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Originally published In Press as doi:10.1074/jbc.M003882200 on June 23, 2000

J. Biol. Chem., Vol. 275, Issue 37, 28893-28901, September 15, 2000
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Profilin Is Required for Sustaining Efficient Intra- and Intercellular Spreading of Shigella flexneri*

Hitomi MimuroDagger , Toshihiko SuzukiDagger , Shiro Suetsugu§, Hiroaki Miki§, Tadaomi Takenawa§, and Chihiro SasakawaDagger ||

From the Dagger  Division of Bacterial Infection, Department of Microbiology and Immunology, § Department of Biochemistry, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 and the  Department of Bacterial Toxicology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan

The ability of Shigella to mediate actin-based motility within the host cell is a prominent pathogenic feature of bacillary dysentery. The ability is dependent on the interaction of VirG with neural Wiskott-Aldrich syndrome protein (N-WASP), which in turn mediates recruitment of Arp2/3 complex and several actin-related proteins. In the present study, we show that profilin I is essential to the rapid movement of Shigella in epithelial cells, for which the capacity of profilin to interact with G-actin and N-WASP is critical. In COS-7 cells overexpressing either mutated profilin H119E, which failed to bind G-actin, or H133S, which is unable to interact with poly-L-proline, Shigella motility was significantly inhibited. Similarly, depletion of profilin from Xenopus egg extracts resulted in a decrease in bacterial motility that was completely rescued by adding back profilin I but not H119E or H133S. In COS-7 cells overexpressing a N-WASP mutant lacking the proline-rich domain (Delta p) unable to interact with profilin, the actin tail formation of intracellular Shigella was inhibited. In N-WASP-depleted extracts, addition of Delta p but not full-length N-WASP was unable to restore the bacterial motility. Furthermore, in a plaque formation assay with Madin-Darby canine kidney cell monolayers infected by Shigella, Madin-Darby canine kidney cells stably expressing H119E, H133S, or Delta p reduced the bacterial cell-to-cell spreading. These results indicate that profilin I associated with N-WASP is an essential host factor for sustaining efficient intra- and intercellular spreading of Shigella.

* This work was supported by the "Research for the Future" Program of the Japan Society for the Promotion of Science, and a grant-in-aid for Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan. Tel.: 81-3-5449-5252; Fax: 81-3-5449-5405; E-mail: sasakawa@ims.u-tokyo.ac.jp.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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