II.P.1 Search for Rv2998A protein partners using molecular techniques

Mycobacterium tuberculosis (M.tb) is one of the oldest known infectious factor, causing tuberculosis. This is still one of the major causes of mortality. The success of M. tuberculosis as a pathogen is connected to its ability to adapt to a wide range of living conditions inside as well as outside the human host. This serious pathogen has spread intensively worldwide and recent years have seen an increase in the number of multi-drug resistant M. tuberculosis strains. Comparing to the genetically linked two component signal transduction systems from mycobacteria very little is known about the orphaned elements of the signaling cascade in these bacteria. A typical TCSS composes of membrane histidine sensor kinase and cytosolic response regulator. One of the Mycobacterium tuberculosis gene, rv2998A is coding for an putative orphane kinase – Rv2998A, whose function was not yet identified. To better understanding the adaptation of M. tuberculosis to environmental changes, Rv2998A was used in functional analysis, which was based on mass spectrometry (MS/MS), pull-down and bacterial two hybrid assays. Rv2998A tagged to green fluorescence protein (GFP) was expressed in M.tb as a bait protein, purified together with its putative partners and identified by MS/MS. The identified partners-interactions were confirmed by using bacterial two hybrid assay consist of reconstitution of active cyclic AMP synthase in E. coli host. Further, the selected proteins were expressed in E. coli system and purified to be used in pull-down in vitro assay to confirm an identified interactions. Better understanding of the signal transduction pathways could lead to development of specific drugs for efficiently blocking some of the crucial elements of the signaling cascade. This in turn will prevent bacteria from multiplication and would result in improvement in curability of tuberculosis disease.