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A report of rifampin-resistant leprosy from northern and eastern India: identification and in silico analysis of molecular interactions

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Abstract

Presence of point mutations within the drug resistance determining regions of Mycobacterium leprae (M. leprae) genome confers molecular basis of drug resistance to dapsone, rifampin and ofloxacin in leprosy. This study is focused on the identification of mutations within the rpoB gene region of M. leprae that are specific for rifampin interaction, and further in silico analysis was carried out to determine the variations in the interactions. DNA and RNA were isolated from slit skin scrapings of 60 relapsed leprosy patients. PCR targeting rpoB gene region and amplicon sequencing was performed to determine point mutations. mRNA expression levels of rpoB and high-resolution melt analysis of mutants were performed using Rotor Gene Q Realtime PCR. Molecular docking was performed using LigandFit Software. Ten cases having point mutations within the rpoB gene region were identified and were clinically confirmed to be resistant to rifampin. A new mutation at codon position Gln442His has been identified. There is a 9.44-fold upregulation in the mRNA expression of rpoB gene in mutant/resistant samples when compared with the wild/sensitive samples. In silico docking analysis of rifampin with wild-type and Gln442His mutant RpoB proteins revealed a variation in the hydrogen-bonding pattern leading to a difference in the total interaction energy and conformational change at position Asp441. These preliminary downstream functional observations revealed that the presence of point mutations within the rifampin resistance determining regions of rpoB gene plays a vital role in conferring genetic and molecular basis of resistance to rifampin in leprosy.

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References

  1. Walker SL, Lockwood DN (2006) The clinical and immunological features of leprosy. Br Med Bull 77–78:103–121

    Article  PubMed  Google Scholar 

  2. Williams DL, Gillis TP (2004) Molecular detection of drug resistance in Mycobacterium leprae. Lepr Rev 75(2):118–130

    PubMed  Google Scholar 

  3. Maeda S, Matsuoka M, Nakata N, Kai M, Maeda Y, Hashimoto K et al (2001) Multidrug resistant Mycobacterium leprae from patients with leprosy. Antimicrob Agents Chemother 45(12):3635–3639

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Singal A, Sonthalia S (2013) Leprosy in post-elimination era in India: difficult journey ahead. Indian J Dermatol 58(6):443–446

    Article  PubMed Central  PubMed  Google Scholar 

  5. Lopez-Roa RI, Fafutis-Morris M, Masanori M (2006) A drug-resistant leprosy case detected by DNA sequence analysis from a relapsed Mexican leprosy patient. Rev Latinoam Microbiol 48(3–4):256–259

    PubMed  Google Scholar 

  6. Li W, Matsuoka M, Kai M, Thapa P, Khadge S, Hagge DA et al (2012) Real-time PCR and high-resolution melt analysis for rapid detection of Mycobacterium leprae drug resistance mutations and strain types. J Clin Microbiol 50(3):742–753

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Turankar RP, Lavania M, Singh M, Siva Sai KS, Jadhav RS (2012) Dynamics of Mycobacterium leprae transmission in environmental context: deciphering the role of environment as a potential reservoir. Infect Genet Evol 12(1):121–126

    Article  PubMed  Google Scholar 

  8. Donoghue HD, Holton J, Spigelman M (2001) PCR primers that can detect low levels of Mycobacterium leprae DNA. J Med Microbiol 50(2):177–182

    CAS  PubMed  Google Scholar 

  9. Cambau E, Chauffour-Nevejans A, Tejmar-Kolar L, Matsuoka M, Jarlier V (2012) Detection of antibiotic resistance in leprosy using GenoType LepraeDR, a novel ready-to-use molecular test. PLoS Negl Trop Dis 6(7):e1739

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Vassylyev DG, Sekine S, Laptenko O, Lee J, Vassylyeva MN, Borukhov S et al (2002) Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 A resolution. Nature 417(6890):712–719

    Article  CAS  PubMed  Google Scholar 

  11. Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234(3):779–815

    Article  CAS  PubMed  Google Scholar 

  12. Fiser A, Do RK, Sali A (2000) Modeling of loops in protein structures. Protein Sci 9(9):1753–1773

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 26(2):283–291

    Article  CAS  Google Scholar 

  14. Spassov VZ, Yan L, Flook PK (2007) The dominant role of side-chain backbone interactions in structural realization of amino acid code. ChiRotor: a side-chain prediction algorithm based on side-chain backbone interactions. Protein Sci 16(3):494–506

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Feyfant E, Sali A, Fiser A (2007) Modeling mutations in protein structures. Protein Sci 16(9):2030–2041

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Venkatachalam CM, Jiang X, Oldfield T, Waldman M (2003) LigandFit: a novel method for the shape-directed rapid docking of ligands to protein active sites. J Mol Graph Model 21(4):289–307

