A trans-Translation Inhibitor is Potentiated by Zinc and Kills Mycobacterium tuberculosis and Nontuberculous Mycobacteria

Abstract

Mycobacterium tuberculosis poses a serious challenge for human health, and new antibiotics with novel targets are needed. Here we demonstrate that an acylaminooxadiazole, MBX-4132, specifically inhibits the trans-translation ribosome rescue pathway to kill M. tuberculosis. Our data demonstrate that MBX-4132 is bactericidal against multiple pathogenic mycobacterial species and kills M. tuberculosis in macrophages. We also show that acylaminooxadiazole activity is antagonized by iron but is potentiated by zinc. Our transcriptomic data reveal dysregulation of multiple metal homeostasis pathways after exposure to MBX-4132. Furthermore, we see differential expression of genes related to zinc sensing and efflux when trans-translation is inhibited. Taken together, these data suggest that there is a link between disturbing intracellular metal levels and acylaminooxadiazole-mediated inhibition of trans-translation. These findings provide an important proof-of-concept that trans-translation is a promising antitubercular drug target.

Keywords: Mycobacterium tuberculosis; antibiotic resistance; metal homeostasis; nontuberculous mycobacteria; oxadiazole; trans-translation.

Fig 1.. MBX-4132 inhibits M. tuberculosis trans-translation in vitro.

A) Structures of KKL-35 and MBX-4132. B) A gene encoding DHFR without a stop codon (DHFR-ns) was expressed in the presence of M. tuberculosis tmRNA-SmpB and varying concentrations of MBX-4132. Synthesized protein was detected by incorporation of ³⁵S-methionine followed by SDS-PAGE and phosphorimaging. Bands corresponding to DHFR-ns with tagged DHFR, which is the product of trans-translation are indicated, and the average percentage of DHFR protein found in the tagged band for three repeats is shown with the standard deviation. C) Data from gels as in (B) were plotted and fit with a sigmoidal function to determine the IC₅₀. D) in vitro translation was assayed from the expression of a gene encoding DHFR with a stop codon (DHFR-stop) in the presence of DMSO, 20 μM chloramphenicol (CHL), or 20 μM MBX-4132, and a representative experiment is shown. The percentage of DHFR with respect to the amount in the DMSO-treated control is shown as the average from two independent repeats with the standard deviation.

Fig 2.. tmRNA-SmpB competes with MBX-4132 in vitro.

A) in vitro trans-translation assays as in Figure 1 containing different concentrations of tmRNA-SmpB and MBX-4132. B) Reactions treated with 8.2 μM tmRNA-SmpB suppressed the inhibition of trans-translation by 10 μM MBX-4132. The inhibition was rescued by 20 μM MBX-4132. Data from at least two experiments are shown as the average with error bars indicating the standard deviation.

Fig 3.. Iron antagonizes the activity of MBX-4132.

A) in vitro trans-translation assays as in Figure 1 containing 150 μM Fe₂(SO₄)₃, 150 μM TPEN, 15 μM MBX-4132, or 15 μM KKL-35 as indicated. The average percentage tagging from two independent reactions is shown with the standard deviation. B) Data from gels as in (A) were plotted to show the average from two experiments with error bars indicating the standard deviation. C) in vitro trans-translation assays as in Figure 1 containing 150 μM Fe₂(SO₄)₃, 150 μM FeSO₄, 15 μM MBX-4132, or KKL-35 as indicated. D) Data from gels as in (C) were plotted to show the average from two experiments with error bars indicating the standard deviation.

Fig 4.. Transcriptional responses of M. tuberculosis H37Rv to MBX-4132 treatment and tmRNA knockdown.

A) Venn diagrams showing numbers of genes significantly up- and down-regulated in the MBX-4132 study, the ssr KD study, or both. B) Differential expression of genes in response to MBX-4132 exposure, with vertical and horizontal dashed lines representing log₂-fold change cutoff of ±1 and adjusted p-value cutoff of 0.05, respectively. C) Growth of M. tuberculosis H37Rv ssr KD and NTC strains upon induction in HZMM. Data represents the average OD₆₀₀ of biological triplicates with error bars denoting the standard deviation. D) Inhibition of MBX-4132 against M. tuberculosis H37Rv ssr KD and NTC strains after 21 days of treatment in MM. Data represent geometric means and geometric standard deviations for 3 biological replicates. E) Differential expression of genes as a result of ssr KD, with dashed lines denoting cutoffs as described in (B).

Fig 5.. Changes of fitness of a saturated M. tuberculosis H37Rv transposon library after MBX-4132 treatment.

A) Volcano plot showing insertions conferring significant gain (red) or loss (blue) of fitness as determined by the TRANSIT resampling method. Horizontal and vertical dash lines denote the adjusted p-value and log₂-fold change thresholds of 0.05 and ±1, respectively. B) Fitness of each transposon insertion mutant calculated by comparing its expansion factor relative to the rest of the population in the absence and presence of MBX-4132. Select mutants with insertions in genes of interest are noted. Dashed line denotes the line of correlation for untreated and MBX-4132 treated mutants showing comparable fitness values, dotted lines denote cutoff for ±0.2 gain or loss of fitness.

Fig 6.. Susceptibility of M. tuberculosis H37Rv ΔC− to MBX-4132.

M. tuberculosis H37Rv (wild type, black) and the ΔC− mutant (light blue) were cultured in (A) HZMM and (B) MM and exposed to MBX-4132 for 14 days. Data represent geometric means and geometric standard deviations for 3 biological replicates.

Fig 7.. M. tuberculosis H37Rv is susceptible to KKL-35 and MBX-4132 killing in RAW 264.7 macrophages.

A-B) Resting and C) IFN-γ activated RAW 264.7 macrophages were infected with M. tuberculosis H37Rv and subsequently treated with (A) KKL-35 or (B and C) MBX-4132 with isoniazid (INH) as a control. D) Intracellular M. tuberculosis survival under all 3 KKL-35 or MBX-4132 treatment conditions. Data represent geometric means with error bars indicating the geometric standard deviation for 3 biological replicates.