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In high-TB incidence countries, TB control relies on diagnosis which is mainly based on clinical symptoms or laboratory diagnosis using sputum smear microscopy. TB smear microscopy is highly insensitive for HIV-co-infected individuals and for children due to the reduced pulmonary bacillary loads in these patients. TB diagnosis by smear microscopy is usually further confirmed by culture. However, this requires extended incubation times and is significantly more expensive than smears, requiring specialized equipment and highly trained personnel (Parsons et al., 2004; Storla et al., 2008). Thus, there is a basic need for the

Using recombinant DNA techniques, synthetic peptides, antigen-specific antibodies and T cells, several major antigens of *M. tuberculosis* have been identified which include hsp60, hsp70, Ag85, ESAT-6 and CFP10 (Mustafa, 2001). In addition, Hsp65, Hsp71, 14-kDa Hsp and GroE proteins can play an important role in the diagnosis of TB. The identification of these markers can contribute to the clinical diagnosis of TB and may also provide additional insight into the pathogenesis of TB (Kashyap et al., 2010). sHsp18 has been shown to be a major immunodominant antigen of *M. leprae* (Lini et al., 2008). Hsp65 has been shown to be an attractive marker for TB (Bothamley et al., 1992; Haldar et al., 2010; Lee et al., 1994; Rambukkana et al., 1991). The 65 kDa heat shock protein is detected even in the cerebrospinal fluid of tuberculous meningitis patients, indicating its potential use as a diagnostic marker for tuberculous meningitis (Mudaliar et al., 2006). In case of TB ascites, the ascitic fluid has shown the presence of Hsp65, Hsp71 and Hsp14 as very useful diagnostic markers (Kashyap et al., 2010). A multiplex PCR against Hsp65 gene coding for 65 kDa antigen for early detection has been tested. The technique was able to distinguish between strains of the *M. tuberculosis* complex and non-tuberculous mycobacteria

The response to recombinant 10-kDa heat shock protein of *M. leprae* was evaluated by indirect ELISA in sera from leprosy patients, household contacts, tuberculosis patients and healthy controls. However, this test seems to have a low sensitivity and specificity for leprosy detection and tuberculosis patients sera cross-reacted with *M. leprae* antigen as well

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**Mammalian Heme Peroxidases and** 

*Department of Biophysics, All India Institute of Medical Sciences,* 

Amit K. Singh, Nisha Pandey, Mau Sinha, Sujata Sharma and Tej P. Singh

Tuberculosis (TB) is a lethal infectious disease which is caused by *Mycobacterium tuberculosis*. The alarming rate at which the incidence of bacterial resistance to known antibiotics has been rising is a serious cause of concern. At present, the two well known anti-tuberculosis drugs, isonicotinic acid hydrazide (INH, isoniazid) and pyrazinamide (PZA, pyrazin-2 carboxamide) which are important components of the current course of the first-line TB chemotherapy suffer from increasing bacterial resistance. The other drugs of the combination therapy include rifampicin and ethambutol. It may be noted that both INH and PZA are prodrugs and require specific enzymes to convert them into drugs. INH is activated by a bacterial heme enzyme catalase peroxidase (*Mt*CP) into a free radical form (Scheme I) (Zhang et al., 1992). The structure of unliganded *Mt*CP is known (Bertrand et al., 2004) and detailed information is available about the substrate-binding site and the residues that might be involved in the binding and conversion of INH into a beneficial product. However, a precise mode of binding and the mechanism of action are not yet clearly understood because the structure of INH bound *Mt*CP is not yet determined. On the other hand, PZA is metabolized into its active form pyrazinoic acid (POA) by amidase activity of the *Mycobacterium tuberculosis* nicotinamidase/pyrazinamidase (PncA) (Scheme II) (Konno et al., 1967). Although the crystal structure of pyrazinamidase in complex with POA is known but the structure of the complex with the original compound PZA is not yet determined. Therefore, the mode of binding of PZA with PncA has not so far been revealed. As shown by the crystal structure of the complex of LPO with INH, the binding of INH to lactoperoxidase (LPO) occurs through the distal heme cavity where INH interacts with a conserved water molecule W1 which is hydrogen bonded to ferric iron (Singh et al., 2010). Similarly, as revealed by the structure determination of the complex formed between LPO and PZA, PZA has been located in the substrate-binding site and interacts with substrate recognition residues of LPO (PDB ID: 3R4X) indicating a possible role of LPO in the conversion of PZA into an active form. Although the crystal structure of the PZA bound PncA is not known but a piece of information is available on the possible mode of ligand binding based on the molecular modeling data (Petrella et al., 2011). Therefore, it is of great interest that both prodrugs, INH and PZA bind to LPO specifically at the substrate-binding site on the distal heme side as the substrates bind to LPO (Singh et al., 2009) so that these

**1. Introduction** 

*Mycobacterium tuberculosis* 

*New Delhi, India* 

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