Structural Implications of Mutations Conferring Rifampin Resistance in Mycobacterium leprae

Abstract

The rpoB gene encodes the β subunit of RNA polymerase holoenzyme in Mycobacterium leprae (M. leprae). Missense mutations in the rpoB gene were identified as etiological factors for rifampin resistance in leprosy. In the present study, we identified mutations corresponding to rifampin resistance in relapsed leprosy cases from three hospitals in southern India which treat leprosy patients. DNA was extracted from skin biopsies of 35 relapse/multidrug therapy non-respondent leprosy cases, and PCR was performed to amplify the 276 bp rifampin resistance-determining region of the rpoB gene. PCR products were sequenced, and mutations were identified in four out of the 35 cases at codon positions D441Y, D441V, S437L and H476R. The structural and functional effects of these mutations were assessed in the context of three-dimensional comparative models of wild-type and mutant M. leprae RNA polymerase holoenzyme (RNAP), based on the recently solved crystal structures of RNAP of Mycobacterium tuberculosis, containing a synthetic nucleic acid scaffold and rifampin. The resistance mutations were observed to alter the hydrogen-bonding and hydrophobic interactions of rifampin and the 5′ ribonucleotide of the growing RNA transcript. This study demonstrates that rifampin-resistant strains of M. leprae among leprosy patients in southern India are likely to arise from mutations that affect the drug-binding site and stability of RNAP.

Computational Saturation Mutagenesis to predict structural consequences of systematic mutations on protein stability and rifampin interactions in the β subunit of RNA polymerase inMycobacterium leprae

“…Comparative modelling, quality assessment and model refinement: A model for RNAP holoenzyme of M. leprae was built using Modeller 9.21 based using templates from M. tuberculosis (PDB ID:5UH5 (96% identity, 3.8A resolution) containing RNAP and nucleic acid scaffold with DNA and three nucleotides of RNA complementary to the template DNA strand and PDB ID: 5UHC (96% identity, 4.0A resolution) containing all the elements similar to 5UH5 and rifampin) as described earlier by us (Vedithi et al, 2018). The quality of the generated model was assessed using Molprobity (Davis et al, 2004) and atomic clashes were removed by minimizing the energy of the model by 100 steps using Steepest Decent (step size = 0.02 A ) and by 10 steps (step size = 0.02A ) using conjugate gradient algorithms.…”

“…Substitutions to arginine predominate mutations that destabilize  subunit-rifampin affinity: Systematic mutations in the set of 70 residues that lie 10 A from the rifampin binding site reveal that highly destabilizing mutations are primarily arginine and glutamate substitutions (mCSM-lig). In the binding site R173, R454, R465 and R613 form hydrogen bonds and a network of interatomic interactions with rifampin that stabilize the molecule in the binding site (Vedithi et al, 2018). Introduction of additional arginine residues by mutations may influence the stability and orientation of rifampin in the binding site.…”

“…We have noted that any mutation at rifampin-interacting residues S437, H451, R454, S456, L458, G459, R465, P489, P492 and N493 destabilize protein ligand affinity (mCSM-lig). Of these we have chosen wild-type residues H451 and P489, which are experimentally identified mutations, and wild-type residues S437 and G459, which are computationally predicted (only one mutation was experimentally identified at residue position S437L as reported by us earlier (Vedithi et al, 2018), and this has destabilizing effects on the overall stability and affinity to rifampin).…”

“…While mutations within the -subunit of RNA polymerase contribute to clinical resistance to rifampin, the associated structural changes can complicate the transcription process in bacteria by modulating various complex physiological processes (Vedithi et al, 2018), the knowledge of which is essential for novel drug discovery or alternative therapies to treat rifampin resistant strains of M. leprae. In the absence of an artificial culture system to propagate and study the molecular mechanisms of resistance, it is exceptionally challenging to define an experimental phenotype for rifampin resistance in leprosy.…”

“…Mutations contribute to disruption of protein-ligand and protein-nucleic acid interactions resulting in drug resistance in mycobacterial diseases (Portelli et al, 2018;Karmakar et al, 2018). Changes in affinity for the ligand can result from both orthosteric and allosteric mechanism leading to various resistance phenotypes (Vedithi et al, 2018).The -subunit of RNA Polymerase in M. leprae is encoded by the rpoB gene (ML1891) whose product is 1178 amino acids in length. The rifampin resistance determining region (RRDR) is located between residue positions 410 and 480.…”