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Biochemical Characterization of VapC46 Toxin from Mycobacterium tuberculosis

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Abstract

Emergence of multidrug resistant strains and extremely drug resistant strains of Mycobacterium tuberculosis is due to its ability to form persister cells. The formation of persister cells is assumed to be triggered due to the presence of large number of toxin–antitoxin (TA) systems in its genome. Mtb genome encodes 47 VapBC TA systems. In this work, we aim to biochemically characterize VapC46 toxin of the VapBC46 TA operon from Mycobacterium tuberculosis. Heterologous expression of VapC46 in E. coli is shown to exhibit bacteriostasis and toxicity alters the surface morphology of the E. coli cells. VapC46 is shown to possess ribonuclease activity in a magnesium-dependent manner. Using FRET and pull down assay, VapC46 is shown to interact with VapB46 antitoxin. A model of VapC46 is shown to resemble PIN domain family of proteins and reveals the putative active site required for its ribonuclease activity.

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References

  1. Mwinga, A., & Bernard Fourie, P. (2004). Prospects for new tuberculosis treatment in Africa. Tropical Medicine & International Health,9, 827–832.

    Article  CAS  Google Scholar 

  2. Gupta, S., & Chatterji, D. (2005). Stress responses in mycobacteria. IUBMB Life,57, 149–159.

    Article  CAS  Google Scholar 

  3. Buts, L., Lah, M.-H., Dao-Thi, J., Wyns, L., & Loris, R. (2005). Toxin-antitoxin modules as bacterial metabolic stress managers. Trends in Biochemical Sciences,30, 672–679.

    Article  CAS  Google Scholar 

  4. Gerdes, K., Christensen, S. K., & Løbner-Olesen, A. (2005). Prokaryotic toxin-antitoxin stress response loci. Nature Reviews Microbiology,3, 371–382.

    Article  CAS  Google Scholar 

  5. Bukowski, M., Rojowska, A., & Wladyka, B. (2011). Prokaryotic toxin-antitoxin systems-the role in bacterial physiology and application in molecular biology. Acta Biochimica Polonica,58, 1–9.

    Article  CAS  Google Scholar 

  6. Gerdes, K., & Maisonneuve, E. (2012). Bacterial persistence and toxin-antitoxin loci. Annual Review of Microbiology,66, 103–123.

    Article  CAS  Google Scholar 

  7. Hayes, F. (2003). Toxins-antitoxins: Plasmid maintenance, programmed cell death, and cell cycle arrest. Science,301, 1496–1500.

    Article  CAS  Google Scholar 

  8. Ramage, H. R., Connolly, L. E., & Cox, J. S. (2009). Comprehensive functional analysis of Mycobacterium tuberculosis toxin-antitoxin systems: Implications for pathogenesis, stress responses, and evolution. PLoS Genetics,5, e1000767.

    Article  Google Scholar 

  9. Arcus, V. L., McKenzie, J. L., Robson, J., & Cook, G. M. (2011). The PIN-domain ribonucleases and the prokaryotic VapBC toxin-antitoxin array. Protein Engineering, Design & Selection,24, 33–40.

    Article  CAS  Google Scholar 

  10. Clissold, P. M., & Ponting, C. P. (2000). PIN domains in nonsense-mediated mRNA decay and RNAi. Current Biology,10, R888–R890.

    Article  CAS  Google Scholar 

  11. Min, A. B., Miallau, L. M., Sawaya, R., Habel, J., Cascio, D., & Eisenberg, D. (2012). The crystal structure of the Rv0301-Rv0300 VapBC-3 toxin-antitoxin complex from M. tuberculosis reveals a Mg2+ ion in the active site and a putative RNA-binding site. Protein Science,21, 1754–1767.

    Article  CAS  Google Scholar 

  12. Cruz, J. W., Sharp, J. D., Hoffer, E. D., Maehigashi, T., Vvedenskaya, I. O., Konkimalla, A., et al. (2015). Growth-regulating Mycobacterium tuberculosis VapC-mt4 toxin is an isoacceptor-specific tRNase. Nat Commun.,6, 7480.

