全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...
PLOS ONE  2013 

Identification of Shigella flexneri IcsA Residues Affecting Interaction with N-WASP, and Evidence for IcsA-IcsA Co-Operative Interaction

DOI: 10.1371/journal.pone.0055152

Full-Text   Cite this paper   Add to My Lib

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.

References

[1]  Philpott DJ, Edgeworth JD, Sansonetti PJ (2000) The pathogenesis of Shigella flexneri infection: lessons from in vitro and in vivo studies. Philos Trans R Soc Lond B Biol Sci 355: 575–586.
[2]  Bernardini ML, Mounier J, d’Hauteville H, Coquis-Rondon M, Sansonetti PJ (1989) Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin. Proc Natl Acad Sci U S A 86: 3867–3871.
[3]  Suzuki T, Sasakawa C (2001) Molecular basis of the intracellular spreading of Shigella. Infect Immun 69: 5959–5966.
[4]  Lett MC, Sasakawa C, Okada N, Sakai T, Makino S, et al. (1989) virG, a plasmid-coded virulence gene of Shigella flexneri: identification of the virG protein and determination of the complete coding sequence. J Bacteriol 171: 353–359.
[5]  Makino S, Sasakawa C, Kamata K, Kurata T, Yoshikawa M (1986) A genetic determinant required for continuous reinfection of adjacent cells on large plasmid in S. flexneri 2a. Cell 46: 551–555.
[6]  Sansonetti PJ, Arondel J, Fontaine A, d’Hauteville H, Bernardini ML (1991) OmpB (osmo-regulation) and icsA (cell-to-cell spread) mutants of Shigella flexneri: vaccine candidates and probes to study the pathogenesis of shigellosis. Vaccine 9: 416–422.
[7]  Cossart P (2000) Actin-based motility of pathogens: the Arp2/3 complex is a central player. Cell Microbiol 2: 195–205.
[8]  Goldberg MB (2001) Actin-based motility of intracellular microbial pathogens. Microbiol Mol Biol Rev 65: 595–626, table of contents.
[9]  Pantaloni D, Le Clainche C, Carlier MF (2001) Mechanism of actin-based motility. Science 292: 1502–1506.
[10]  Pallen MJ, Chaudhuri RR, Henderson IR (2003) Genomic analysis of secretion systems. Curr Opin Microbiol 6: 519–527.
[11]  Henderson IR, Navarro-Garcia F, Desvaux M, Fernandez RC, Ala’Aldeen D (2004) Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev 68: 692–744.
[12]  Jain S, Goldberg MB (2007) Requirement for YaeT in the outer membrane assembly of autotransporter proteins. J Bacteriol 189: 5393–5398.
[13]  Suzuki T, Lett MC, Sasakawa C (1995) Extracellular transport of VirG protein in Shigella. J Biol Chem 270: 30874–30880.
[14]  Brandon LD, Goehring N, Janakiraman A, Yan AW, Wu T, et al. (2003) IcsA, a polarly localized autotransporter with an atypical signal peptide, uses the Sec apparatus for secretion, although the Sec apparatus is circumferentially distributed. Mol Microbiol 50: 45–60.
[15]  Peterson JH, Tian P, Ieva R, Dautin N, Bernstein HD (2010) Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment. Proc Natl Acad Sci U S A 107: 17739–17744.
[16]  Wells TJ, Totsika M, Schembri MA (2010) Autotransporters of Escherichia coli: a sequence-based characterization. Microbiology 156: 2459–2469.
[17]  Klemm P, Vejborg RM, Sherlock O (2006) Self-associating autotransporters, SAATs: functional and structural similarities. Int J Med Microbiol 296: 187–195.
[18]  Meng G, Spahich N, Kenjale R, Waksman G, St Geme JW 3rd (2011) Crystal structure of the Haemophilus influenzae Hap adhesin reveals an intercellular oligomerization mechanism for bacterial aggregation. EMBO J 30: 3864–3874.
[19]  May KL, Grabowicz M, Polyak SW, Morona R (2012) Self-association of the Shigella flexneri IcsA autotransporter protein. Microbiology 158: 1874–1883.
[20]  May KL, Morona R (2008) Mutagenesis of the Shigella flexneri autotransporter IcsA reveals novel functional regions involved in IcsA biogenesis and recruitment of host neural Wiscott-Aldrich syndrome protein. J Bacteriol 190: 4666–4676.
[21]  Kuhnel K, Diezmann D (2011) Crystal structure of the autochaperone region from the Shigella flexneri autotransporter IcsA. J Bacteriol 193: 2042–2045.
[22]  Yarar D, To W, Abo A, Welch MD (1999) The Wiskott-Aldrich syndrome protein directs actin-based motility by stimulating actin nucleation with the Arp2/3 complex. Curr Biol 9: 555–558.
