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Infectious Disorders - Drug Targets

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ISSN (Print): 1871-5265
ISSN (Online): 2212-3989

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Research Article

Simultaneous Detection of Drug-resistant Mutations in Mycobacterium tuberculosis and Determining their Role through In Silico Docking

Author(s): Somanna Ajjamada Nachappa, Sumana M. Neelambike, Ahmad Sarikhani and Nallur B. Ramachandra*

Volume 21, Issue 1, 2021

Published on: 18 March, 2020

Page: [134 - 141] Pages: 8

DOI: 10.2174/1871526520666200318111140

Price: $65

Abstract

A molecular method for diagnosis of drug-resistant Tuberculosis is Multiplex allele-specific PCR (MAS-PCR), which is more time-efficient, and its accuracy is studied using DNA sequencing. Also, understanding the role of mutations, when translated to protein, in causing resistance helps in better drug designing.

Aim: To study MAS-PCR in the detection of drug resistance in comparison to DNA sequencing in Mycobacterium tuberculosis, and understand the mechanism of interaction of drugs with mutant proteins.

Methods: MAS-PCR was used for the detection of drug-resistant mutations and validation was done through DNA sequencing. MAS-PCR targeted four genes, iniA for the drug Ethambutol, rpsL and rrs for Streptomycin, and gyrA for Fluoroquinolone resistance, respectively. Further, the sequence data was analysed and modeled for in silico docking to study the effect on the interaction of the anti-TB drug molecule with the target protein.

Results: We identified drug-resistant mutations in four out of 95 isolates with one of them carrying a mutation at codon iniA501, two at gyrA94, and one for both iniA501 and gyrA94 using MASPCR. DNA sequencing confirmed drug-resistant mutations in only two isolates, whereas two others had mutation adjacent to the target allele. Drug-protein docking showed Estimated Free Energy of Binding to be higher for Fluoroquinolone binding with GyrA D94V mutant. Both wild and mutant IniA interact with EMB but had no significant effect on binding energy.

Conclusion: DNA sequencing-based drug resistance detection of TB is more accurate than MASPCR. Understanding the role of mutations in influencing the drug-protein interaction will help in designing effective drug alternatives.

Keywords: Tuberculosis, resistance, ethambutol, fluoroquinolone, docking, drug-resistant, mutations.

