Mutagenesis of the Shigella flexneri autotransporter IcsA reveals novel functional regions involved in IcsA biogenesis and recruitment of host N-WASP

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

^* To whom correspondence should be addressed. Email: renato.morona{at}adelaide.edu.au.

	Abstract

The IcsA (VirG) protein of S. flexneri is a polarly localised, outer membrane protein that is essential for virulence. Within host cells, IcsA activates the host actin regulatory protein, N-WASP, which in turn recruits the Arp2/3 complex that 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 icsA. Mutants were characterised for IcsA protein production, cell surface-expression and localisation, intercellular spreading, F-actin comet tail formation, and N-WASP recruitment. Using this approach we have identified a putative auto-chaperone region required for IcsA biogenesis and our data suggests an additional region, not previously identified, is required for N-WASP recruitment.