title: Identification of Shigella flexneri IcsA residues affecting interaction with N-WASP, and evidence for IcsA-IcsA co-operative interaction.
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.
authors: Discipline of Microbiology and Immunology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia.
authors: Min Yan
authors: MY
authors: Teh
authors: Renato
authors: R
authors: Morona
mesh_terms: D051378:Actin-Related Protein 3 / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D054730:Protein Interaction Domains and Motifs; D012763:Shigella flexneri / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000235:genetics / Q000378:metabolism*
chemical_list: D051378:Actin-Related Protein 3; D000199:Actins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D008070:Lipopolysaccharides; D014157:Transcription Factors; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; C058483:virG protein, Shigella flexneri
references: Microbiology (Reading). 2012 Nov;158(Pt 11):2835-2850
references: 22936034
references: EMBO J. 1998 May 15;17(10):2767-76
references: 9582270
references: EMBO J. 2011 Aug 12;30(18):3864-74
references: 21841773
references: J Mol Biol. 2007 Oct 19;373(2):367-81
references: 17825319
references: Infect Immun. 1985 Apr;48(1):124-9
references: 3884506
references: Cell Microbiol. 2000 Jun;2(3):195-205
references: 11207576
references: Microbiol Mol Biol Rev. 2004 Dec;68(4):692-744
references: 15590781
references: Microbiology (Reading). 2012 Jul;158(Pt 7):1874-1883
references: 22516224
references: J Biol Chem. 2003 Dec 5;278(49):49316-22
references: 14506253
references: Cell. 1986 Aug 15;46(4):551-5
references: 3524856
references: Science. 2001 May 25;292(5521):1502-6
references: 11379633
references: Annu Rev Biochem. 2010;79:707-35
references: 20533885
references: Microbiology (Reading). 2003 Apr;149(Pt 4):925-939
references: 12686635
references: Microbiology (Reading). 2010 Aug;156(Pt 8):2459-2469
references: 20447993
references: Infect Immun. 2001 Oct;69(10):5959-66
references: 11553531
references: J Biochem. 2003 Sep;134(3):309-13
references: 14561714
references: Proc Natl Acad Sci U S A. 1989 May;86(10):3867-71
references: 2542950
references: Microbiology (Reading). 1999 Feb;145 ( Pt 2):367-378
references: 10075419
references: J Biol Chem. 1995 Dec 29;270(52):30874-80
references: 8537341
references: Nat Cell Biol. 2000 Jul;2(7):441-8
references: 10878810
references: J Bacteriol. 2007 Jul;189(14):5393-8
references: 17513479
references: J Biol Chem. 1994 Dec 30;269(52):32800-6
references: 7806503
references: Proc Natl Acad Sci U S A. 2010 Oct 12;107(41):17739-44
references: 20876094
references: Gene. 1977;2(2):75-93
references: 344136
references: J Bacteriol. 1989 Jan;171(1):353-9
references: 2644195
references: J Bacteriol. 2008 Jul;190(13):4666-76
references: 18456802
references: Philos Trans R Soc Lond B Biol Sci. 2000 May 29;355(1397):575-86
references: 10874731
references: Microb Pathog. 2003 Jul;35(1):11-8
references: 12860454
references: Science. 2005 Feb 4;307(5710):727-31
references: 15576571
references: Int J Med Microbiol. 2006 Aug;296(4-5):187-95
references: 16600681
references: Mol Microbiol. 2003 Oct;50(1):45-60
references: 14507362
references: Mol Cell. 2008 Nov 7;32(3):426-38
references: 18995840
references: Microbiol Mol Biol Rev. 2001 Dec;65(4):595-626, table of contents
references: 11729265
references: Vaccine. 1991 Jun;9(6):416-22
references: 1887672
references: Nat Cell Biol. 2001 Oct;3(10):897-904
references: 11584271
references: Mol Microbiol. 1997 Feb;23(4):765-75
references: 9157247
references: Cell Microbiol. 2002 Apr;4(4):223-33
references: 11952639
references: Curr Opin Microbiol. 2003 Oct;6(5):519-27
references: 14572546
references: Curr Biol. 1999 May 20;9(10):555-8
references: 10339430
references: Gene. 1991 Jun 15;102(1):75-8
references: 1840539
references: FEBS Lett. 1975 Oct 15;58(1):254-8
references: 773686
references: Methods Enzymol. 2000;326:430-40
references: 11036656
references: J Bacteriol. 2011 Apr;193(8):2042-5
references: 21335457
references: Mol Microbiol. 2003 Mar;47(5):1367-83
references: 12603741
references: J Bacteriol. 1995 Feb;177(4):1059-68
references: 7532168