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J. Biol. Chem., Vol. 280, Issue 40, 33953-33959, October 7, 2005

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Functional Analysis of Early Secreted Antigenic Target-6, the Dominant T-cell Antigen of Mycobacterium tuberculosis, Reveals Key Residues Involved in Secretion, Complex Formation, Virulence, and Immunogenicity^*

Priscille Brodin12, Marien I. de Jonge1, Laleh Majlessi1, Claude Leclerc, Michael Nilges¶, Stewart T. Cole, and Roland Brosch3

From the Unité de Génétique Moléculaire Bactérienne, Unité de Biologie des Regulations Immunitaires, INSERM E352, ^¶Unité de Bioinformatique Structurale, Institut Pasteur, Paris 75724, France

Proteins of the 6-kDa early secreted antigenic target (ESAT-6) secretion system-1 of Mycobacterium tuberculosis are not only strongly involved in the anti-mycobacterial Th1-host immune response but are also key players for virulence. In this study, protein engineering together with bioinformatic, immunological, and virulence analyses allowed us to pinpoint regions of the ESAT-6 molecule that are critical for its biological activity in M. tuberculosis. Mutation of the Trp-Xaa-Gly motif, conserved in a wide variety of ESAT-6-like proteins, abolished complex formation with the partner protein CFP-10, induction of specific T-cell responses, and virulence. Replacement of conserved Leu residues interfered with secretion, coiled-coil formation, and virulence, whereas certain mutations at the extreme C terminus did not affect secretion but caused attenuation, possibly because of altered ESAT-6 targeting or trafficking. In contrast, the mutation of several residues on the outer surface of the four-helical bundle structure of the ESAT-6·CFP-10 complex showed much less effect. Construction of recombinant BCG expressing ESAT-6 with a C-terminal hexahistidine tag allowed us to co-purify ESAT-6 and CFP-10, experimentally confirming their strong interaction both in and outside of the mycobacterial cell. The strain induced potent, antigen-specific T-cell responses and intermediate in vivo growth in mice, suggesting that it remained immunogenic and biologically active despite the tag. Together with previous NMR data, the results of this study have allowed a biologically relevant model of the ESAT-6·CFP-10 complex to be constructed that is critical for understanding the structure-function relationship in tuberculosis pathogenesis.

Received for publication, March 31, 2005 , and in revised form, June 23, 2005.

^* This work was supported by the Institut Pasteur (GPH5, PTR110), the Ministère de la Recherche et Nouvelles Technologies (ACI Microbiologie) the European Community (QLK2-CT-2001-02018 and LSHP-CT-2003-503367), and the Association Française Raoul Follereau. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3 and Table S1.

¹ These authors have contributed equally to this work.

² Recently joined the Institut National de la Santé et de la Recherche Médicale.

³ To whom correspondence should be addressed. Unité deGénétique Moléculaire Bactérienne, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex, France. Tel.: 33-1-45688449; Fax: 33-1-40613583; E-mail: rbrosch{at}pasteur.fr.

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