Vinay K. Nandicoori
Indian Institute of Technology, Mumbai, India
Indian Institute of Sciences, Bangalore, India
Texas A & M University, College Station, Texas, USA
University of Virginia, Charlottesville, Virginia, USA
Deciphering kinase-mediated signaling networks in Mycobacterium tuberculosis (Mtb). Investigating the role of Mtb protein kinases in regulating cellular events of the pathogen and host-pathogen interactions.
Dr. Savita Lochab, Ms. Divya Arora, Ms. Prabhjot Kaur, Ms. Preeti Jain, Ms. Basanti Malakar, Ms. Mehak Zahoor Khan, Mr. Yogesh Chawla, Mr. Vijay Soni, Ms. Saba Naz, Mr. Mahesh Ray, Mr. Babulal Nepali
Summary of Research
Cell-signaling is a consortium of cellular events wherein the transmission of environmental signals to cells sets off a cascade of reactions that ultimately culminate in changes in gene expression and activity. One of the major mechanisms through which cell signaling pathways operate is the reversible phosphorylation of cellular proteins. Eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb regulate a number of cellular processes including cell division. There are 11 eukaryotic-like STPKs in Mtb, and our lab has been working on analyzing the functional roles of the phosphorylation events mediated by these kinases.
Protein kinases A and B are essential genes that play a role in modulating cell shape, and possibly cell division. Overexpressing of PknA in M. tuberculosis results in a deviation from normal cell morphology with the cells forming an elongated and branched structure, while overexpression of PknB in M. tuberculosis results in the formation of widened and bulging cells. The Mtb protein kinase B (PknB) comprises an intracellular kinase domain, connected through a transmembrane domain to an extracellular region that contains four PASTA domains. We performed comprehensive analysis of different domains of PknB in the context of viability in avirulent and virulent mycobacteria and showed that although the presence of the C-terminal PASTA domain is dispensable in the avirulent M. smegmatis, all four PASTA domains are essential in Mtb. Work from the lab showed that the stringent regulation of PknB expression is necessary for cell survival, with depletion or overexpression of PknB leading to cell death. Recently we showed that Protein kinase A is essential for in vitro growth as well as for the survival of the pathogen in the host. Although PknA and PknB are expressed as part of the same operon, they appear to be regulating cellular processes through divergent signaling pathways.
Protein kinase G (PknG) has been shown to play a role in the survival of the pathogen in host macrophages by modulating phagosome-lysosome fusion (lysosomal transfer) after macrophages phagocytose mycobacterium, and PknG kinase activity is essential for mediating its effect on lysosomal transfer. We have investigated the role of PknG and PknG mutants in the pathogenesis of mycobacteria in mice, and showed for the first time that the expression of PknG in non-pathogenic mycobacteria allows the continued existence of these bacteria in host tissues. We were the first to characterize PknK and show that PknK modulates the activation of transcription from the promoter of mycobacterial monooxygenase operon, through phosphorylation of the transcriptional regulator VirS.
The identification and characterization of target substrates for kinases is extremely important to decipher the function of the pathway within the cell. We have identified enzymes N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) to be novel substrates of PknA as well as PknB and demonstrated that while PknB mediated phosphorylation of GlmU does not affect its uridyltransferase activity, it significantly modulates the protein’s acetyltransferase activity. In collaboration with Dr. Balaji Prakash, we determined the structure of GlmUMtb in complex with substrates bound at the acetyltransferase active site, and uncovered unique features in the carboxy terminus of GlmUMtb. Recently we have comprehensively dissected the role of GlmUMtb in arbitrating the survival of pathogen both in vitro and in vivo. In collaboration with Dr. D. Sriram, we developed a novel oxazolidine derivative targeting the uridyltransferase allosteric site, which specifically inhibited GlmUMtbin vitro and ex vivo. Work from our lab showed that phosphorylation of InhA, the primary target of the first-line anti-tuberculosis drug isoniazid and a key enzyme of the Fatty Acid Synthase-II system involved in mycolic acid biosynthesis in Mtb, impacts mycobacterial growth and survival.
Though all the Mtb STPKs have been biochemically characterized and a number of substrates identified, many questions remain to be answered. The role of the extracellular environment on activation/inactivation of kinases has not yet been investigated. Despite the fact that many of the substrates were initially identified to be substrates for one specific kinase or other, most are actually phosphorylated by multiple STPKs. Thus, a key feature amongst all these kinase-substrate interactions is the redundancy of signalling, suggesting a complicated temporal and spatial control over key mycobacterial processes. It is conceivable that many or all of the signalling molecules described here may in fact regulate each other, similar to the cross-talk seen in most eukaryotic signalling pathways. Our ultimate aim to decipher the signaling pathways in Mtb and determine how these signaling molecules influence the survival in the host upon infection.