    Article  Google Scholar 

  13. Miallau, L. M., Chiang, J., Arbing, M., Guo, F., Cascio, D., & Eisenberg, D. (2009). Structure and proposed activity of a member of the VapBC family of toxin-antitoxin systems. VapBC-5 from Mycobacterium tuberculosis. Journal of Biological Chemistry,284, 276–283.

    Article  CAS  Google Scholar 

  14. Deep, A., Kaundal, S., Agarwal, S., Singh, R., & Thakur, K. G. (2017). Crystal structure of Mycobacterium tuberculosis VapC20 toxin and its interactions with cognate antitoxin, VapB20, suggest a model for toxin–antitoxin assembly. FEBS Journal,284, 4066–4082.

    Article  CAS  Google Scholar 

  15. Gonçalves, R., Jardim, P., Cristina, I., & De Freitas, M. (2016). Crystal structure of VapC21 from Mycobacterium tuberculosis at 1.31 Å resolution. Biochemical and Biophysical Research Communications,478, 1370–1375.

    Article  Google Scholar 

  16. Lee, I.-G., Lee, S. J., Chae, S., Lee, K.-Y., Kim, J.-H., & Lee, B.-J. (2015). Structural and functional studies of the Mycobacterium tuberculosis VapBC30 toxin-antitoxin system: Implications for the design of novel antimicrobial peptides. Nucleic Acids Research.,43, 7624–7637.

    Article  CAS  Google Scholar 

  17. Kang, S., Kim, D., Lee, K., Park, S. J., Yoon, H., Lee, J., et al. (2017). Functional details of the Mycobacterium tuberculosis VapBC26 toxin-antitoxin system based on a structural study: Insights into unique binding and antibiotic peptides. Nucleic Acids Research,45, 8564–8580.

    Article  CAS  Google Scholar 

  18. Deep, A., Tiwari, P., Agarwal, S., Kaundal, S., Kidwai, S., Singh, R., et al. (2018). Structural, functional and biological insights into the role of Mycobacterium tuberculosis VapBC11 toxin–antitoxin system: targeting a tRNase to tackle mycobacterial adaptation. Nucleic Acids Research,30, 11639–11655.

    Article  Google Scholar 

  19. Gupta, A. (2009). Killing activity and rescue function of genome-wide toxin-antitoxin loci of Mycobacterium tuberculosis. FEMS Microbiology Letters,290, 45–53.

    Article  CAS  Google Scholar 

  20. Gupta, A., Venkataraman, B., Vasudevan, M., & Bankar, K. G. (2017). Co-expression network analysis of toxin-antitoxin loci in Mycobacterium tuberculosis reveals key modulators of cellular stress. Scientific Report,19, 5868.

    Article  Google Scholar 

  21. Lopes, A. P. Y., Lopes, L. M., Fraga, T. R., Chura-Chambi, R. M., Sanson, A. L., Cheng, E., et al. (2014). VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA. PLoS ONE,9, e101678.

    Article  Google Scholar 

  22. Wang, H., Cheng, H., Wang, F., Wei, D., & Wang, X. (2010). An improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay for evaluating the viability of Escherichia coli cells. Journal of Microbiol Methods,82, 330–333.

    Article  CAS  Google Scholar 

  23. Gupta, M., Nayyar, N., Chawla, M., Sitaraman, R., Bhatnagar, R., & Banerjee, N. (2016). The chromosomal parDE2 toxin-antitoxin system of Mycobacterium tuberculosis H37Rv: Genetic and functional characterization. Frontier Microbiology,7, 886.

    Google Scholar 

  24. Lakowicz, J. R. (1999). Principles of fluorescence spectroscopy. New York: Kluwer Academic.

    Book  Google Scholar 

  25. Li, Z., Zhao, B., Wang, P., Chen, F., Dong, Z., Yang, H., et al. (2010). Structural insights into the YAP and TEAD complex. Genes & Development,24, 235–240.

    Article  CAS  Google Scholar 

  26. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., & Sternberg, M. J. E. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols,10, 845–858.

