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Journal of Bacteriology, July 2008, p. 4666-4676, Vol. 190, No. 13
0021-9193/08/$08.00+0     doi:10.1128/JB.00093-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mutagenesis of the Shigella flexneri Autotransporter IcsA Reveals Novel Functional Regions Involved in IcsA Biogenesis and Recruitment of Host Neural Wiscott-Aldrich Syndrome Protein{triangledown} ,{dagger}

Kerrie L. May and Renato Morona*

Australian Bacterial Pathogenesis Program, Discipline of Microbiology and Immunology, School of Molecular and Biomedical Science, University of Adelaide, South Australia, Australia

Received 17 January 2008/ Accepted 28 April 2008

The IcsA (VirG) protein of Shigella flexneri is a polarly localized, outer membrane protein that is essential for virulence. Within host cells, IcsA activates the host actin regulatory protein, neural Wiskott-Aldrich syndrome protein (N-WASP), which in turn recruits the Arp2/3 complex, which nucleates host actin to form F-actin comet tails and initiate bacterial motility. Linker insertion mutagenesis was undertaken to randomly introduce 5-amino-acid in-frame insertions within IcsA. Forty-seven linker insertion mutants were isolated and expressed in S. flexneri {Delta}icsA strains. Mutants were characterized for IcsA protein production, cell surface expression and localization, intercellular spreading, F-actin comet tail formation, and N-WASP recruitment. Using this approach, we have identified a putative autochaperone region required for IcsA biogenesis, and our data suggest an additional region, not previously identified, is required for N-WASP recruitment.

* Corresponding author. Mailing address: School of Molecular and Biomedical Science, University of Adelaide, South Australia 5005, Australia. Phone: 61-8-83034151. Fax: 61-8-83037532. E-mail: renato.morona{at}adelaide.edu.au

{triangledown} Published ahead of print on 2 May 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, July 2008, p. 4666-4676, Vol. 190, No. 13
0021-9193/08/$08.00+0     doi:10.1128/JB.00093-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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