*Mycobacterium tuberculosis* **RD-1 Secreted Antigens as Protective and Risk Factors for Tuberculosis**

Niladri Ganguly1,2 and Pawan Sharma1

*1International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 2Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, Dundee, 1India 2UK* 

### **1. Introduction**

116 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

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*Mycobacterium tuberculosis* (Mtb) infects about 8 million people every year and causes death of about 2-3 million (Raviglione, 2003). In recent times, there has been a wider spread of tuberculosis, mainly due to emergence of multi drug resistance (MDR) bacilli and enhanced susceptibility to the disease by patients infected with human immunodeficiency virus (HIV) (Elliot et al., 1995; Chintu and Mwinga, 1999). Transmission of the infection by Mtb bacilli is air borne and occurs through inhalation of aerosol containing the bacilli exhaled by coughing, sneezing or spitting by patients suffering from pulmonary tuberculosis. The inhaled bacilli are engulfed by the alveolar macrophages, where the bacilli are able to persist successfully in a latent or proliferating state. This persistence is achieved by modulation of several intracellular signaling pathways in order to create a suitable environment for the bacilli. The interplay of mycobacteria with host signaling pathways is a complex and dynamic process that is not clearly understood. Mtb secretes several molecules that modulate the signaling pathways (Koul et al., 2004). Most of these molecules commonly target macrophages, which helps the bacilli to evade innate immune response and propagate throughout the system (Rosenberger and Finlay, 2003).

The proteins secreted by Mtb have gained attention in recent years as putative vaccine and diagnostic candidates (Harboe et al., 1996; Colangeli et al., 2000). But there have been recent reports about their role in modulation of macrophage signaling pathways leading to compromise of macrophage functions (Trajkovic et al., 2002; Pym et al., 2003; Guinn et al., 2004). Thus the secretory proteins can act as risk or virulent factors too. This notion is also supported by the fact that only live but not dead bacilli can down regulate macrophage functions (Malik et al., 2001). In this chapter, we focus our discussion on the role of the

*Mycobacterium tuberculosis* RD-1 Secreted

**and vaccine** 

al., 2005).

**and ESAT6** 

**5. Macrophage subversion by CFP10** 

**4. Use of CFP10 and ESAT6 as tools for diagnosis** 

Antigens as Protective and Risk Factors for Tuberculosis 119

CFP-10 and ESAT-6 were identified in a screen to identify the proteins present in culture filtrates of Mtb and *M. bovis BCG*, which could induce T cell mediated response. (Andersen et al., 1991a; Andersen et al., 1991b; Andersen et al., 1994; Weldingh et al., 1998; Weldingh et al., 1999). The screen yielded six low molecular weight antigens *viz*. Rv3871, Rv3872, Rv3873, CFP-10, ESAT-6 and Rv3878. These antigens when expressed and purified as recombinant proteins gave strong humoral response in tuberculous guinea pigs while only two antigens i.e. CFP-10 and ESAT-6 showed strong delayed type hypersensitivity (DTH) reaction in the guinea pigs (Weldingh et al., 1999). CFP10 and ESAT6 are potent T cell antigens and induce strong T cell response. In mice infected with Mtb, CFP-10 specific T cells were observed at quite early stage of infection in lungs. These T cells were activated by CFP-10 epitopes and were recruited in large numbers (Kamath et al., 2004). This resulted in production of large amounts of interferon- (IFN-). Recombinant CFP-10 has also been shown to be a potent T cell antigen, inducing T cell proliferation and IFN- production in peripheral blood mononuclear cells in about 70% of purified protein derivative (PPD) positive asymptomatic individuals. CFP-10 was also shown to induce delayed type hypersensitivity (DTH) in Mtb infected guinea pigs but not in *M. bovis* BCG infected guinea pigs (Colangeli et al., 2000). ESAT-6 is also a RD-1 antigen inducing robust levels of IFN- by T cells in early stages of *M. tuberculosis* infection (Porsa et al., 2006; Ravn et al., 1999; Skjot et al., 2000; de Jong et al., 2006). Two different T cell epitopes were observed in mice, which were recognized by different MHCII molecules under different circumstances (Dietrich et

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

proteins secreted by region of difference-1 (RD-1), the region that is deleted in all the strains of *M. bovis* BCG.
