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. 2016 Nov 9;7:13339.
doi: 10.1038/ncomms13339.

Chaperone Addiction of Toxin-Antitoxin Systems

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Chaperone Addiction of Toxin-Antitoxin Systems

Patricia Bordes et al. Nat Commun. .
Free PMC article

Abstract

Bacterial toxin-antitoxin (TA) systems, in which a labile antitoxin binds and inhibits the toxin, can promote adaptation and persistence by modulating bacterial growth in response to stress. Some atypical TA systems, known as tripartite toxin-antitoxin-chaperone (TAC) modules, include a molecular chaperone that facilitates folding and protects the antitoxin from degradation. Here we use a TAC module from Mycobacterium tuberculosis as a model to investigate the molecular mechanisms by which classical TAs can become 'chaperone-addicted'. The chaperone specifically binds the antitoxin at a short carboxy-terminal sequence (chaperone addiction sequence, ChAD) that is not present in chaperone-independent antitoxins. In the absence of chaperone, the ChAD sequence destabilizes the antitoxin, thus preventing toxin inhibition. Chaperone-ChAD pairs can be transferred to classical TA systems or to unrelated proteins and render them chaperone-dependent. This mechanism might be used to optimize the expression and folding of heterologous proteins in bacterial hosts for biotechnological or medical purposes.

