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Summer Research Fellowship Programme of India's Science Academies

Cloning, Expression and Purification of Mycobacterial Thioredoxin (Trx) in Escherichia coli and antibody generation against pe-domain of M.tb PE2 protein

Raksha L

3rd year MBBS, Bangalore Medical College and Research Institute, Fort, K.R. Road, Bangalore, Karnataka 560002.

Prof. Sharmistha Banerjee

Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India 500046

Abstract

Tuberculosis is an aerosolic infectious disease caused by Mycobacterium tuberculosis. India has the highest TB burden in the world, where approximately 2 out of 5 individuals are infected with TB and prevalence of disease is 195 per 100,000 people. Treatment of tuberculosis is complicated due to the emergence of strains that are resistant to anti-tubercular drugs (MDR-TB, XDR-TB and TDR-TB). Mycobacterium tuberculosis is an intracellular pathogen residing inside macrophages. Mycobacterium tuberculosis makes use of several strategies to evade host innate immune response. The lipid rich mycolate layer present in the cell wall of mycobacteria forms a dense waxy hydrophobic outer membrane which protects the bacteria from environmental stress and antibiotic therapies. This thick hydrophobic layer makes it difficult for the bacteria to secrete their proteins across the membrane; hence they use specialized mechanisms for transport through their cell membrane, called secretion system. Mycobacterium tuberculosis has five secretion systems of which ESX-1, ESX-3 and ESX-5 secretion systems play a major role in virulence. There are many reports and ongoing investigations on mycobacterial protein secretion systems including structural and functional characterization of the secretory proteins. Still their function and the mechanisms by which they interact with host cells is unclear. One such secreted protein is thioredoxin from Mycobacterium tuberculosis. The genome of M. tuberculosis encodes three thioredoxins and bears a single copy of trxR, the gene encoding thioredoxin reductase. The Trx redox system has been implicated in the resistance of Mycobacterium tuberculosis to phagocytosis. Trx is able to reduce a variety of target substrates and reactive oxygen species (ROS) within the host environment. Thioredoxins and TrxR are considered to be attractive targets for antitubercular drug design studies. Hence, my project would focus on understanding the functional characterization of probable thioredoxin (Rv0526) and their role in mycobacterial survival and persistence. Towards achieving this, the cloning of Mycobacterial thioredoxin (Rv0526) is in progress. The coming four weeks of my project will include expression and purification of mycobacterial Thioredoxins for better understanding of mechanisms which help in intracellular survival of mycobacteria inside host macrophages.

Keywords: Secretion systems Multidrug resistance bacteria.

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