The antibiotic ADEP reprogrammes ClpP, switching it from a regulated to an uncontrolled protease

Abstract

A novel class of antibiotic acyldepsipeptides (designated ADEPs) exerts its unique antibacterial activity by targeting the peptidase caseinolytic protease P (ClpP). ClpP forms proteolytic complexes with heat shock proteins (Hsp100) that select and process substrate proteins for ClpP-mediated degradation. Here, we analyse the molecular mechanism of ADEP action and demonstrate that ADEPs abrogate ClpP interaction with cooperating Hsp100 adenosine triphosphatases (ATPases). Consequently, ADEP treated bacteria are affected in ClpP-dependent general and regulatory proteolysis. At the same time, ADEPs also activate ClpP by converting it from a tightly regulated peptidase, which can only degrade short peptides, into a proteolytic machinery that recognizes and degrades unfolded polypeptides. In vivo nascent polypeptide chains represent the putative primary target of ADEP-activated ClpP, providing a rationale for the antibacterial activity of the ADEPs. Thus, ADEPs cause a complete functional reprogramming of the Clp-protease complex.

Figure 1. ADEP activates ClpP to degrade casein with reduced processivity

A. Degradation of FITC-labelled casein (100 nM) by E. coli ClpA/ClpP, ClpP/ADEP or ClpP only. B. Degradation of casein by the indicated E. coli (A–C) and B. subtilis (D) components was monitored by SDS–PAGE. (B) 1 µM casein, C, D. 20 µM casein. A protein standard is given. The positioning of casein and the respective proteins is indicated. E.c. indicates from E. coli and B.s. indicates from B. subtilis. Casein degradation products of intermediate size are labelled with ‘*’.

Figure 2. ADEP binding triggers oligomerization of B. subtilis ClpP

1. The oligomeric state of ClpP in the absence (solid line) and presence (dashed line) of ADEP (0.5 µg/ml) was monitored by size exclusion chromatography using a Superdex 75 column. Elution positions of protein standards are given. Samples of both eluted peak fractions (as indicated in the chromatogram: 1: ClpP oligomer and 2: ClpP monomer) were incubated with 1 µM casein and tested for proteolytic activity by SDS–PAGE analysis.

2. The association state of 10 µM ClpP in the presence of 12 µM ADEP was analysed by analytical ultracentrifugation. The left panel shows the sedimentation velocity at 40,000 rpm and 20 °C (scans taken after every 20 min intervals depicted). The calculated sedimentation velocity was s(app) = 11.5 S. The right panel displays the data of a sedimentation equilibrium experiment after 60 h at 6,000 rpm and 20 °C. In several independent experiments, the molecular mass of ClpP was determined over the range of 280–320 kDa, fitting well to the size of a double heptameric structure of ClpP. The offset of A_{230 nm} = 0.23 in both sedimentation velocity and equilibrium experiments arises from non-bound ADEP, which has been used in a slight molar excess over ClpP.

Figure 3. ADEP abrogates the cooperation of ClpP and its Hsp100 partner proteins

1. Degradation and unfolding of the N-end rule model substrate FR-linker-GFP (100 nM) was followed by monitoring the decrease of GFP fluorescence in the presence of the indicated components (+/− ADEP).

2. Degradation and unfolding of GFP-SsrA (100 nM) was followed by monitoring the decrease of GFP fluorescence in the presence of the indicated components (+/− ADEP). The rate of GFP-SsrA degradation and unfolding determined in the absence of ADEP was set as 100%.

3. In vitro degradation of MecA by ClpC/ClpP and ComK by ClpC/ClpP/MecA and Spx by either ClpC/ClpP/MecA or ClpX/ClpP in the absence and presence of ADEP. Substrate degradation was analysed by SDS–PAGE (all proteins were used at 1 µM).

4. Analysis of the in vivo stability of MurAA in B. subtilis cells (wild type (wt) and ΔclpP mutant). ADEP (final concentration: 1.6 µg/ml) was added upon entry of cells into stationary phase and cells were incubated for an additional period of 2 h prior to harvesting. MurAA levels were determined by immunoblot analysis using MurAA-specific antibodies.

