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Infection and Immunity, March 2003, p. 1551-1556, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1551-1556.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Effects of Ectopically Expressed Neuronal Wiskott-Aldrich Syndrome Protein Domains on Rickettsia rickettsii Actin-Based Motility

Ronald S. Harlander,¹ Michael Way,² Qun Ren,¹ Dale Howe,^1, Scott S. Grieshaber,^1, and Robert A. Heinzen^1*

Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-3944,¹ Cell Motility Laboratory, Cancer Research UK, London, United Kingdom²

Received 1 October 2002/ Returned for modification 26 November 2002/ Accepted 13 December 2002

Neuronal Wiskott-Aldrich syndrome protein (N-WASP) and the actin-related protein 2/3 (Arp2/3) complex have emerged as critical host proteins that regulate pathogen actin-based motility. Actin tail formation and motility in Listeria monocytogenes require the Arp2/3 complex but bypasses N-WASP signaling. Motility of Shigella flexneri and vaccinia virus requires both N-WASP and the Arp2/3 complex. Functional roles for these cytoskeletal regulatory proteins in actin-based motility of Rickettsia rickettsii have not been established. In this study, functional domains of N-WASP tagged with green fluorescent protein that have characterized effects on Shigella and vaccinia virus actin-based motility were ectopically expressed in HeLa cells infected with R. rickettsii to assess their effects on rickettsial motility. S. flexneri-infected cells were used as a control. Expressed N-WASP domains did not localize to R. rickettsii or their actin tails. Expression of N-WASP missing the VCA domain (for "verprolin homology, cofilin homology, and acidic domains"), which acts as a dominant-negative form of N-WASP, completely inhibited actin-based motility of S. flexneri while only moderately inhibiting motility of R. rickettsii. Similarly, expression of the VCA domain, which acts as a dominant-negative with respect to Arp2/3 complex function, severely inhibited actin-based motility of S. flexneri (no motility observed in the majority of expressing cells) but only moderately inhibited R. rickettsii motility. These results, taken together with the differential effects on motility observed upon expression of other N-WASP domains, suggest that actin-based motility of R. rickettsii is independent of N-WASP and the Arp2/3 complex.

Editor: F. C. Fang

Present address: Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, MT 59840.

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