Awards / Fellowships
Fellow, The National Academy of Sciences (India), Allahabad, (2014)
National Bioscience Award for Career Development (2010)
Elected Member, Guha Research Conference, India (GRC) (2009)
NASI-Scopus Young Scientist Award (2009)
M. Sreenivasaya Award for the Best Thesis, IISc, Bangalore, India (1999)
- Sharma, G., Upadhyay, S.,Srilalitha, M., Nandicoori, V. K. & Khosla, S. (2015) The interaction of mycobacterial protein Rv2966c with host chromatin is mediated through non-CpG methylation and Histone H3/H4 binding. Nucleic Acids Res. 43, 3922-37.
- Nagarajan, S. N., Upadhyay, S., Chawla, Y., Khan, S., Naz, S., Subramanian, J., Gandotra, S. & Nandicoori, V. K.* (2015) Protein kinase A (PknA) of Mycobacterium tuberculosis is independently activated and is critical for growth in vitro and survival of the pathogen in the host . J Biol Chem. 290, 9626-9645.
- Rajanala, K., Sarkar, A., Jhingan, G. D., Priyadarshini, R., Jalan, M., Sengupta, S. & Nandicoori, V. K.* (2014) Phosphorylation of nucleoporin Tpr governs its differential localization and is required for its mitotic function. J Cell Science. 127, 3505-3520.
- Chawla, Y., Upadhyay, S., Khan, S., Nagarajan, S. N., Forti, F. & Nandicoori, V.K.* (2014) Protein Kinase B (PknB) of Mycobacterium tuberculosis is essential for growth of the pathogen in vitro as well as for survival within the host. J Biol Chem. 289, 13858 - 13875
- Parikh, A., Kumar, D., Chawla, Y., Kurthkoti, K., Khan, S., Varshney, U. & Nandicoori, V. K.* (2013) Development of new generation of vectors for gene expression, gene replacement, and protein-protein interaction studies in mycobacteria. Appl Environ Microbiol. 79, 1718-1729.
- Jagtap, P. K. A., Soni, V., Vithani, N., Jhingan, G.D., Bais, V. S., Nandicoori, V. K.* & Prakash, B.* (2012) Substrate bound crystal structure reveals feature unique to Mycobacterium tuberculosis N-acetyl glucosamine-1-phosphate uridyltransferase and a catalytic mechanism for acetyltransfer. J. Biol. Chem. 287, 39524-37.
- Rajanala, K. & Nandicoori, V.K.* (2012) Localization of nucleoporin Tpr to the nuclear pore complex is essential for Tpr mediated regulation of the export of unspliced RNA. Plos One 7, e29921.
- Khan, S., Nagarajan, S. N, Parikh, A., Samantaray, S., Singh, A., Kumar, D., Roy, R.P., Bhatt, A. & Nandicoori, V.K.* (2010) Phosphorylation of enoyl-ACP reductase InhA impacts mycobacterial growth and survival. J. Biol. Chem. 285, 37860-37871.
- Tiwari, D., Singh, R. K., Goswami, K., Verma, S. K., Prakash, B. & Nandicoori, V. K.* (2009) Key residues in Mycobacterium tuberculosis protein kinase G play a role in regulating kinase activity and survival in the host. J Biol. Chem. 284, 27467-27479.
- Kumar, P., Kumar, D., Parikh, A., Rananaware, D., Gupta, M., Singh, Y. & Nandicoori, V. K. (2009) The Mycobacterium tuberculosis protein kinase K modulates activation of transcription from the promoter of mycobacterial monooxygenase operon through phosphorylation of transcriptional regulator VirS. J. Biol. Chem. 284, 11090-11099.
- Parikh, A., Verma, S.K. Khan,S., Prakash, B.* & Nandicoori, V. K.* (2009) PknB-mediated phosphorylation of novel substrate, N-Acetylglucosamine-1-Phosphate Uridyltransferase, modulates its acetyltransferase activity. J. Mol. Biol. 386, 451-464.
- Sajish, M., Kalayil, S., Verma, S. K., Nandicoori, V. K.* & Prakash, B.* (2009) The significance of ExDD and RxKD motif conservation in Rel proteins. J. Biol. Chem 284, 9115-9123
- Vomastek, T., Iwanicki, M. P., Burack, W. R., Tiwari, D., Kumar, D., Parsons, J. T., Weber, M. J.* & Nandicoori, V. K.* (2008) ERK2 phosphorylation sites and docking domain on the Nuclear Pore Complex protein Tpr cooperatively regulates ERK2-Tpr interaction. Mol. Cell Biol. 22, 6954-6966.
- Samantaray, S., Marathe, U., Dasgupta, S., Nandicoori, V. K. & Roy, R. P. (2008) Peptide-sugar ligation catalyzed by transpeptidase sortase: a facile approach to neoglycoconjugate synthesis. (2008) J Am Chem Soc. 130, 2132-3.
- Sajish, M., Tiwari, D., Rananaware, D., Nandicoori, V. K. and Prakash, B. (2007) A Charge Reversal Differentiates (p)ppGpp Synthesis by Monofunctional and Bifunctional Rel Proteins. J. Biol. Chem. 282, 34977-83
* Represents corresponding author