Graphical Abstract

[1]
Virupakshaiah,D.; Ahmed, M.; Patil, S.T.; Kelmani, C. Molecular Docking Studies of Mycobacterium tuberculosis RNA Polymerase [Beta] Subunit (rpoB) Receptor. World Academy of Science, Engineering and Technology (WASET)., 2013, 7(5).
[2]
Floyd, K.; Glaziou, P.; Zumla, A.; Raviglione, M. The global tuberculosis epidemic and progress in care, prevention, and research: an overview in year 3 of the End TB era. Lancet Respir. Med., 2018, 6(4), 299-314.
[http://dx.doi.org/10.1016/S2213-2600(18)30057-2] [PMID: 29595511]
[3]
Organization, W.H. Global tuberculosis report 2018; World Health Organization, 2018.
[4]
Programme, R.N.T.C. TB India 2018 annual status report; India Offset Press, 2018.
[5]
Chia, B-S.; Lanzas, F.; Rifat, D.; Herrera, A.; Kim, E.Y.; Sailer, C.; Torres-Chavolla, E.; Narayanaswamy, P.; Einarsson, V.; Bravo, J.; Pascale, J.M.; Ioerger, T.R.; Sacchettini, J.C.; Karakousis, P.C. Use of multiplex allele-specific polymerase chain reaction (MAS-PCR) to detect multidrug-resistant tuberculosis in Panama. PLoS One, 2012, 7(7), e40456.
[http://dx.doi.org/10.1371/journal.pone.0040456] [PMID: 22792333]
[6]
Colangeli, R.; Helb, D.; Sridharan, S.; Sun, J.; Varma-Basil, M.; Hazbón, M.H.; Harbacheuski, R.; Megjugorac, N.J.; Jacobs, W.R., Jr; Holzenburg, A.; Sacchettini, J.C.; Alland, D. The Mycobacterium tuberculosis iniA gene is essential for activity of an efflux pump that confers drug tolerance to both isoniazid and ethambutol. Mol. Microbiol., 2005, 55(6), 1829-1840.
[http://dx.doi.org/10.1111/j.1365-2958.2005.04510.x] [PMID: 15752203]
[7]
Jaber, A.A.; Ahmad, S.; Mokaddas, E. Minor contribution of mutations at iniA codon 501 and embC-embA intergenic region in ethambutol-resistant clinical Mycobacterium tuberculosis isolates in Kuwait. Ann. Clin. Microb. Anti., 2009, 8, 2.
[8]
Cuevas-Córdoba, B.; Cuellar-Sánchez, A.; Pasissi-Crivelli, A.; Santana-Álvarez, C.A.; Hernández-Illezcas, J.; Zenteno-Cuevas, R. rrs and rpsL mutations in streptomycin-resistant isolates of Mycobacterium tuberculosis from Mexico. J. Microbiol. Immunol. Infect., 2013, 46(1), 30-34.
[http://dx.doi.org/10.1016/j.jmii.2012.08.020] [PMID: 23040237]
[9]
Jagielski, T.; Ignatowska, H.; Bakuła, Z.; Dziewit, Ł.; Napiórkowska, A.; Augustynowicz-Kopeć, E.; Zwolska, Z.; Bielecki, J. Screening for streptomycin resistance-conferring mutations in Mycobacterium tuberculosis clinical isolates from Poland. PLoS One, 2014, 9(6), e100078.
[http://dx.doi.org/10.1371/journal.pone.0100078] [PMID: 24937123]
[10]
Springer, B.; Kidan, Y.G.; Prammananan, T.; Ellrott, K.; Böttger, E.C.; Sander, P. Mechanisms of streptomycin resistance: selection of mutations in the 16S rRNA gene conferring resistance. Antimicrob. Agents Chemother., 2001, 45(10), 2877-2884.
[http://dx.doi.org/10.1128/AAC.45.10.2877-2884.2001] [PMID: 11557484]
[11]
Li, J.; Gao, X.; Luo, T.; Wu, J.; Sun, G.; Liu, Q.; Jiang, Y.; Zhang, Y.; Mei, J.; Gao, Q. Association of gyrA/B mutations and resistance levels to fluoroquinolones in clinical isolates of Mycobacterium tuberculosis. Emerg. Microbes Infect., 2014, 3(3), e19.
[http://dx.doi.org/10.1038/emi.2014.21] [PMID: 26038513]
[12]
Yokoyama, K.; Kim, H.; Mukai, T.; Matsuoka, M.; Nakajima, C.; Suzuki, Y. Impact of amino acid substitutions in B subunit of DNA gyrase in Mycobacterium leprae on fluoroquinolone resistance. PLoS Negl. Trop. Dis., 2012, 6(10), e1838.
[http://dx.doi.org/10.1371/journal.pntd.0001838] [PMID: 23071850]
[13]
Wang, X.; Jiao, J.; Xu, W.; Chai, X.; Li, Z.; Wang, Q. A simple, rapid and economic method for detecting multidrug-resistant tuberculosis. Braz. J. Infect. Dis., 2013, 17(6), 667-671.
[http://dx.doi.org/10.1016/j.bjid.2013.04.008] [PMID: 24029439]
[14]
Kant, L. Translational research in tuberculosis: bridging the long journey from bench to bedside. Indian J. Tuberc., 2005, 52(3), 117-119.
[15]
Ye, J.; Coulouris, G.; Zaretskaya, I.; Cutcutache, I.; Rozen, S.; Madden, T.L. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics, 2012, 13, 134.
[http://dx.doi.org/10.1186/1471-2105-13-134] [PMID: 22708584]
[16]
Rice, P.; Longden, I.; Bleasby, A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet., 2000, 16(6), 276-277.
[http://dx.doi.org/10.1016/S0168-9525(00)02024-2] [PMID: 10827456]
[17]
Arjomandzadegan, M.; Titov, L.; Farnia, P.; Owlia, P.; Ranjbar, R.; Sheikholeslami, F.; Surkova, L. Molecular detection of fluoroquinolone resistance-associated gyrA mutations in ofloxacin-resistant clinical isolates of Mycobacterium tuberculosis from Iran and Belarus. Int. J. Mycobacteriol., 2016, 5(3), 299-305.
[http://dx.doi.org/10.1016/j.ijmyco.2016.07.004] [PMID: 27847014]
[18]
Nisha, J.; Shanthi, V. Characterization of Ofloxacin Interaction with Mutated (A91V) Quinolone Resistance Determining Region of DNA Gyrase in Mycobacterium Leprae through Computational Simulation. Cell Biochem. Biophys., 2018, 76(1-2), 125-134.
