**5. Macrophage subversion by CFP10 and ESAT6**

Despite their well-known role as T cell antigens, CFP10 and ESAT6 modulate several pathways inside the macrophage, thereby creating a suitable environment to persist inside the host cell. Studies from our lab have shown that CFP-10 and ESAT-6 downregulates the production of reactive oxygen species (ROS) inside the macrophages; which in turn dampens the NF-ĸB transactivation property (Ganguly et al., 2008a, Ganguly et al., 2008b). The inhibition of ROS production was greater with the CFP10:ESAT6 complex compared to the individual proteins. Most of the effects of these proteins seem to be mediated by Toll-like receptors (TLR). Analysis of global phosphoproteome in CFP-10 treated J774.1 macrophages showed that CFP-10 caused dephosphorylation of a large number of macrophage proteins (Basu et al., 2006; Basu et al., 2009). The de-phsophorylation occurs due to increase in activity of membrane tyrosine phosphatases SHP-1 and SHP-2 (Src homology domain proteins). The increased phosphatase activity is due to reduction in production of ROS inside the macrophages. The ROS production in macrophages occurs through NADPH oxidase pathway. These observations suggest that upon binding of CFP-10 and ESAT-6 to macrophage surface, Mtb is able to reduce the burst of ROS inside the cell which contributes to bactericidal activity. Thus it might be one of the survival strategies of the bacilli. Mtb contains several enzymes to deal with the ROS/oxidative burst like catalase, peroxidase (Kat) (Sherman et

*Mycobacterium tuberculosis* RD-1 Secreted

**7. Conclusions** 

function.

**8. Acknowledgement** 

Biotechnology-DBT- (Government of India), New Delhi

Antigens as Protective and Risk Factors for Tuberculosis 121

attenuation could be due to the inhibition of CFP10/ESAT6 secretion, which further highlights the role of these two proteins in modulation of macrophage function. A study has shown that one of the components of ESX-1 secretion system, Rv3871 binds to the Cterminus of CFP-10, and this facilitates the secretion of both CFP-10 and ESAT-6. Mutations at the C-terminus resulted in loss of binding to CFP-10 and impaired secretion of the two proteins (Champion et al., 2006). This suggests that CFP-10 and ESAT-6 might be secreted in the form of heterodimeric 1:1 complex out of the cell. The ESX-1 system has four paralogues in Mtb and some of them have been shown to be essential for invitro growth of the bacilli (Simeone et al., 2009). In the ESX-1 system, Rv3868, Rv3869, Rv3870, Rv3871 and Rv3877 have been shown to be essential for CFP-10/ESAT-6 secretion while loss of Rv3865 and partial loss of Rv3866 did not affect protein secretion, rather it caused attenuation of the bacilli (Brodin et al., 2006). Thus Rv3865/3866 might be some virulence factor that does not control ESAT-6 secretion. Studies with mutant bacilli showed that ESX-1 system is required for the induction of type I IFN induction that in turn contributes to the spread of the bacilli (Stanley et al., 2007). In *Mycobacterium marinum*, the CFP-10/ESAT-6 secretion manipulates the phagosome-lysosome fusion. Mutations in this secretion system results in enhanced phagosome-lysosome fusion and reduced survival of mycobacteria (Tan et al., 2006; Majlessi et al., 2005; Champion et al., 2006; Xu et al., 2007; Lee et al., 2001). Analyses of deletion mutants of ESAT-6 have identified the key amino acids in complex formation, virulence and secretion (Brodin et al., 2005). The Trp-X-Gly motif on ESAT-6 is involved in complex formation with CFP-10, virulence and induction of specific T cell responses whereas mutations in the six amino acids at the C-terminus had no effect on secretion but caused attenuation. At acidic pH (as normally found in phagosomes), ESAT-6 dissociated from its complexing partner CFP-10 and bound to liposomes, which caused lysis of the liposomes (de Jonge et al., 2007). This could be a mechanism for the Mtb to escape degradation within the phagosome. In dendritic cells, Mtb translocated from phagolysosome to cytoplasm, which is dependent upon the CFP-10/ESAT-6 secretion

(van der Wel et al., 2007). This translocation resulted in the death of the host cell.

The interaction between mycobacteria and the host macrophage or dendritic cells is very complex and dependent on multiple factors. In this review, we have focused mainly on the modulating activities of CFP10/ESAT6, the molecules which are being evaluated as vaccine candidates, indicating that they may act like double-edged sword generating a favorable response from the host immune system. Apart from CFP-10/ESAT-6, several other protein antigens are also being reported which modulate the macrophage response to Mtb. Further studies are undergoing in our lab to elucidate the finer mechanisms by which these proteins

Work reported from the Sharma lab has been generously funded by the Department of

al., 1995; Manca et al., 1999; Ng et al., 2004) as well as superoxide dismutases Sod A and Sod C (Piddington et al., 2001; Zhang et al., 1991). ESAT-6 was also found to inhibit mitogen activated kinase/extracellular signal regulated kinases 1/2 (MAPK/ERK1/2). This occurs due to some phosphatase activity in the nucleus which dephosphorylates ERK1/2. This resulted in reduction in lipopolysaccharide (LPS) induced expression of transcription factor c-myc (Ganguly et al., 2007). ESAT-6 also reduced the LPS-induced expression of several genes like *IL-1β*, *Bax*, *Icam-1* and *tnfr-1*. Recent studies have shown that ESAT-6 binds to toll-like receptor-2 (TLR2) on the macrophage surface; and the six amino acids at the C-terminus of the protein are critical for its TLR2 binding (Pathak et al., 2007). This binding caused inactivation of transcription factors interferon regulatory factors (IRF) and NF-ĸB. Recent observations from our lab have shown that ESAT-6 down-regulates IFN- inducible expression of type I and type IV isomers of MHC class II transactivator (CIITA) in macrophages (Kumar et al., 2011). Interestingly, the downregulation of type I CIITA was independent of TLR-2 while the effect on type IV CIITA was mediated through TLR-2. This suggests that ESAT-6 may bind to other TLRs or some other receptor on macrophages. Another study has shown that ESAT-6 was able to induce apoptosis in human monocytic cell line THP-1 through activation of caspases (Derrick et al., 2007). It was also shown that ESAT-6 could induce pore formation on the surface of some cell types.

Apart from macrophages, CFP-10 and ESAT-6 also modulate functions in dendritic cells (Natarajan et al., 2011; Trajkovic et al., 2004). Studies have shown that CFP-10 induced differentiation of bone marrow cells into dendritic cells (DC) and this involved activation of NF-ĸB (Latchumanan et al., 2002). CFP-10 also induced maturation of DCs, which caused downregulation of pro-inflammatory cytokines like interleukin-2 (IL-2) and IFN- (Natarajan et al., 2003). The CFP-10-differentiated and CFP-10-matured DCs when cultured with the Mtb whole-cell-extract primed T cells, showed reduced levels of pro-inflammatory cytokines IL-12p40 and IFN- along with elevated levels of anti-inflammatory cytokines IL-10 and transforming growth factor (TGF-) (Balkhi et al., 2004). Therefore CFP-10 primes DCs to have reduced efficacy to eliminate Mtb. CFP-10 also reduced ROS production during differentiation of DCs compared to the positive stimulator granulocyte macrophage colonyan stimulating factor (GM-CSF). This downregulation of ROS resulted in increased survival of *M. bovis* BCG in these DCs (Sinha et al., 2006). The CFP-10 differentiated DCs also had reduced levels of chemokines RANTES and IP-10 upon infection by mycobacteria (Salam et al., 2008).
