through the cytoplasm at rates up to 1.5 ,um/sec (9, 10, 12-14). Next, the bacteria are often seen in long projections extending from the cell, which may facilitate cell-to-cell spread. The ability of L. monocytogenes to nucleate host actin filaments is similar to the unrelated Gramnegative pathogens Shigellae (15) and Rickettsiae (16, 32)....