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.

Figure 1

[a] RNAP holoenzyme of M. leprae in complex with synthetic nucleic acid scaffold and rifampin. [b] Heterocyclic structure of rifampin (orange) with the piperazine moiety and naphthoquinone core that is spanned by an aliphatic chain.

Figure 2

[a] Interatomic interactions of rifampin (orange) with the β subunit of RNAP. All the interacting and proximal residues were depicted. The black dashed lines indicate hydrogen bonding interactions and green dashed lines indicate hydrophobic interactions. [b] Hydrogen bonds (black dashed lines) between rifampin and sidechains of R454, R465 and N493, and the mainchain of Q438 and F439.

Figure 3

[a] Interatomic interactions of D441 (green) with the residue environment in wild-type complex. D441, located at a distance of 3.43 Å from rifampin (orange), makes hydrogen bonds with side chains of S447, R454 and N443 (black dashed lines), a carbonyl interaction with G448 (red dashed line) and proximal hydrophobic interactions with rifampin, and H451 (green dashed lines). [b] Mutant Y441 interactions with the residue environment. Y441 makes hydrogen bonds (black dashed lines), hydrophobic interactions (green dashed lines), carbon-π interactions (grey dashed lines) and donor-π interactions (blue dashed lines). [c] Mutant V441 interactions with the residue environment. Hydrogen bonding, hydrophobic and carbonyl interactions are depicted.

Figure 4

[a] Interatomic interactions of S437 (green) with the residue environment in the wild-type complex. Hydrogen bonding interactions with S434, G432 and R173 (black dashed lines) were depicted. [b] Mutant L437 with hydrogen bonding and hydrophobic interactions with the surrounding residues.

Figure 5

[a] Interactions of H476 with the residue environment in the wild-type complex include hydrogen bonds ((black dashed lines), hydrophobic interactions (green dashed lines), ionic interactions (yellow dashed lines), donor-π interactions (blue dashed lines), carbon-π interactions (grey dashed lines). [b] Mutant R476 interactions with the residue environment.

Figure 6

[a–d] Difference graphs indicating distortion in RMSD (in Å) for each normal mode in the wild-type and in each of the mutant models.