    Article  CAS  PubMed  Google Scholar 

  17. Campbell EA, Korzheva N, Mustaev A, Murakami K, Nair S, Goldfarb A et al (2001) Structural mechanism for rifampicin inhibition of bacterial rna polymerase. Cell 104(6):901–912

    Article  CAS  PubMed  Google Scholar 

  18. Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M (1983) CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J Comput Chem 4(2):187–217

    Article  CAS  Google Scholar 

  19. Momany FA, Rone R (1992) Validation of the general purpose QUANTA ®3.2/CHARMm® force field. J Comput Chem 13(7):888–900

    Article  CAS  Google Scholar 

  20. Calvori C, Frontali L, Leoni L, Tecce G (1965) Effect of rifamycin on protein synthesis. Nature 207(995):417–418

    Article  CAS  PubMed  Google Scholar 

  21. Honore N, Cole ST (1993) Molecular basis of rifampin resistance in Mycobacterium leprae. Antimicrob Agents Chemother 37(3):414–418

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Feklistov A, Mekler V, Jiang Q, Westblade LF, Irschik H, Jansen R et al (2008) Rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNA polymerase active center. Proc Natl Acad Sci USA 105(39):14820–14825

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Cambau E, Bonnafous P, Perani E, Sougakoff W, Ji B, Jarlier V (2002) Molecular detection of rifampin and ofloxacin resistance for patients who experience relapse of multibacillary leprosy. Clin Infect Dis 34(1):39–45

    Article  CAS  PubMed  Google Scholar 

  24. de Knegt GJ, Bruning O, ten Kate MT, de Jong M, van Belkum A, Endtz HP et al (2013) Rifampicin-induced transcriptome response in rifampicin-resistant Mycobacterium tuberculosis. Tuberculosis (Edinb) 93(1):96–101

    Article  Google Scholar 

  25. Williams DL, Gillis TP (2012) Drug-resistant leprosy: monitoring and current status. Lepr Rev 83(3):269–281

    PubMed  Google Scholar 

  26. Heep M, Brandstatter B, Rieger U, Lehn N, Richter E, Rusch-Gerdes S et al (2001) Frequency of rpoB mutations inside and outside the cluster I region in rifampin-resistant clinical Mycobacterium tuberculosis isolates. J Clin Microbiol 39(1):107–110

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Verhagen CE, de Boer T, Smits HH, Verreck FA, Wierenga EA, Kurimoto M et al (2000) Residual type 1 immunity in patients genetically deficient for interleukin 12 receptor beta1 (IL-12Rbeta1): evidence for an IL-12Rbeta1-independent pathway of IL-12 responsiveness in human T cells. J Exp Med 192(4):517–528

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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Acknowledgments

At the outset, we extend our special thanks to all the participants who volunteered for the study. We would like to thank Dr. Sunil Anand, Director of The Leprosy Mission (TLM) Trust India, Dr. Annamma John—Research Coordinator—TLM and the entire associated medical and research staff who have been a continuous source of support throughout the study. Our Special thanks to all the research staff at the Bio-Medical Informatics Centre, supported by ICMR (Indian Council of Medical Research), Department of Biophysics, All India Institute of Medical Sciences, New Delhi who have contributed to the Molecular Docking Experiments. Finally, we would like to thank all the other support staff at Stanley Browne Laboratory and Department of Biophysics—All India Institute of Medical Sciences for their immense support and encouragement throughout the work.

Conflict of interest

All the authors declare that they do not have any conflict of interest.

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Authors and Affiliations

  1. Stanley Browne Laboratory, The Leprosy Mission Community Hospital, Nand Nagri, New Delhi, 110093, India

    Sundeep Chaitanya Vedithi, Mallika Lavania, Ravindra P. Turankar, Itu Singh, Astha Nigam & Utpal Sengupta

  2. Department of Biophysics, All India Institute of Medical Sciences, Aurobindo Marg, Ansari Nagar, New Delhi, 110029, India

    Manoj Kumar & Punit Kaur

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  1. Sundeep Chaitanya Vedithi
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  2. Mallika Lavania
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  3. Manoj Kumar
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  4. Punit Kaur
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  5. Ravindra P. Turankar
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  6. Itu Singh
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  7. Astha Nigam
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  8. Utpal Sengupta
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Correspondence to Utpal Sengupta.

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Vedithi, S.C., Lavania, M., Kumar, M. et al. A report of rifampin-resistant leprosy from northern and eastern India: identification and in silico analysis of molecular interactions. Med Microbiol Immunol 204, 193–203 (2015). https://doi.org/10.1007/s00430-014-0354-1

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  • DOI: https://doi.org/10.1007/s00430-014-0354-1

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