Mutagenesis of the Shigella flexneri autotransporter IcsA reveals novel functional regions involved in IcsA biogenesis and recruitment of host neural Wiscott-Aldrich syndrome protein.
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.
May
Kerrie L
KL
School of Molecular and Biomedical Science, University of Adelaide, South Australia 5005, Australia.
Morona
Renato
R
D000199:Actins / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D016254:Mutagenesis, Insertional; D012763:Shigella flexneri / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014357:Trypsin / Q000378:metabolism; D051316:Wiskott-Aldrich Syndrome Protein / Q000378:metabolism*
D000199:Actins; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D051316:Wiskott-Aldrich Syndrome Protein; C058483:virG protein, Shigella flexneri; D014357:Trypsin