The IcsA autotransporter (AT) is a key virulence protein of Shigella flexneri, a human pathogen that causes bacillary dysentery through invasion of colonic epithelium. IcsA is a polarly distributed, outer membrane protein that confers motility to intracellular bacteria by engaging the host actin regulatory protein, neural Wiskott-Aldrich syndrome protein (NWASP). The activated N-WASP in turn activates Arp2/3 complex, which initiates de novo actin nucleation and polymerisation to form F-actin comet tails and allow actin-based motility (ABM). The N-terminal surface-exposed IcsA passenger -domain (aa 53-758) is responsible for N-WASP interaction, where multiple IcsA regions: aa 185-312 (N-WASP interacting region [IR] I), aa 330-382 (N-WASP IR II) and aa 508-730 (N-WASP IR III), have been suggested to be interacting with N-WASP from previous linker-insertion mutagenesis (IcsAi). A putative autochaperone (AC) region (aa 634-735) located at the C-terminal end of IcsA passenger domain, which forms part of the self-associating AT (SAAT) domain, has been suggested to be required for IcsA biogenesis. IcsAi proteins with linker insertion mutations within the AC region had a significant reduction in production when expressed in smooth lipopolysaccharide (S-LPS) S. flexneri. This thesis investigated the biogenesis of IcsA, seeking to identify factors that affect IcsA AC mutant production in the S-LPS background. IcsAi AC mutant production was restored to a wild-type comparable levels in the rough LPS (R-LPS) S. flexneri (that lack the O-antigen component). The same phenotypes were observed in S. flexneri (both S-LPS and R-LPS) expressing site-directed mutagenised IcsA AC protein (aa 716-717). Various approaches were performed to identify the factors that caused different IcsA AC mutant production between SLPS and R-LPS S. flexneri. Both LPS Oag and DegP (a periplasmic chaperone/protease) were identified to affect IcsA AC mutant production in S-LPS strain, as the IcsA AC mutant production was restored in S. flexneri ΔdegP S-LPS strain. In addition, site-directed mutagenesis of residues Y716 and D717 within the AC region showed that these residues are critical for IcsA production and/or stability in the S-LPS background but not in the R-LPS background. Another aim of this work was to further define N-WASP IRs II and III via site-directed mutagenesis of specific amino acids. Mutant IcsA protein production level, N-WASP recruitment and F-actin comet tail formation by S-LPS and R-LPS S. flexneri were characterised. Residues 330-331, and residue 382 within N-WASP IR II, and residues 716- 717 within N-WASP IR III, were identified to be involved in N-WASP recruitment. It was shown for the first time that N-WASP activation involves interaction with different regions on different IcsA molecules and hence that oligomeric IcsA is needed for this interaction. Various “GFP-N-WASP” sub-domain proteins and IcsA protein were over-expressed, purified and used in protein binding assays. These provided preliminary data for protein binding assays which investigate the relationship between N-WASP and IcsA. Another S. flexneri virulence protein, IcsB, which is involved in preventing autophagy activation by intracellular bacteria was investigated. An S. flexneri 2457T ΔicsB mutant was created and characterised in this study. In this background, the icsB mutation had a less of an effect on plaque formation than reported for another S. flexneri background.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201