Figures

Figure 1
Figure 1. Mtb-SecBTA interacts with the C-terminal region of Mtb-HigA1.
(a) Schematic representation of Mtb-HigA1. The dark blue box represents the helix-turn-helix (HTH) motif and the blue hachures the TAC-specific C-terminal extension. The deletion in ΔC42 is indicated with an arrow. (b) In vivo interaction between Mtb-SecBTA and Mtb-HigA1. In vivo pulldowns of His-tagged Mtb-SecBTA and the different Mtb-HigA1 (wild type), Mtb-HigA1W108A/W137A (W108A/W137A) and Mtb-HigA1ΔC42 (ΔC42) proteins were revealed using anti-Mtb-HigA1 or anti-Mtb-SecBTA antibodies. (c) In vivo interaction between Mtb-SecBTA and the C-terminal extension of Mtb-HigA1 (aa 104–149) wild type (Cter) or mutant (Cter W108A/W137A) fused with luciferase (Luc). In vivo pulldowns of His-tagged Mtb-SecBTA and the pK6-Luc constructs were revealed with anti-luciferase or anti-Mtb-SecBTA antibodies. Full blots for b and c are shown in Supplementary Fig. 6. (d) In vitro native PAGE separation of complexes between Mtb-HigA1 or Mtb-HigA1W108A/W137A, at 2, 4 and 8 μM and Mtb-SecBTA (16 μM). Full gel for d is shown in Supplementary Fig. 7. (e) Suppression of Mtb-HigB1 toxicity by Mtb-HigA1 and Mtb-SecBTA in E. coli. Strains W3110 ΔsecB containing the plasmid pSE (−) or pSE-Mtb-SecBTA (+; with Mtb-SecBTA under control of Ptrc promoter) were transformed with pK6-based plasmids harbouring Mtb-HigA1, Mtb-HigA1ΔC42 or Mtb-HigA1W108A/W137A under control of PBAD promoter, grown to mid-log phase, serially diluted and spotted on LB–ampicillin–kanamycin agar plates without IPTG and with arabinose as indicated. Plates were incubated at 37 °C overnight.
Figure 2
Figure 2. The chaperone facilitates the folding of Mtb-HigA1 and other unrelated proteins only in the presence of ChAD.
(a) Folding of newly translated Mtb-HigA1 relies on Mtb-SecBTA when the ChAD extension of Mtb-HigA1 is present. Mtb-HigA1, Mtb-HigA1ΔC42 and Mtb-HigA1W108A/W137A were independently expressed in a cell-free translation system with or without Mtb-SecBTA. Translation products were labelled with [35S]methionine. Mtb-SecBTA concentrations were 8 μM otherwise stated (+a indicates 4 μM) and reactions were performed for 1 h at 37 °C. When indicated, 0.4 mM of puromycin was added, either from the start of the translation reaction (+c) or 30 min after (+b), to release ribosome-bound nascent chains for 1 h, and the chaperone was then added for 1 h at 37 °C. After translation, the total (t) and soluble (s) fractions were separated on SDS–PAGE and quantified by phosphorimager. The numbers below the electrophoretic pattern represent the mean solubility values (%), calculated by the ratio of the amount of translation products in the soluble (s) and total (t) fractions obtained from three different translation experiments. The s.d. is indicated. (b) In vitro aggregation kinetics of urea-denatured Mtb-HigA1 and Mtb-HigA1W108A/W137A (4 μM) followed at 25 °C by monitoring light scattering at 355 nm. (c) Effect of Mtb-SecBTA on the solubility of nascent luciferase (Luc) and chimeric luciferase containing C-terminal ChAD (Luc-ChAD) or N-terminal ChAD-luciferase (ChAD-Luc) and (d) on the solubility of nascent GFP and chimeric GFP-C-terminal ChAD (GFP-ChAD) and N-terminal ChAD-GFP (ChAD-GFP), as performed in a, with or without Mtb-SecBTA (8 μM). Full phosphorimager images for a,c and d are shown in Supplementary Fig. 8.
Figure 3
Figure 3. Primary Mtb-SecBTA chaperone-binding site of ChAD.
(a) Thirteen-mer peptides (C1–C15) covering the entire ChAD sequence are depicted on top of the amino-acid sequence. The rectangles displayed under specific regions of the ChAD sequence represents the amino acids (single or triple) that have been changed for alanine in the experiment described in d. The grey rectangles represent mutations without apparent phenotype, the orange rectangles represent mutations with partial inactivation and the red rectangles represent mutations with the most severe phenotype, see d below. (b) DSF analysis of Mtb-HigA1 peptide binding to Mtb-SecBTA. The thermal stability of Mtb-SecBTA was monitored in the presence of each peptide. Tm values were deduced from the fluorescence curves recorded using a temperature gradient from 15 to 90 °C. Shifts in melting temperature are shown at molar ratios of 12.5:1.0 (dark blue bars) and 60:1.0 (light blue bars) of peptides:Mtb-SecBTA chaperone. The means and s.e.m.'s of three replicates are shown. (c) Representative DSF curves (fluorescence versus temperature gradient) for a temperature gradient from 15 to 90 °C for Mtb-SecBTA alone (red) or in the presence of C4 peptide at molar ratios of 12.5:1.0 (dark blue curve) and 60:1.0 (light blue curve) of peptides: Mtb-SecBTA. (d) Suppression of Mtb-HigB1 toxicity by Mtb-HigA1 and its triple and single alanine mutant derivatives with or without Mtb-SecBTA chaperone. Plates were incubated at 37 °C overnight. (e) In vivo pulldown of His-tagged Mtb-SecBTA and Mtb-HigA1 (wild type) or Mtb-HigA1Y114A (Y114) proteins was revealed using anti-Mtb-HigA1 or anti-Mtb-SecBTA antibodies. Full blots for e are shown in Supplementary Fig. 6. (f) In vitro native PAGE separation of complexes between Mtb-HigA1 or Mtb-HigA1Y114A, at 2, 4 and 8 μM and Mtb-SecBTA (16 μM), as performed in Fig. 1. Full gel for f is shown in Supplementary Fig. 7. (g) Mtb-HigA1Y114A expressed in a cell-free translation system is not solubilized by Mtb-SecBTA. Experiments were performed as described in Fig. 2a with or without chaperone (8 μM). Full phosphorimager images for g are shown in Supplementary Fig. 8.
Figure 4
Figure 4. SecB/ChAD addiction modules of TAC systems are specific.
(a) Mtb-SecBTA, VchoA-SecBTA, Mmet-SecBTA and Glov-SecBTA chaperone specificity towards their cognate TA pair. Strain W3110 ΔsecB was co-transformed with plasmid pK6-Mtb-HigBA1, pK6-VchoA-MqsRA, pK6-Mmet-HicAB or pK6-Glov-HigBA and pSE-based plasmids harbouring different SecB-like chaperones as indicated. Double transformants were grown to mid-log phase, serially diluted and spotted on LB–ampicillin–kanamycin agar plates containing arabinose and IPTG inducers as indicated. Plates were incubated at 37 °C overnight. (b) Functional inter-species transfer of SecB/ChAD-specific pairs within TAC systems using antitoxin chimeras. Strain W3110 ΔsecB was co-transformed with pK6-based plasmids containing chimeric TA systems in which the putative ChAD regions have been swapped, and pSE derivatives harbouring different SecB-like chaperones as indicated, and analysed by spot tests as in a.
Figure 5
Figure 5. Chaperone addiction module can be transferred to classic two-component TA systems.
(a) In vivo Mtb-SecBTA addiction of classical two-component TA systems belonging to three different TA families in which antitoxins have been fused to the Mtb-ChAD. Strain DLT1900 was co-transformed with plasmid pSE vector (−) or pSE-Mtb-SecBTA (+) and pK6-Eco-MqsRA, pK6-Eco-MqsRA-ChAD, pK6-Vcho-HigBA2, pK6-Vcho-HigBA2-ChAD, pK6-Tde-HicAB or pK6-Tde-HicAB-ChAD. Double transformants were grown to mid-log phase, serially diluted and spotted on LB–ampicillin–kanamycin agar plates with arabinose inducer as indicated. Plates were incubated at 37 °C overnight. (b) Effects of Mtb-SecBTA on the solubility of Eco-MqsA, Vcho-HigA2, Tde-HicB wild type or chimeras containing the Mtb-ChAD region, namely, Eco-MqsA-Mtb-ChAD (MqsA-ChAD), Vcho-HigA2-Mtb-ChAD (HigA2-ChAD) and Tde-HicB-Mtb-ChAD (HicB-ChAD), synthesized using a reconstituted cell-free translation system with or without Mtb-SecBTA chaperone (8 μM) as performed in Fig. 2a. Full phosphorimager images for b are shown in Supplementary Fig. 8.

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