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A nucleotidyltransferase toxin inhibits growth of Mycobacterium tuberculosis through inactivation of tRNA acceptor stems

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Science Advances  29 Jul 2020:
Vol. 6, no. 31, eabb6651
DOI: 10.1126/sciadv.abb6651
  • Fig. 1 Analysis of the four TA systems with NTase-like toxins encoded by the M. tuberculosis genome.

    (A) Scaled representation of the four M. tuberculosis TA systems containing NTase-like toxin genes with original and revised nomenclature (left), and corresponding toxicity and antitoxicity assays in M. smegmatis (right). For toxicity and antitoxicity assays, cotransformants of M. smegmatis mc2 155 containing pGMC-vector, -MenT1, -MenT2, -MenT3, or -MenT4 (toxins) and pLAM-vector, -MenA1, -MenA2, -MenA3, or -MenA4 (antitoxins) were plated on LB-agar in the presence or absence of anhydrotetracycline (Atc; 100 ng ml−1) and acetamide (Ace; 0.2%) inducers for toxin and antitoxin expression, respectively. Plates were incubated for 3 days at 37°C. “T” and “A” denote toxin and antitoxin, respectively. “−” and “+” represent absence or presence of inducer, respectively. (B) M. smegmatis strain mc2 155 transformed with plasmid pGMCS-TetR-P1-RBS1-MenT3 was grown in complete 7H9 medium with Sm. At time 0, the culture was divided into two. Half was kept in the same medium (pale blue bars) and half was additionally treated with Atc (200 ng ml−1) (dark blue bars). Samples were harvested at the indicated times, washed, diluted, and plated on LB-agar with Sm but without Atc. Colonies were counted after 3 days at 37°C. Shown values are the average of three biological replicates with SD. CFU, colony-forming unit. (C) Samples of the same cultures as in (B) were harvested after 8 or 24 hours, labeled with the LIVE/DEAD BacLite dyes [Syto 9; propidium iodide (PI)], and analyzed by fluorescence-activated cell sorting. The percentage of PI-positive cells is shown for each sample (pale blue bars, no Atc; dark blue bars, 200 ng ml−1 Atc). Shown values are the average of three biological replicates with SD. (D) M. tuberculosis wild-type (WT) H37Rv or mutant strain H37Rv Δ(menA3-menT3)::dif6 were transformed with 100 ng of plasmids expressing either menA3, menT3, or menA3-menT3. These plasmids encode a consensus Shine-Dalgarno sequence (RBS1), except for “Weak-RBS-menT3,” which encodes a near-consensus sequence (RBS4) to weaken expression. After phenotypic expression, half of the transformation mix was plated on 7H11 oleic acid–albumin-dextrose-catalase (OADC) plates with Sm, and the other half was plated on 7H11 OADC Sm plates supplemented with Atc (200 ng ml−1). Plates were imaged after 20 days at 37°C; data are representative of three independent experiments. (E) Mutant strain H37Rv Δ(menA3-menT3)::dif6 was transformed with 100 ng of plasmids expressing either menT3 WT or mutant alleles introducing the D80A, K189A, or D211A substitutions. After phenotypic expression, half of the transformation mix was plated on 7H11 OADC plates with Sm, and the other half was plated on 7H11 OADC Sm plates supplemented with Atc (200 ng ml−1). Pictures were taken after 20 days at 37°C; data are representative of three independent experiments.

  • Fig. 2 Crystal structure of the MenT3 and MenT4 toxins.

    (A) Structure of monomeric MenT3 toxin, with views from front and back, shown as cyan cartoon representations. (B) Surface electrostatics of MenT3, viewed as in (A), with red for electronegative and blue for electropositive potential. (C) Structure of monomeric MenT4, with views from front and back, shown as salmon cartoon representations. (D) Surface electrostatics of MenT4, viewed as in (C), colored as per (B). (E) Superposition of MenT4 onto MenT3, viewed and colored as per (A) and (C). (F) Tilted close-up view of the toxin active sites, as indicated by the boxed region of (E). MenT3 residues S78 (phosphorylated), D80, K189, and D211 are indicated, along with the homologous MenT4 residues S67, D69, K171, and D186. (G) Alignment of JHP933 (PDB: 4O8S) as orange cartoon representation, against MenT3 viewed and colored as per (A, left).

  • Fig. 3 RNase PH suppresses MenT3 toxicity and inhibition of translation.

    (A) The E. coli K-12 genomic region containing the rph gene is shown. Suppressor plasmids that counteract MenT3 toxicity encoded rph, as depicted by small arrows under the adjacent genes pyrE, yicC, and dinD. The positions in base pair of the ends of each suppressor fragment, in relation to the E. coli K-12 chromosome, are indicated between brackets. (B) Overexpression of E. coli RNase PH partially suppresses MenT3 toxicity. E. coli DLT1900 strains containing either pK6-vector (−) or pK6-MenT3 (+) were cotransformed with p29SEN-vector (−) or p29SEN-Rph (RNase PH) (+). The resulting cotransformants were serially diluted, spotted onto LB-agar plates in the presence or absence of l-ara (0.1%) and IPTG (200 μM) inducers, and incubated at 37°C. (C) Deletion of rph further increases MenT3 toxicity. Transformants of E. coli DLT1900 WT and ∆rph mutant strains containing plasmid pK6-MenT3 were serially diluted, spotted onto LB-agar plates with or without l-ara (0.01%), and incubated at 37°C. (D) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the absence or presence of increasing concentrations of MenT3 toxin. Samples were separated by SDS–polyacrylamide gel electrophoresis and stained with InstantBlue. (E) For in vivo assays, transformants of E. coli BL21 (λDE3) containing plasmid pET-MenT3 or the empty vector were grown in M9M at 37°C. Following overexpression of MenT3, tRNAs were extracted, separated, and visualized by Northern blot using specific radiolabeled probes against tRNATrp. For in vitro assays, purified MenT3 (10 μM) was added to transcription/translation assays producing GatZ protein. After 2 hours at 37°C, tRNAs were extracted, separated, and visualized by Northern blot as performed for the in vivo samples. All images are representative of triplicate data.

