My Ssec Capstone Project Tuberculosis


Tuberculosis (TB) is one of the major infectious diseases caused by Mycobacterium tuberculosis. The World Health organization (WHO) reported that 10.4 million peoples were infected and 1.7 million peoples were died because of TB (WHO, 2016). TB control is difficult due to emergence of multi-drug resistant (MDR) and extremely drug resistant (XDR) TB and HIV-TB co-infections (Kaufmann and Parida 2007). Though, we have one approved TB vaccine Mycobacterium bovis Bacillus Calmette-Geurin (BCG), it gives protection only for childhood not for adulthood. Researchers using different approaches to replace older BCG vaccine strain by substitution of new genes in BCG or by construction new attenuated live M. tuberculosis vaccine.
Mycobacterium tuberculosis has several unique features like complex cell wall, evading mechanism from immune reactions and able to survive in dormant stage for several decades. The cell division and cell wall synthesis of Mycobacterium tuberculosis is a co-linked process which occurs simultaneously during cell division. The peptidoglycan biosynthesis of Mycobacterium tuberculosis regulated by both Serine/Threonine Protein Kinases (STPKs) and Penicillin Binding Proteins (PBPs) (Narayan et al., 2007; Molle and Kremer, 2010). The link between STPK dependent phosphorylation and PG biosynthesis in mycobacteria is also supported by the fact that DacB1, a probable PBP, is phosphorylated by PknH in M. tuberculosis (Zheng et al., 2007).
The DacB2 (rv2911) is a low molecular mass (LMM) D,D-carboxypeptidase which was inhibited by meropenem (Kumar et al., 2012). As like other STPKs, PknI (rv2914c) also has kinase domain, N-terminal cytosolic domain and C-terminal extracellular domain (Narayan. A et al., 2007). It has been reported that PknI located in between DacB2, ftsY and ffh genes and predicted to play role in cell wall synthesis and cell division (Av-gay and Everett, 2000). In our lab, we have extensively characterized functions of PknI and DacB2 individually.
We found that PknI has a kinase activity and it was dependent on Mn2+ for autophosphorylation at serine and threonine residues (Gopalaswamy et al., 2004). The lysine41 present in the active site of PknI which was essential for kinase activity (Kandasamy et al., 2014). We also found that PknI and DacB2 overexpressed strains caused similar phenotypes like growth retardation and altered colony morphology in M. smegmatis mc2. We constructed single knockout strains of ?PknI and ?DacB2 found that both exhibited virulent phenotype both in vitro and in vivo models (Gopalaswamy et al., 2008; Bourai et al., 2012).
The construction of attenuated M. tuberculosis knockout strain potentially elicits highly protective immune response than recombinant protein candidate or viral vectors. In order to develop new live attenuated vaccine strain, we constructed ?PknI/?DacB2 double knockout (DKO) strain and characterized its phenotypes by several approaches found that both genes play an important role in the maintenance of colony morphology, cell wall permeability and integrity of M. tuberculosis (Kandasamy and Narayanan, 2015). Hence, the focus of the present study is to evaluated the growth and vaccine potential of DKO strain in guinea pig model.