National Institute for Research in Tuberculosis and 1

Chetput, India

National Institute for Research in Tuberculosis and 1

Chetput, India
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Refaya A.K.,National Institute for Research in Tuberculosis and 1 | Sharma D.,National JALMA Institute for Leprosy and other Mycobacterial Diseases | Kumar V.,National JALMA Institute for Leprosy and other Mycobacterial Diseases | Bisht D.,National JALMA Institute for Leprosy and other Mycobacterial Diseases | Narayanan S.,National Institute for Research in Tuberculosis and 1
Microbiological Research | Year: 2016

Mycobacterium tuberculosis adapts itself to various environmental stress conditions to thrive inside the phagosome for establishing a chronic infection. Serine/threonine protein kinases (STPKs) play a major role in the physiology and pathogenesis of Mycobacterium tuberculosis. Some of these STPKs are involved in regulating the growth of the mycobacterium under nutrient stress and starvation conditions. In this study, we have investigated the role of PknL, a STPK in the adaptive responses of M. tuberculosis by conditional inactivation of the gene using antisense technology. The inhibition of PknL in the knockdown strain was validated by RT-PCR. The in vitro growth kinetics of M. tuberculosis strain following inhibition of PknL was found to be bacteriostatic. The knock down strain of PknL exhibited a better survival in pH 5.5 when compared to its growth in pH 7.0. Similarly, it also exhibited more resistance to both SDS(0.01%) and Lysozyme stress (2.5 mg/ml), indicating that loss of PknL enhances the growth of mycobacterium under stress conditions. SEM pictographs also represent an increase in the cell length of the knock down strain compared to Wild type stressing its role in cellular integrity. Lastly, the proteome analysis of differentially expressing PknL strains by 2D gel electrophoresis and mass spectrometry identified 19 differentially expressed proteins. Our findings have shown that PknL plays an important role in sensing the host environment and adapting itself in slowing down the growth of the pathogen and persisting within the host. © 2016 Elsevier GmbH.


Venkatesan A.,National Institute for Research in Tuberculosis and 1 | Hassan S.,National Institute for Research in Tuberculosis and 1 | Palaniyandi K.,National Institute for Research in Tuberculosis and 1 | Narayanan S.,National Institute for Research in Tuberculosis and 1
Journal of Molecular Graphics and Modelling | Year: 2015

Protein-protein interactions control the diverse and essential molecular processes inside the cell. To maintain the cellular physiology, protein kinases not only signal their substrates through reversible phosphorylation, but they also physically interact with them. PknI, a serine/threonine protein kinase of Mycobacterium tuberculosis is known to be important for cellular homoeostasis. In this study, we have identified the interacting proteins for PknI. We screened for proteins interacting with PknI using an in vitro assay, Far-western blot. This protein kinase specifically interacts with two peroxidase proteins of M. tuberculosis, Rv2159c and Rv0148. The PknI-Rv2159c interaction pair was further studied for the critical amino acid residues in Rv2159c that are responsible for the interaction. Rv2159c, a hypothetical protein is predicted to be an antioxidant with peroxidase activity. We performed homology modelling of Rv2159c protein and molecular docking using multiple docking servers such as Z-Dock and ClusPro. Further, the most favorable conformation of PknI-Rv2159c interaction was obtained using molecular dynamics simulation. The critical amino acid residues of the Rv2159c involved in interaction with PknI were identified. Mutation and docking analysis showed that the Ala1-Gly2-Trp3 residues in Rv2159c structure are responsible for the interaction. The free binding energy between the wild type and mutant complexes using MM-GBSA has provided insight about the stability of PknI-Rv2159c interaction. We propose that, PknI physically interacts with Rv2159c both in vitro and in silico studies. © 2015 Published by Elsevier Inc.


PubMed | National Institute for Research in Tuberculosis and 1
Type: | Journal: Journal of molecular graphics & modelling | Year: 2015

Protein-protein interactions control the diverse and essential molecular processes inside the cell. To maintain the cellular physiology, protein kinases not only signal their substrates through reversible phosphorylation, but they also physically interact with them. PknI, a serine/threonine protein kinase of Mycobacterium tuberculosis is known to be important for cellular homoeostasis. In this study, we have identified the interacting proteins for PknI. We screened for proteins interacting with PknI using an in vitro assay, Far-western blot. This protein kinase specifically interacts with two peroxidase proteins of M. tuberculosis, Rv2159c and Rv0148. The PknI-Rv2159c interaction pair was further studied for the critical amino acid residues in Rv2159c that are responsible for the interaction. Rv2159c, a hypothetical protein is predicted to be an antioxidant with peroxidase activity. We performed homology modelling of Rv2159c protein and molecular docking using multiple docking servers such as Z-Dock and ClusPro. Further, the most favorable conformation of PknI-Rv2159c interaction was obtained using molecular dynamics simulation. The critical amino acid residues of the Rv2159c involved in interaction with PknI were identified. Mutation and docking analysis showed that the Ala1-Gly2-Trp3 residues in Rv2159c structure are responsible for the interaction. The free binding energy between the wild type and mutant complexes using MM-GBSA has provided insight about the stability of PknI-Rv2159c interaction. We propose that, PknI physically interacts with Rv2159c both in vitro and in silico studies.

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