N-methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis

Abstract

The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-β-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.

Keywords: antimicrobial resistance; arabinogalactan synthesis; methyltransferase; transcription factor.

Fig. 1.

Activity of 14 and the Rv2887 mutations identified in 14-resistant Mtb clones. (A) Structure of 14. (B) Kinetics of bactericidal activity of 14 against Mtb as measured by cfu counts on postexposure days 1, 3, 5, 8, and 15. (C) Phyre 2 generated homology model of Rv2887 highlighting the 16 unique mutations identified in Mtb clones resistant to 14. The secondary structure elements are color coded as described: blue, α1 helix; light blue, α2 helix; light green, α3 helix; dark green, α4 helix; orange, β1–loop–β2 region; dark red, α5 helix; and red, α6 helix. The dashed line demarcates the predicted dimerization and DNA-binding domains.

Fig. 2.

Role of rv2887 in resistance to 14 and genes regulated by Rv2887 in Mtb. (A) Fold changes in IC₉₀ of 14 relative to wild-type Mtb H37Rv (WT) are shown. The strains tested are two clones resistant to 14, 1A and 8A; wild-type strains carrying the two Rv2887 mutations, R81Q and C-terminal deletion, found in clones 1A and 8A; and three strains, WT, 1A, and 8A, that express rv2887 constitutively (WT o/e rv2887, 1A o/e rv2887, and 8A o/e rv2887). o/e, overexpression. (B) Relative change in expression of rv0560c, rv0559c, rv0558, and rv0557 in wild-type Mtb H37Rv (WT) treated with 3.9 μM of 14 for 4 h and two resistant clones, 1A and 8A, that were vehicle-treated for 4 h. The baseline of 1 indicates expression level in Mtb H37Rv treated with vehicle control. (C) Native PAGE gels of wild-type Rv2887 or mutant Rv2887, with the point mutation R81Q, incubated with a 70-bp DNA sequence spanning the promoter of rv0560c (560c-prom).

Fig. 3.

Role of Rv0560c in resistance of Mtb toward 14. (A) IC₉₀ curve of 14 and rifampin (RIF) against wild-type Mtb strain that constitutively expresses rv0560c (pMV261-rv0560c) or the vector control strain (pMV261). (B) Michaelis–Menten kinetics of the SAM-dependent methylation of 14 by wild-type Rv0560c (WT) protein and S140A-Rv0560c protein in the absence of coupling enzymes. Table lists the steady-state kinetic parameters of the reactions. (C) Overlay of the 1D ¹H spectrum and the NOESY spectra of me-14 with a schematic highlighting the site of methylation. (D) Activity of purified methylated 14 (me-14), 14, and the inactive analog, 14-IA, which is methylated at the N-5 position. (E) Normalized (relative to wild-type Mtb H37Rv, Rv) quantification of 14 and me-14 in the two 14-resistant clones, 1A and 8A, in the wild-type strain that constitutively expresses rv0560c (Rv O/E rv0560c), in Δrv0560c, its wild-type background strain, H37Rv London Pride (LP), and Δrv0560c complemented with a constitutive expression vector for rv0560c (Δrv0560c O/E rv0560c). All strains were treated with 7.8 μM 14 for 24 h. (*P < 0.05; **P value < 0.01, with two-tailed unpaired t test analysis.)

Fig. 4.

Compound 14 targets Mtb DprE1. (A) IC₉₀ curve of 14 against two resistant clones, R Clone 1 and 2, isolated in the Δrv0560c background, which is referred to as WT-rv0560c KO. Activity against the recombineered H37Rv strain carrying only the DprE1 P116S point mutation (P116S DprE1) and its wild-type control strain (WT-reco) are also shown. (B) Concentration-dependent activity of 14 against DprE1 as detected by a fluorescence-based assay is depicted by grey symbols. Activity of 14 against DprE1 after preincubation with 10 μM Rv0560c and 300 μM SAM for 6.5 h is depicted by clear symbols, and the control without SAM is shown by black symbols. (C) Activity of 14, the known DprE1 inhibitors TCA1 and BTZ-043, ethambutol (EMB), rifampin (RIF), and isoniazid (INH) against Mtb H37Rv strains carrying the listed point mutations in DprE1. Mutations highlighted in red were introduced into the wild-type Mtb strain by oligonucleotide-based recombineering.