Figure 4. ADEP prevents complex formation between ClpP and cooperating Hsp100 proteins both in vitro and in bacterial cells

1. A complex of ClpC-DWB, MecA and ClpP was preformed in the presence of 5 mM ATP at 37 °C for 10 min. ADEP (0.5 µg/ml) was subsequently added and the sample was further incubated for an additional 10 min. As a control, a sample without the addition of ADEP was analysed in parallel. The association and dissociation of the respective complexes were analysed by size exclusion chromatography using a Superose 6 column. The chromatogram of ClpC-DWB, MecA and ClpP is shown by a solid line and the chromatogram of the ADEP including sample by a dashed line. The resulting peaks (as indicated in the chromatogram) could be assigned as follows: 1: ClpC-DWB₆MecA₆/(ClpP₇)₂/ClpC-DWB₆MecA₆; 2: ClpC-DWB₆/MecA₆; 3: (ClpP₇)₂-ADEP and 4: ClpC_DWB/MecA heterodimer. The elution profile of ClpP is shown for both samples as a Coomassie-stained SDS gel. Elution positions of protein standards are given.

2. ADEP causes a delocalization of ClpP in bacterial cells. Localization of ClpP-GFP in B. subtilis 168 in the absence (upper row) or presence of ADEP (0.4 and 1.6 µg/ml middle and bottom row, respectively) is shown. ADEP was added during the logarithmic growth phase (30 °C, OD_600nm = 1) and ClpP-GFP localization was monitored 120 and 360 min after ADEP addition by fluorescence microscopy. A scale bar of 5 µm length is depicted.

3. ADEP does not affect ClpC and ClpX localization in bacterial cells. Localization of ClpC-GFP and ClpX-YFP in B. subtilis 168 in the absence or presence of ADEP. Cells were treated as described in (B) and localization of fluorescent fusion proteins was analysed prior to and 360 min after the addition of ADEP. Scale bars are depicted for the left (1 µm) and right columns (2 µm).

Figure 5. ADEP-activated ClpP degrades nascent polypeptides in vitro

[³⁵S] methionine-labelled model polypeptides (a ribosome-arrested ICDH fragment of 45 kDa, a ribosome-arrested 3m10-SecM chain of 26 kDa (A) and non-arrested full-length ICDH (46 kDa), SH3 and m10 (B and C) were synthesized in an E. coli-based transcription/translation system and analysed for degradation at 37 °C. Polypeptides were separated on tricine gels and visualized by autoradiography. Only the full-length products are depicted. A. ClpP, ADEP and DMSO were added to the translation system after the generation of ribosome-arrested nascent chains. B. ClpP, ADEP and DMSO were added to the translation system before the synthesis of full-length, non-arrested polypeptides. C. ClpP, ADEP and DMSO added after the translation of full-length, released polypeptides. D, E. Quantification of the degradation experiments shown in (B) and (C), respectively. The amount of full-length product at the end of translation under control conditions (DMSO without ClpP addition) was set at 100%. The data correspond to one representative experiment out of three and a second experiment, performed using another purification of the translation extract, is depicted in the Supporting Information Fig 1.

Figure 6. ADEP leads to the degradation of newly synthesized proteins prior to folding in bacterial cells

1. ADEP causes the degradation of ribosome-arrested nascent polypeptides in vivo. The degradation of two ribosome-arrested nascent chains derived from ICDH (Strep₃-ICDH66-SecM, Strep₃-ICDH318-SecM) was monitored in ER2566 ΔacrA::kan cells at 37 °C. Two hours after induction (i) of the model constructs, translation was stopped with chloramphenicol and ADEP or DMSO was added (0 min). At the indicated time points, cells were harvested and proteins separated by SDS–PAGE followed by Western blots against the N-terminal Strep-tag (upper panel) and against ICDH, DnaK, Trigger Factor and L23 as controls (lower panel). Only full-length products are depicted.

2. The degradation of newly synthesized proteins is increased in the presence of ADEP in vivo. Total protein degradation was determined in HN818 ΔacrA::kan cells in the exponential phase at 37 °C. ADEP1 and DMSO were either added before (left panel) or after (right panel) [³⁵S] methionine pulse labelling. At the denoted time points, aliquots were TCA precipitated and the radioactivity in the TCA-soluble and insoluble fractions was determined by scintillation counting. The amount of degradation is given as percentage of the total cellular radioactivity. Mean values and standard errors of the mean of four to five independent experiments are shown.