Abstract
Type III secretion systems (T3SS) are highly conserved virulence factors employed by a large number of pathogenic gram-negative bacteria. Like many T3SS translocators, recombinant expression of the hydrophobic Shigella protein IpaB requires the presence of its cognate chaperone IpgC. Chaperone-bound IpaB is maintained in a nonfunctional state, which has hampered in vitro studies aimed at understanding molecular structure and function of this important class of T3SS proteins. Herein, we describe an expression and purification protocol that utilizes mild detergents to produce highly purified, homogeneous IpaB of defined oligomeric states.
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References
Diepold A, Wagner S (2014) Assembly of the bacterial type III secretion machinery. FEMS Microbiol Rev 38(4):802–822. doi:10.1111/1574-6976.12061
Hueck CJ (1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62(2):379–433
Schroeder GN, Hilbi H (2008) Molecular pathogenesis of Shigella spp.: controlling host cell signaling, invasion, and death by type III secretion. Clin Microbiol Rev 21(1):134–156. doi:10.1128/CMR.00032-07
Ashida H, Mimuro H, Sasakawa C (2015) Shigella manipulates host immune responses by delivering effector proteins with specific roles. Front Immunol 6:219. doi:10.3389/fimmu.2015.00219
Stensrud KF, Adam PR, La Mar CD, Olive AJ, Lushington GH, Sudharsan R, Shelton NL, Givens RS, Picking WL, Picking WD (2008) Deoxycholate interacts with IpaD of Shigella flexneri in inducing the recruitment of IpaB to the type III secretion apparatus needle tip. J Biol Chem 283(27):18646–18654. doi:10.1074/jbc.M802799200
Olive AJ, Kenjale R, Espina M, Moore DS, Picking WL, Picking WD (2007) Bile salts stimulate recruitment of IpaB to the Shigella flexneri surface, where it colocalizes with IpaD at the tip of the type III secretion needle. Infect Immun 75(5):2626–2629. doi:10.1128/IAI.01599-06
Blocker A, Gounon P, Larquet E, Niebuhr K, Cabiaux V, Parsot C, Sansonetti P (1999) The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes. J Cell Biol 147(3):683–693
Adam PR, Patil MK, Dickenson NE, Choudhari S, Barta M, Geisbrecht BV, Picking WL, Picking WD (2012) Binding affects the tertiary and quaternary structures of the Shigella translocator protein IpaB and its chaperone IpgC. Biochemistry 51(19):4062–4071. doi:10.1021/bi300243z
Birket SE, Harrington AT, Espina M, Smith ND, Terry CM, Darboe N, Markham AP, Middaugh CR, Picking WL, Picking WD (2007) Preparation and characterization of translocator/chaperone complexes and their component proteins from Shigella flexneri. Biochemistry 46(27):8128–8137. doi:10.1021/bi700099c
Senerovic L, Tsunoda SP, Goosmann C, Brinkmann V, Zychlinsky A, Meissner F, Kolbe M (2012) Spontaneous formation of IpaB ion channels in host cell membranes reveals how Shigella induces pyroptosis in macrophages. Cell Death Dis 3, e384. doi:10.1038/cddis.2012.124
Hilbi H, Moss JE, Hersh D, Chen Y, Arondel J, Banerjee S, Flavell RA, Yuan J, Sansonetti PJ, Zychlinsky A (1998) Shigella-induced apoptosis is dependent on caspase-1 which binds to IpaB. J Biol Chem 273(49):32895–32900
Zychlinsky A, Kenny B, Menard R, Prevost MC, Holland IB, Sansonetti PJ (1994) IpaB mediates macrophage apoptosis induced by Shigella flexneri. Mol Microbiol 11(4):619–627
Dickenson NE, Choudhari SP, Adam PR, Kramer RM, Joshi SB, Middaugh CR, Picking WL, Picking WD (2013) Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon. Protein Sci 22(5):614–627. doi:10.1002/pro.2245
Adam PR, Dickenson NE, Greenwood JC II, Picking WL, Picking WD (2014) Influence of oligomerization state on the structural properties of invasion plasmid antigen B from Shigella flexneri in the presence and absence of phospholipid membranes. Proteins 82(11):3013–3022. doi:10.1002/prot.24662
Barta ML, Dickenson NE, Patil M, Keightley A, Wyckoff GJ, Picking WD, Picking WL, Geisbrecht BV (2012) The structures of coiled-coil domains from type III secretion system translocators reveal homology to pore-forming toxins. J Mol Biol 417(5):395–405. doi:10.1016/j.jmb.2012.01.026
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Barta, M.L., Adam, P.R., Dickenson, N.E. (2017). Recombinant Expression and Purification of the Shigella Translocator IpaB. In: Nilles, M., Condry, D. (eds) Type 3 Secretion Systems. Methods in Molecular Biology, vol 1531. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6649-3_15
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DOI: https://doi.org/10.1007/978-1-4939-6649-3_15
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