[23]  Snapper SB, Takeshima F, Anton I, Liu CH, Thomas SM, et al. (2001) N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility. Nat Cell Biol 3: 897–904.
[24]  Suzuki T, Miki H, Takenawa T, Sasakawa C (1998) Neural Wiskott-Aldrich syndrome protein is implicated in the actin-based motility of Shigella flexneri. EMBO J 17: 2767–2776.
[25]  Miki H, Takenawa T (2003) Regulation of actin dynamics by WASP family proteins. J Biochem 134: 309–313.
[26]  Suzuki T, Mimuro H, Suetsugu S, Miki H, Takenawa T, et al. (2002) Neural Wiskott-Aldrich syndrome protein (N-WASP) is the specific ligand for Shigella VirG among the WASP family and determines the host cell type allowing actin-based spreading. Cell Microbiol 4: 223–233.
[27]  Ogawa M, Yoshimori T, Suzuki T, Sagara H, Mizushima N, et al. (2005) Escape of intracellular Shigella from autophagy. Science 307: 727–731.
[28]  Teh MY, Tran EN, Morona R (2012) Absence of O-antigen suppresses Shigella flexneri IcsA autochaperone region mutations. Microbiology 158: 2835–2850.
[29]  Morona R, Daniels C, Van Den Bosch L (2003) Genetic modulation of Shigella flexneri 2a lipopolysaccharide O antigen modal chain length reveals that it has been optimized for virulence. Microbiology 149: 925–939.
[30]  Baker SJ, Gunn JS, Morona R (1999) The Salmonella typhi melittin resistance gene pqaB affects intracellular growth in PMA-differentiated U937 cells, polymyxin B resistance and lipopolysaccharide. Microbiology 145 (Pt 2): 367–378.
[31]  Morona R, van den Bosch L, Manning PA (1995) Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri. J Bacteriol 177: 1059–1068.
[32]  Van Den Bosch L, Manning PA, Morona R (1997) Regulation of O-antigen chain length is required for Shigella flexneri virulence. Mol Microbiol 23: 765–775.
[33]  Lugtenberg B, Meijers J, Peters R, van der Hoek P, van Alphen L (1975) Electrophoretic resolution of the “major outer membrane protein” of Escherichia coli K12 into four bands. FEBS Lett 58: 254–258.
[34]  Cull MG, Schatz PJ (2000) Biotinylation of proteins in vivo and in vitro using small peptide tags. Methods in enzymology 326: 430–440.
[35]  Oaks EV, Wingfield ME, Formal SB (1985) Plaque formation by virulent Shigella flexneri. Infect Immun 48: 124–129.
[36]  Bitto E, McKay DB (2003) The periplasmic molecular chaperone protein SurA binds a peptide motif that is characteristic of integral outer membrane proteins. J Biol Chem 278: 49316–49322.
[37]  Xu X, Wang S, Hu YX, McKay DB (2007) The periplasmic bacterial molecular chaperone SurA adapts its structure to bind peptides in different conformations to assert a sequence preference for aromatic residues. J Mol Biol 373: 367–381.
[38]  Padrick SB, Rosen MK (2010) Physical mechanisms of signal integration by WASP family proteins. Annu Rev Biochem 79: 707–735.
[39]  Padrick SB, Cheng HC, Ismail AM, Panchal SC, Doolittle LK, et al. (2008) Hierarchical regulation of WASP/WAVE proteins. Mol Cell 32: 426–438.
[40]  Ohnishi Y, Nishiyama M, Horinouchi S, Beppu T (1994) Involvement of the COOH-terminal pro-sequence of Serratia marcescens serine protease in the folding of the mature enzyme. J Biol Chem 269: 32800–32806.
[41]  Oliver DC, Huang G, Nodel E, Pleasance S, Fernandez RC (2003) A conserved region within the Bordetella pertussis autotransporter BrkA is necessary for folding of its passenger domain. Mol Microbiol 47: 1367–1383.
[42]  Moreau V, Frischknecht F, Reckmann I, Vincentelli R, Rabut G, et al. (2000) A complex of N-WASP and WIP integrates signalling cascades that lead to actin polymerization. Nat Cell Biol 2: 441–448.
[43]  Van Den Bosch L, Morona R (2003) The actin-based motility defect of a Shigella flexneri rmlD rough LPS mutant is not due to loss of IcsA polarity. Microb Pathog 35: 11–18.
[44]  Bolivar F, Rodriguez RL, Betlach MC, Boyer HW (1977) Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9. Gene 2: 75–93.
[45]  Bartolome B, Jubete Y, Martinez E, de la Cruz F (1991) Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives. Gene 102: 75–78.

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133