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Journal of Bacteriology, August 2007, p. 6028-6034, Vol. 189, No. 16
0021-9193/07/$08.00+0     doi:10.1128/JB.00469-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

ESAT-6 from Mycobacterium tuberculosis Dissociates from Its Putative Chaperone CFP-10 under Acidic Conditions and Exhibits Membrane-Lysing Activity{triangledown}

Marien I. de Jonge,1,{dagger} Gérard Pehau-Arnaudet,2 Marjan M. Fretz,3 Felix Romain,4 Daria Bottai,1 Priscille Brodin,1 Nadine Honoré,1 Gilles Marchal,5 Wim Jiskoot,3 Patrick England,6 Stewart T. Cole,1 and Roland Brosch1*

Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, Paris, France,1 Plate-Forme de Cryomicroscopie Moléculaire, Institut Pasteur, Paris, France,2 Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands,3 Unité Postulante de Dynamique Structurale des Macromolécules, Institut Pasteur, Paris, France,4 Laboratoire Immunothérapie, Institut Pasteur, Paris, France,5 Plate-Forme de Biophysique des Macromolécules et de Leurs Interactions, Institut Pasteur, Paris, France6

Received 28 March 2007/ Accepted 31 May 2007

The 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10 of Mycobacterium tuberculosis are secreted by the ESX-1 system into the host cell and thereby contribute to pathogenicity. Although different studies performed at the organismal and cellular levels have helped to explain ESX-1-associated phenomena, not much is known about how ESAT-6 and CFP-10 contribute to pathogenesis at the molecular level. In this study we describe the interaction of both proteins with lipid bilayers, using biologically relevant liposomal preparations containing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol, and cholesterol. Using floatation gradient centrifugation, we demonstrate that ESAT-6 showed strong association with liposomes, and in particular with preparations containing DMPC and cholesterol, whereas the interaction of CFP-10 with membranes appeared to be weaker and less specific. Most importantly, binding to the biomembranes no longer occurred when the proteins were present as a 1:1 ESAT-6·CFP-10 complex. However, lowering of the pH resulted in dissociation of the protein complex and subsequent protein-liposome interaction. Finally, cryoelectron microscopy revealed that ESAT-6 destabilized and lysed liposomes, whereas CFP-10 did not. In conclusion, we propose that one of the main features of ESAT-6 in the infection process of M. tuberculosis is the interaction with biomembranes that occurs after dissociation from its putative chaperone CFP-10 under acidic conditions typically encountered in the phagosome.

* Corresponding author. Mailing address: Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, Paris, France. Phone: 33 1 45688449. Fax: 33 1 40613583. E-mail: rbrosch{at}pasteur.fr

{triangledown} Published ahead of print on 8 June 2007.

{dagger} Present address: Department of Bacteriological R&D, Nobilon International B.V., Boxmeer, The Netherlands.

Journal of Bacteriology, August 2007, p. 6028-6034, Vol. 189, No. 16
0021-9193/07/$08.00+0     doi:10.1128/JB.00469-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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