Abstract
Gram-negative bacteria utilize a dedicated membrane-embedded apparatus, the type III secretion system (T3SS), to inject proteins into host cells. The passage of the proteins across the target membrane is accomplished by a proteinaceous pore—the translocon—formed within the host-cell cytoplasmic membrane. Translocators bound to their chaperones can be expressed in Escherichia coli and subsequently dissociated from the chaperone by guanidine treatment. The pore formation properties of the translocators can then be studied by an in-vitro liposome leakage assay. Sulforhodamine-B is encapsulated within lipid vesicles during liposome preparation. At high concentration, this fluorochrome exhibits self-quenching limiting fluorescence emission. Upon pore formation, liposome leakage leads to the dilution of Sulforhodamine-B in the medium and fluorescence emission increases. Alternatively, fluorochromes coupled to large dextran molecules can be encapsulated in order to estimate pore dimensions. Here we describe protein expression and purification, dye-liposome preparation, and leakage assay conditions.
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References
Parker MW, Feil SC (2005) Pore-forming protein toxins: from structure to function. Prog Biophys Mol Biol 88:91–142
Tilley SJ, Saibil HR (2006) The mechanism of pore formation by bacterial toxins. Curr Opin Struct Biol 16:230–236
Mueller CA, Broz P, Cornelis GR (2008) The type III secretion system tip complex and translocon. Mol Microbiol 68:1085–1095
Cornelis GR (2006) The type III secretion injectisome. Nat Rev Microbiol 4:811–825
Faudry E, Job V, Dessen A et al (2007) Type III secretion system translocator has a molten globule conformation both in its free and chaperone-bound forms. FEBS J 274:3601–3610
Faudry E, Vernier G, Neumann E et al (2006) Synergistic pore formation by type III toxin translocators of Pseudomonas aeruginosa. Biochemistry 45:8117–8123
Romano FB, Rossi KC, Savva CG et al (2011) Efficient isolation of Pseudomonas aeruginosa type III secretion translocators and assembly of heteromeric transmembrane pores in model membranes. Biochemistry 50:7117–7131
Schoehn G, Di Guilmi AM, Lemaire D et al (2003) Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas. EMBO J 22:4957–4967
Wager B, Faudry E, Attree I, et al. (2012) Pore properties of the type III secretion system translocon of Pseudomonas aeruginosa, Submitted.
Dalla Serra M, Menestrina G (2003) Liposomes in the study of pore-forming toxins. Methods Enzymol 372:99–124
Lasic DD (1988) The mechanism of vesicle formation. Biochem J 256:1–11
Delcour AH, Martinac B, Adler J et al (1989) Modified reconstitution method used in patch-clamp studies of Escherichia coli ion channels. Biophys J 56:631–636
Meleard P, Bagatolli LA, Pott T (2009) Giant unilamellar vesicle electroformation from lipid mixtures to native membranes under physiological conditions. Methods Enzymol 465:161–176
Agafonov A, Gritsenko E, Belosludtsev K et al (2003) A permeability transition in liposomes induced by the formation of Ca2+/palmitic acid complexes. Biochim Biophys Acta 1609:153–160
Galloux M, Vitrac H, Montagner C et al (2008) Membrane Interaction of botulinum neurotoxin A translocation (T) domain. The belt region is a regulatory loop for membrane interaction. J Biol Chem 283:27668–27676
Acknowledgments
The work in our lab is supported in part by CEA-DSV programs and the French cystic fibrosis association “Vaincre la Mucoviscidose.” The authors are grateful to Michel Ragno for optimization of expression and purification procedures, and to Vincent Forge and Grégory Vernier for helpful discussions and technical help to set up the vesicle protocol.
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Faudry, E., Perdu, C., Attrée, I. (2013). Pore Formation by T3SS Translocators: Liposome Leakage Assay. In: Delcour, A. (eds) Bacterial Cell Surfaces. Methods in Molecular Biology, vol 966. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-245-2_11
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DOI: https://doi.org/10.1007/978-1-62703-245-2_11
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