Medical Microbiology and Immunology

, Volume 204, Issue 2, pp 193–203 | Cite as

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

  • Sundeep Chaitanya Vedithi
  • Mallika Lavania
  • Manoj Kumar
  • Punit Kaur
  • Ravindra P. Turankar
  • Itu Singh
  • Astha Nigam
  • Utpal Sengupta
Original Investigation
First Online:
Received:
Accepted:

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.

Keywords

Rifampin resistance Mycobacterium leprae Point mutations Multidrug therapy Relapse Leprosy 
This is a preview of subscription content, log in to check access.

Notes

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.

References

  1. 1.
    Walker SL, Lockwood DN (2006) The clinical and immunological features of leprosy. Br Med Bull 77–78:103–121CrossRefPubMedGoogle Scholar
  2. 2.
    Williams DL, Gillis TP (2004) Molecular detection of drug resistance in Mycobacterium leprae. Lepr Rev 75(2):118–130PubMedGoogle Scholar
  3. 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):3635–3639CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Singal A, Sonthalia S (2013) Leprosy in post-elimination era in India: difficult journey ahead. Indian J Dermatol 58(6):443–446CrossRefPubMedCentralPubMedGoogle Scholar
  5. 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):256–259PubMedGoogle Scholar
  6. 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):742–753CrossRefPubMedCentralPubMedGoogle Scholar
  7. 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):121–126CrossRefPubMedGoogle Scholar
  8. 8.
    Donoghue HD, Holton J, Spigelman M (2001) PCR primers that can detect low levels of Mycobacterium leprae DNA. J Med Microbiol 50(2):177–182PubMedGoogle Scholar
  9. 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):e1739CrossRefPubMedCentralPubMedGoogle Scholar
  10. 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):712–719CrossRefPubMedGoogle Scholar
  11. 11.
    Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234(3):779–815CrossRefPubMedGoogle Scholar
  12. 12.
    Fiser A, Do RK, Sali A (2000) Modeling of loops in protein structures. Protein Sci 9(9):1753–1773CrossRefPubMedCentralPubMedGoogle Scholar
  13. 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):283–291CrossRefGoogle Scholar
  14. 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):494–506CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Feyfant E, Sali A, Fiser A (2007) Modeling mutations in protein structures. Protein Sci 16(9):2030–2041CrossRefPubMedCentralPubMedGoogle Scholar
  16. 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):289–307CrossRefPubMedGoogle Scholar
  17. 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):901–912CrossRefPubMedGoogle Scholar
  18. 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):187–217CrossRefGoogle Scholar
  19. 19.
    Momany FA, Rone R (1992) Validation of the general purpose QUANTA ®3.2/CHARMm® force field. J Comput Chem 13(7):888–900CrossRefGoogle Scholar
  20. 20.
    Calvori C, Frontali L, Leoni L, Tecce G (1965) Effect of rifamycin on protein synthesis. Nature 207(995):417–418CrossRefPubMedGoogle Scholar
  21. 21.
    Honore N, Cole ST (1993) Molecular basis of rifampin resistance in Mycobacterium leprae. Antimicrob Agents Chemother 37(3):414–418CrossRefPubMedCentralPubMedGoogle Scholar
  22. 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):14820–14825CrossRefPubMedCentralPubMedGoogle Scholar
  23. 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):39–45CrossRefPubMedGoogle Scholar
  24. 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):96–101CrossRefGoogle Scholar
  25. 25.
    Williams DL, Gillis TP (2012) Drug-resistant leprosy: monitoring and current status. Lepr Rev 83(3):269–281PubMedGoogle Scholar
  26. 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):107–110CrossRefPubMedCentralPubMedGoogle Scholar
  27. 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):517–528CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Sundeep Chaitanya Vedithi
    • 1
  • Mallika Lavania
    • 1
  • Manoj Kumar
    • 2
  • Punit Kaur
    • 2
  • Ravindra P. Turankar
    • 1
  • Itu Singh
    • 1
  • Astha Nigam
    • 1
  • Utpal Sengupta
    • 1
  1. 1.Stanley Browne LaboratoryThe Leprosy Mission Community HospitalNew DelhiIndia
  2. 2.Department of BiophysicsAll India Institute of Medical SciencesNew DelhiIndia

Personalised recommendations