**3. Structural biology of CFP-10 and ESAT-6**

The ORFs Rv3874 and Rv3875 encoding CFP10 and ESAT6 respectively are cotranscribed into a single RNA product (Cole et al., 1998). Nuclear magnetic resonance (NMR) spectroscopy showed that CFP10 exhibits very little secondary structure and consists mostly of random coils, which are unstructured. The ESAT6, on the other hand has 75% secondary structure in the form of -helices (Renshaw et al., 2002). One interesting study showed that CFP10 and ESAT6 forms a tight 1:1 complex where both proteins adopt more stable and folded configuration than the native moieties (Renshaw et al., 2002; Lightbody et al., 2004). The complex formation between the two proteins is hydrophobic in nature and led to a significant increase in the helical content of the two proteins. Inside the core of the complex, helix-turn-helix motifs of the two proteins form a quad-helix bundle (Renshaw et al., 2005). Within the complex, the flexible C-terminus of CFP-10 is involved in binding to the cell surface. This was confirmed by the fact that deletion 87 amino acids at C-terminus of CFP-10 inhibited the binding of complex to the cell surface, while deletion of the same in ESAT-6 had no effect (Renshaw et al., 2005). The complex formation between the two proteins was reversible and the complex broke down to individual proteins at 53.40C. The complex was also shown to be more stable to proteolytic digestion by trypsin (Meher at al., 2006). The enhanced stability to proteolytic digestion caused lower T cell activation compared to ESAT-6 alone (Marei et al., 2005). ESAT-6 protein was also found to have auto-proteolytic activity; it can self cleave-off six amino acids at the C-terminus which are responsible for its binding to the cell surface. A mutant ESAT-6 lacking these six amino acids was unable to bind to cell surface (Pathak et al., 2007).

proteins secreted by region of difference-1 (RD-1), the region that is deleted in all the strains

Comparative genome analysis using DNA microarray, bacterial artificial chromosomes (BAC) and the subtractive hybridization between virulent and attenuated strains of Mtb complex and *M. bovis* BCG identified several regions of difference (RD) (Behr et al., 1999; Gordon et al., 1999; Mahairas et al., 1996). A gene segment of 9.5kb that encompasses nine open reading frames (ORF) of Rv3871-Rv3879c is present in virulent strains of Mtb, and which is deleted consistently in all the strains of *M. bovis* BCG (Cole et al., 1998). This region was designated as RD-1. Two of these ORFs, Rv3874 and Rv3875, encode 10-kDa culture filtrate protein (CFP-10) and 6-kDa early secreted antigenic target (ESAT-6) protein respectively. Interestingly, deletion of the RD-1 fragments from Mtb causes loss of its virulence, while introduction of the RD-1 locus into *M. bovis* BCG or *M. microti* resulted in increased virulence and survival properties (Behr, 2002; Pym et al., 2002; Lewis et al., 2003; Demangel et al., 2005). This review will focus on the role of these two proteins in modulation of the macrophage signaling pathways and macrophage functions for the bacteria to persist for longer time. We also discuss about the potential role of these proteins

The ORFs Rv3874 and Rv3875 encoding CFP10 and ESAT6 respectively are cotranscribed into a single RNA product (Cole et al., 1998). Nuclear magnetic resonance (NMR) spectroscopy showed that CFP10 exhibits very little secondary structure and consists mostly of random coils, which are unstructured. The ESAT6, on the other hand has 75% secondary structure in the form of -helices (Renshaw et al., 2002). One interesting study showed that CFP10 and ESAT6 forms a tight 1:1 complex where both proteins adopt more stable and folded configuration than the native moieties (Renshaw et al., 2002; Lightbody et al., 2004). The complex formation between the two proteins is hydrophobic in nature and led to a significant increase in the helical content of the two proteins. Inside the core of the complex, helix-turn-helix motifs of the two proteins form a quad-helix bundle (Renshaw et al., 2005). Within the complex, the flexible C-terminus of CFP-10 is involved in binding to the cell surface. This was confirmed by the fact that deletion 87 amino acids at C-terminus of CFP-10 inhibited the binding of complex to the cell surface, while deletion of the same in ESAT-6 had no effect (Renshaw et al., 2005). The complex formation between the two proteins was reversible and the complex broke down to individual proteins at 53.40C. The complex was also shown to be more stable to proteolytic digestion by trypsin (Meher at al., 2006). The enhanced stability to proteolytic digestion caused lower T cell activation compared to ESAT-6 alone (Marei et al., 2005). ESAT-6 protein was also found to have auto-proteolytic activity; it can self cleave-off six amino acids at the C-terminus which are responsible for its binding to the cell surface. A mutant ESAT-6 lacking these six amino acids was unable to bind to cell

**2. Genetic architecture of Region of Difference-1 (RD-1)** 

as vaccine candidates owing to their high immunogenicity.

**3. Structural biology of CFP-10 and ESAT-6** 

surface (Pathak et al., 2007).

of *M. bovis* BCG.