    Article  CAS  Google Scholar 

  27. Bunker, R. D., Mckenzie, J. L., Baker, E. N., & Arcus, V. L. (2008). Crystal structure of PAE0151 from Pyrobaculum aerophilum, a PIN-domain (VapC) protein from a toxin-antitoxin operon. Proteins.,72, 510–518.

    Article  CAS  Google Scholar 

  28. Laskowski, R. A., MacArthur, M. W., Moss, D. S., & Thornton, J. M. (1993). PROCHECK: A program to check the stereochemical quality of protein structures. Journal of Applied Crystallography,26, 283–291.

    Article  CAS  Google Scholar 

  29. Land, H., & Humble, M. S. (2018). YASARA: A tool to obtain structural guidance in biocatalytic investigations. Methods in Molecular Biology,1685, 43–67.

    Article  CAS  Google Scholar 

  30. Das, U., Pogenberg, V., Subhramanyam, U. K. T., Wilmanns, M., Gourinath, S., & Srinivasan, A. (2014). Crystal structure of the VapBC-15 complex from Mycobacterium tuberculosis reveals a two-metal ion dependent PIN-domain ribonuclease and a variable mode of toxin-antitoxin assembly. Journal of Structural Biology,188, 249–258.

    Article  CAS  Google Scholar 

  31. Winther, K. S., & Gerdes, K. (2012). Regulation of enteric vapBC transcription: Induction by VapC toxin dimer-breaking. Nucleic Acids Research,40, 4347–4357.

    Article  CAS  Google Scholar 

  32. Holm, L., & Park, J. (2000). DaliLite workbench for protein structure comparison. Bioinformatics,16, 566–567.

    Article  CAS  Google Scholar 

  33. Arcus, V. L., Bäckbro, K., Roos, A., Daniel, E. L., & Baker, E. N. (2004). Distant structural homology leads to the functional characterization of an archaeal PIN domain as an exonuclease. Journal of Biological Chemistry,279, 16471–16478.

    Article  CAS  Google Scholar 

  34. Lioy, V. S., Rey, O., Balsa, D., Pellicer, T., & Alonso, J. C. (2010). A toxin-antitoxin module as a target for antimicrobial development. Plasmid,63, 31–39.

    Article  CAS  Google Scholar 

  35. Dubnau, D., & Losick, R. (2006). Bistability in bacteria. Molecular Microbiology,61, 564–572.

    Article  CAS  Google Scholar 

  36. Rotem, E., Loinger, A., Ronin, I., Levin-reisman, I., & Gabay, C. (2010). Regulation of phenotypic variability by a threshold-based mechanism underlies bacterial persistence. Proceedings of National Academic Science in United States of America,107, 12541–12546.

    Article  CAS  Google Scholar 

  37. Roy, M., Kundu, A., Bhunia, A., Das Gupta, S., De, S., & Das, A. K. (2019). Structural characterization of VapB46 antitoxin from Mycobacterium tuberculosis: Insights into VapB46–DNA binding. FEBS Journal,286, 1174–1190.

    Article  CAS  Google Scholar 

Download references

Acknowledgement

We acknowledge Central research facility IIT Kharagpur for SEM facility. The work was supported by SGBSI Grant IIT/SRIC/BIO/LDO/2014-15/33 of IIT Kharagpur.

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Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology, Kharagpur, India

    Madhurima Roy, Madhuparna Bose, Anirban Kundu & Amit Kumar Das

  2. School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, India

    Kamakshi Bankoti & Santanu Dhara

  3. School of Bioscience, Indian Institute of Technology, Kharagpur, India

    Amit Kumar Das

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  1. Madhurima Roy
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  2. Madhuparna Bose
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Correspondence to Amit Kumar Das.

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Roy, M., Bose, M., Bankoti, K. et al. Biochemical Characterization of VapC46 Toxin from Mycobacterium tuberculosis. Mol Biotechnol 62, 335–343 (2020). https://doi.org/10.1007/s12033-020-00253-z

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