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Papers In Press, published online ahead of print July 29, 2005
Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, Paris 75724
Corresponding Author: rbrosch{at}pasteur.fr
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-X-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 due to altered ESAT-6 targeting or trafficking. In contrast, 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 in spite of 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.
J. Biol. Chem, 10.1074/jbc.M503515200
Submitted on March 31, 2005
Revised on June 23, 2005
Accepted on July 27, 2005
Functional analysis of ESAT-6, the dominant T-cell antigen of mycobacterium tuberculosis, reveals key residues involved in secretion, complex-formation, virulence and immunogenicity
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