[http://dx.doi.org/10.1007/s12013-017-0822-5] [PMID: 28822069]
[19]
Gasteiger, E.; Gattiker, A.; Hoogland, C.; Ivanyi, I.; Appel, R.D.; Bairoch, A. ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res., 2003, 31(13), 3784-3788.
[http://dx.doi.org/10.1093/nar/gkg563] [PMID: 12824418]
[20]
Arnold, K.; Bordoli, L.; Kopp, J.; Schwede, T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics, 2006, 22(2), 195-201.
[http://dx.doi.org/10.1093/bioinformatics/bti770] [PMID: 16301204]
[21]
Biasini, M.; Bienert, S.; Waterhouse, A.; Arnold, K.; Studer, G.; Schmidt, T.; Kiefer, F.; Gallo Cassarino, T.; Bertoni, M.; Bordoli, L.; Schwede, T. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res., 2014, 42(Web Server issue), W252-8.
[http://dx.doi.org/10.1093/nar/gku340] [PMID: 24782522]
[22]
Guex, N.; Peitsch, M.C.; Schwede, T. Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective. Electrophoresis, 2009, 30(S1)(Suppl. 1), S162-S173.
[http://dx.doi.org/10.1002/elps.200900140] [PMID: 19517507]
[23]
Kiefer, F.; Arnold, K.; Künzli, M.; Bordoli, L.; Schwede, T. The SWISS-MODEL Repository and associated resources. Nucleic Acids Res., 2009, 37(Database issue)(Suppl. 1), D387-D392.
[http://dx.doi.org/10.1093/nar/gkn750] [PMID: 18931379]
[24]
Low, H.H.; Löwe, J. A bacterial dynamin-like protein. Nature, 2006, 444(7120), 766-769.
[http://dx.doi.org/10.1038/nature05312] [PMID: 17122778]
[25]
Blower, T.R.; Williamson, B.H.; Kerns, R.J.; Berger, J.M. Crystal structure and stability of gyrase-fluoroquinolone cleaved complexes from Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA, 2016, 113(7), 1706-1713.
[http://dx.doi.org/10.1073/pnas.1525047113] [PMID: 26792525]
[26]
pubchem.ncbi.nlm.nih.gov/compound/ethambutol Ethambutol, compound summary. Available from:
[27]
pubchem.ncbi.nlm.nih.gov/compound/ofloxacin Ofloxacin, compound summary. Available from:
[28]
Bikadi, Z.; Hazai, E. Application of the PM6 semi-empirical method to modeling proteins enhances docking accuracy of AutoDock. J. Cheminform., 2009, 1(1), 15.
[http://dx.doi.org/10.1186/1758-2946-1-15] [PMID: 20150996]
[29]
Rufai, S.B.; Singh, J.; Kumar, P.; Mathur, P.; Singh, S. Association of gyrA and rrs gene mutations detected by MTBDRsl V1 on Mycobacterium tuberculosis strains of diverse genetic background from India. Sci. Rep., 2018, 8(1), 9295.
[http://dx.doi.org/10.1038/s41598-018-27299-z] [PMID: 29915257]
[30]
Arjomandzadegan, M.; Gravand, S. Analysis of rpsL and rrs genes mutations related to streptomycin resistance in Mdr and Xdr clinical isolates of Mycobacterium tuberculosis. Tuberk. Toraks, 2015, 63(4), 235-242.
[http://dx.doi.org/10.5578/tt.6474] [PMID: 26963306]
[31]
Maruri, F.; Sterling, T.R.; Kaiga, A.W.; Blackman, A.; van der Heijden, Y.F.; Mayer, C.; Cambau, E.; Aubry, A. A systematic review of gyrase mutations associated with fluoroquinolone-resistant Mycobacterium tuberculosis and a proposed gyrase numbering system. J. Antimicrob. Chemother., 2012, 67(4), 819-831.
[http://dx.doi.org/10.1093/jac/dkr566] [PMID: 22279180]
[32]
Evans, J.; Segal, H. Novel multiplex allele-specific PCR assays for the detection of resistance to second-line drugs in Mycobacterium tuberculosis. J. Antimicrob. Chemother., 2010, 65(5), 897-900.
[http://dx.doi.org/10.1093/jac/dkq047] [PMID: 20185419]
[33]
Programme, R.N.T.C. TB India 2016 annual status report; India Offset Press, 2016.
[34]
Sreevatsan, S.; Pan, X.; Stockbauer, K.E.; Connell, N.D.; Kreiswirth, B.N.; Whittam, T.S.; Musser, J.M. Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc. Natl. Acad. Sci. USA, 1997, 94(18), 9869-9874.
[http://dx.doi.org/10.1073/pnas.94.18.9869] [PMID: 9275218]
[35]
O’Brien, R.J.; Nunn, P.P. The need for new drugs against tuberculosis. Obstacles, opportunities, and next steps. Am. J. Respir. Crit. Care Med., 2001, 163(5), 1055-1058.
[http://dx.doi.org/10.1164/ajrccm.163.5.2007122] [PMID: 11316634]
[36]
Almeida Da Silva, P.E.; Palomino, J.C. Molecular basis and mechanisms of drug resistance in Mycobacterium tuberculosis: classical and new drugs. J. Antimicrob. Chemother., 2011, 66(7), 1417-1430.
[http://dx.doi.org/10.1093/jac/dkr173] [PMID: 21558086]
[37]
Hazbón, M.H.; Motiwala, A.S.; Cavatore, M.; Brimacombe, M.; Whittam, T.S.; Alland, D. Convergent evolutionary analysis identifies significant mutations in drug resistance targets of Mycobacterium tuberculosis. Antimicrob. Agents Chemother., 2008, 52(9), 3369-3376.
[http://dx.doi.org/10.1128/AAC.00309-08] [PMID: 18591265]
[38]
Aldred, K.J.; McPherson, S.A.; Turnbough, C.L., Jr; Kerns, R.J.; Osheroff, N. Topoisomerase IV-quinolone interactions are mediated through a water-metal ion bridge: mechanistic basis of quinolone resistance. Nucleic Acids Res., 2013, 41(8), 4628-4639.
[http://dx.doi.org/10.1093/nar/gkt124] [PMID: 23460203]

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