  • Fig. 4 Toxin MenT3 adds pyrimidines to the 3′-CCA acceptor stem of tRNA.

    (A) Radiolabeled E. coli tRNATrp was incubated with 1, 0.1, 0.01, or 0.001 μg of MenT3 WT or no toxin (−) for 20 min at 37°C in the presence of unlabeled GTP, ATP, UTP, or CTP. Extended products are indicated with arrowheads throughout all panels. (B) Radiolabeled E. coli tRNATrp was incubated with 1, 0.1, or 0.01 μg of MenT3 WT or MenT3(D80A) with CTP or UTP, as per conditions in (A). (C) Incubation of radiolabeled E. coli tRNATrp with 1, 0.1, 0.01, or 0.001 μg of MenT3 WT or MenT3(K189A), with CTP, UTP, or a mixture of both, as per conditions in (A). (D) Radiolabeled E. coli tRNATrp preparations, made with or without a 3′-CCA motif, were incubated with 1, 0.1, or 0.01 μg of either MenT3 WT, MenT3(K189A), or no toxin (−), for 20 min at 37°C in the presence of unlabeled UTP or CTP. Note that the (−) CCA lanes have been overexposed to equalize intensity to the (+) CCA lanes of the same gel. Assays of the individual WT and MenT3 substitution proteins and tRNATrp ± CCA substrates shown in (A) to (D) were performed between two and four times.

  • Fig. 5 Screening for MenT3 M. tuberculosis tRNA targets.

    (A) Radiolabeled M. tuberculosis tRNAs were incubated with 0.1 μg of MenT3 WT (+) or no toxin (−) for 20 min at 37°C in the presence of unlabeled CTP. E. coli tRNATrp (EcTrp) was used as a positive control. The global screen of all M. tuberculosis tRNA was performed once and the effect of MenT3 tRNASer2 was confirmed twice independently. (B) Schematic diagram of the MenT3 toxin mechanism of action. MenT3 elongates the 3′-CCA motif of specific tRNAs, preventing their charging by aminoacyl-tRNA synthetases (AaRS), thereby interfering with translation and inhibiting bacterial growth.

  • Table 1 Crystallographic data collection and refinement statistics.

    MenT3 nativeMenT3 Se-peakMenT3 Se-high remoteMenT3 Se-inflectionMenT4 native
    Data collection
    PDB ID code6Y5U---6Y56
    BeamlineDiamond I04Diamond I03Diamond I03Diamond I03Diamond I24
    Wavelength (Å)0.97950.97930.96410.97950.9781
    Resolution range (Å)47.70–1.59 (1.65–1.59)*47.78–2.19 (2.26–2.19)47.83–2.05 (2.11–2.05)53.13–2.04 (2.11–2.04)42.23–1.23 (1.27–1.23)
    Space groupP3221P3221P3221P3221P21
    Unit cell, a b c (Å), α β γ (°)95.4 95.4 69.0, 90.0 90.0 120.095.6 95.6 69.2, 90.0 90.0 120.095.7 95.7 69.3, 90.0 90.0 120.095.6 95.6 69.3, 90.0 90.0 120.042.3 57.8 54.7, 90.0 92.3 90.0
    Total reflections98,016 (9668)36,407 (3179)44,514 (3476)47,255 (4637)149,653
    Unique reflections49,008 (4834)19,130 (1646)23,313 (1788)23,628 (2319)75,996 (7206)
    Multiplicity2.01.91.92.02.0
    Completeness (%)99.95 (99.83)100.00 (100.00)100.00 (99.80)100.00 (99.70)98.80 (88.97)
    Mean I/σ(I)16.76.57.68.97.0
    Rmerge0.016 (0.486)0.055 (0.373)0.055 (0.522)0.048 (0.463)0.060 (0.926)
    Rmeas0.022 (0.687)0.077 (0.528)0.078 (0.739)0.068 (0.654)0.085 (1.310)
    CC1/21.0 (0.672)0.995 (0.803)0.997 (0.544)0.997 (0.641)0.996 (0.294)
    Refinement
    Rwork0.2024 (0.2924)---0.1840 (0.3174)
    Rfree0.2242 (0.3108)---0.1950 (0.3352)
    No. of non-hydrogen atoms2494---2649
    Macromolecules2213---2322
    Solvent281---327
    Protein residues288---292
    RMSD (bonds, Å)0.006---0.005
    RMSD (angles, °)0.940---0.830
    Ramachandran favored (%)97.53---98.28
    Ramachandran allowed (%)2.47---1.72
    Ramachandran outliers (%)0.00---0.00
    Average B factor34.1---20.6
    Macromolecules33.4---19.3
    Solvent39.4---29.9

    *Statistics for the highest-resolution shell are shown in parentheses.

    Supplementary Materials

    • Supplementary Materials

      A nucleotidyltransferase toxin inhibits growth of Mycobacterium tuberculosis through inactivation of tRNA acceptor stems

      Yiming Cai, Ben Usher, Claude Gutierrez, Anastasia Tolcan, Moise Mansour, Peter C. Fineran, Ciarán Condon, Olivier Neyrolles, Pierre Genevaux, Tim R. Blower

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      • Figs. S1 to S8
      • Tables S1 and S2

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