Abstract
The Shigella flexneri IcsA (VirG) protein is a polarly distributed outer membrane protein that is a fundamental virulence factor which interacts with neural Wiskott-Aldrich syndrome protein (N-WASP). The activated N-WASP then activates the Arp2/3 complex which initiates de novo actin nucleation and polymerisation to form F-actin comet tails and allows bacterial cell-to-cell spreading. In a previous study, IcsA was found to have three N-WASP interacting regions (IRs): IR I (aa 185–312), IR II (aa 330–382) and IR III (aa 508–730). The aim of this study was to more clearly define N-WASP interacting regions II and III by site-directed mutagenesis of specific amino acids. Mutant IcsA proteins were expressed in both smooth lipopolysaccharide (S-LPS) and rough LPS (R-LPS) S. flexneri strains and characterised for IcsA production level, N-WASP recruitment and F-actin comet tail formation. We have successfully identified new amino acids involved in N-WASP recruitment within different N-WASP interacting regions, and report for the first time using co-expression of mutant IcsA proteins, that N-WASP activation involves interactions with different regions on different IcsA molecules as shown by Arp3 recruitment. In addition, our findings suggest that autochaperone (AC) mutant protein production was not rescued by another AC region provided in trans, differing to that reported for two other autotransporters, PrtS and BrkA autotransporters.
Highlights
Shigella flexneri is a human pathogen that causes bacillary dysentery by infecting and colonising the colonic epithelium [1]
With the restoration of IcsAi production in S. flexneri rough LPS (R-LPS) strains, we have identified that IcsAi633, IcsAi643, IcsAi677, but not IcsAi716, were able to recruit neural Wiskott-Aldrich syndrome protein (N-WASP) and form filamentous actin (F-actin) comet tails [28]
We investigated the ability of IcsA mutants with point mutations within N-WASP interacting regions (IRs) II and III to recruit N-WASP and form F-actin comet tails in both smooth lipopolysaccharide (S-LPS) and R-LPS S. flexneri backgrounds
Summary
Shigella flexneri is a human pathogen that causes bacillary dysentery by infecting and colonising the colonic epithelium [1]. Shigella multiplies and interacts with the host actin regulatory protein neural Wiskott-Aldrich syndrome protein (N-WASP), which in turn activates the Arp2/3 complex that initiates actin polymerisation by recruiting the globular actin monomers to form the filamentous actin (F-actin) comet tails [7,8,9]. IcsA consists of three major domains: an extended N-terminal signal sequence (amino acids [aa] 1–52), a functional passenger adomain (aa 53–758), and a C-terminal translocation b-domain (aa 759–1102) that mediates the translocation of the passenger domain across the OM via the BAM (beta-barrel assembly machine) complex [11,12,13,14,15]. The b-barrel domain is anchored in the OM, while the functional IcsA passenger domain is exposed on the bacterial surface and is responsible for N-WASP recruitment and ABM [3,8]
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