Abstract
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.
Highlights
Independent but complementary studies revealed that ESAT-6 and CFP-10 are secreted via the ESAT-6 system-1 (ESX-1), a dedicated secretion apparatus encoded by genes flanking esxA and esxB in the extended RD1 region (9 –11)
Mutation of Conserved Residues of ESAT-6 Identifies Biologically Important Amino Acids—Sequence alignment of ESAT-6 orthologs from M. tuberculosis and M. leprae revealed that 33 amino acids are conserved between the 2 ESAT-6 molecules, and 13 of these are conserved in ESAT-6 like proteins from the phylogenetically more distant species Corynebacterium diphtheriae (Fig. 1A)
Thirteen residues scattered along the M. tuberculosis ESAT-6 molecule were selected for mutation in pRD1–2F9 (Fig. 1A), and the resultant cosmids were integrated into a M. tuberculosis ⌬RD1 strain (H37Rv⌬RD1) (7)
Summary
This segment is localized close to the origin of replication (4) in all fully virulent members of the complex (5) and harbors genes esxA, coding for the 6-kDa early secreted antigenic target (ESAT-6), and esxB, encoding the 10-kDa culture filtrate protein (CFP-10). ESAT-6 and CFP-10, which have been shown to form a 1:1 complex in vitro (15), belong to a large family of small proteins identified in Gram-positive bacteria. Proteins of this family have a size of ϳ100 amino acids and are characterized by a conserved motif Trp-Xaa-Gly (WXG) (16). We describe the behavior of these mutant strains in immunogenicity and virulence tests relative to control strains and show how the modifications allowed new features of ESAT-6 and the corresponding secretion system to be discovered
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