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ClpP inhibitors are produced by a widespread family of bacterial gene clusters

Abstract

The caseinolytic protease (ClpP) is part of a highly conserved proteolytic complex whose disruption can lead to antibacterial activity but for which few specific inhibitors have been discovered. Specialized metabolites produced by bacteria have been shaped by evolution for specific functions, making them a potential source of selective ClpP inhibitors. Here, we describe a target-directed genome mining strategy for discovering ClpP-interacting compounds by searching for biosynthetic gene clusters that contain duplicated copies of ClpP as putative antibiotic resistance genes. We identify a widespread family of ClpP-associated clusters that are known to produce pyrrolizidine alkaloids but whose connection to ClpP has never been made. We show that previously characterized molecules do not affect ClpP function but are shunt metabolites derived from the genuine product of these gene clusters, a reactive covalent ClpP inhibitor. Focusing on one such cryptic gene cluster from Streptomyces cattleya, we identify the relevant inhibitor, which we name clipibicyclene, and show that it potently and selectively inactivates ClpP. Finally, we solve the crystal structure of clipibicyclene-modified Escherichia coli ClpP. Clipibicyclene’s discovery reveals the authentic function of a family of natural products whose specificity for ClpP and abundance in nature illuminate the role of eco-evolutionary forces during bacterial competition.

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Fig. 1: Genome mining identifies a family of ClpP-associated BGCs.
Fig. 2: Biological activity of an elusive ClpP inhibitor.
Fig. 3: Clipibicyclene in fermentation broth covalently modifies and inhibits ClpP.
Fig. 4: Clipibicyclene is a potent, specific inhibitor of ClpP.
Fig. 5: Cac16/17 provide resistance to clipibicyclene.
Fig. 6: Structure of the ClpPec:clipibicyclene complex.

Data availability

All data generated or analysed during this study are included in this published article and its Supplementary Information. The S. cattleya DSM 46488 whole genome sequence is available in GenBank under accession number NC_017586. The structure of ClpPec in complex with clipibicyclene is available in PDB with accession number 7MK5. Source data are provided with this paper.

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Acknowledgements

We thank M. Tyers for the gift of pSASS vectors and S. cerevisiae SASy31 and SASy35, W. Houry for the gift of E. coli BL21(DE3) 1146D and pET9a-EcClpP, H. Brötz-Oesterhelt for B. subtilis ΔclpP and V. Larionov for S. cerevisiae VL648N. This research was funded by a Canadian Institutes of Health Research grant (FRN-148463), the Ontario Research Fund and a Canada Research Chair to G.D.W. E.J.C. was supported by a CIHR Vanier Canada Graduate Scholarship. D.S. was supported by a CIHR Postdoctoral Fellowship. Protein structural studies were performed using beamline CMCF-BM at the Canadian Light Source at the University of Saskatchewan, which is supported by the Canada Foundation for Innovation, the Natural Sciences and Engineering Research Council (RGPIN-2018-04968), the National Research Council (NRC), the Canadian Institutes of Health Research, the Government of Saskatchewan and the University of Saskatchewan.

Author information

Authors and Affiliations

Authors

Contributions

E.J.C. and G.D.W. conceived the study and designed experiments. A.C.P. and E.J.C. performed target-directed genome mining. G.P. and D.S. performed crystallization, X-ray data collection, processing and model building. D.S. carried out structural analysis, performed peptide mapping, clipibicyclene titration experiments and serine hydrolase profiling. C.H. performed structural elucidation of azabicyclenes and clipibicyclene and purified clipibicyclene. E.J.C. performed all other experiments. E.J.C. and G.D.W. prepared the manuscript.

Corresponding author

Correspondence to Gerard D. Wright.

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The authors declare no competing interests.

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Nature Microbiology thanks Herman Sintim, Kim Lewis and Lynn Silver for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Activation of cac, a pyrrolizidine alkaloid-related BGC.

(a) Structures of previously identified bacterial pyrrolizidine alkaloids and related metabolites. (b) RT–PCR results compare the expression of cac in S. cattleya and heterologous producer S. coelicolor M1154 with various engineered constructs. The cluster-situated XRE family transcriptional activator, Cac15, is overexpressed using the plasmid pIJ10257. RT–PCR was performed in biological triplicate and technical duplicate and normalized to the housekeeping sigma factor hrdB. Bars represent the mean with error bars showing standard deviation and dots individual measurements from biological replicates. (c) Schematic shows the design of the refactored construct pCGW-cac-LHK-apra, which becomes pCGW-cac-LHK after removal of the apramycin resistance cassette.

Source data

Extended Data Fig. 2 LC–MS/MS of azabicyclenes and ClpP active site peptide.

LC–MS/MS spectra of (a) azabicyclene D, (b) azabicyclene C, and (c) azabicyclene B. Exact mass of predicted ions are shown. MS spectra were acquired in positive mode with 10 eV collision energy. (d) LC–MS/MS spectra ClpP active site peptide that is unmodified (m/z = 855.1, left) or modified (m/z = 964.4 right) with clipibicyclene. Ions corresponding to b and y fragments are labelled. Ions y14-16 missing from the modified peptide’s spectra are predicted to be outside of the mass detector’s range. Inset of the low molecular weight range of the modified peptide shows fragments corresponding to masses observed from azabicyclene B and C.

Extended Data Fig. 3 Proposed biosynthesis of azabicyclenes.

See supplementary discussion for a full description. Coloured carbon atoms track those incorporated by each module of the PKS.

Extended Data Fig. 4 In vivo and in vitro activity of azabicyclenes.

(a) Susceptibility testing of various bacteria species and mammalian cells against azabicyclene C and D (AzaC, AzaD) and bohemamines A and B (BohA, BohB). While azabicyclene D has weak growth inhibitory activity (MIC = 64-128 μg/mL), it is equally active against species where ClpP is dispensable (S. aureus, Bacillus subtilis) and clpP knockout strains (B. subtilis ΔclpP), indicating that its activity is independent of ClpP. ND – not determined. (b) Effect of azabicyclenes on ClpP1P2scatt peptidase activity using (S-LLVY)2 Rhodamine 110 as a substrate (left), and ClpPec proteinase activity using 50 μg/mL FITC-casein as a substrate (right). (c) Effect of bohemamines on ClpP1P2scatt peptidase activity using (S-LLVY)2 Rhodamine 110 as a substrate (left), and ClpPec proteinase activity using 50 μg/mL FITC-casein as a substrate (right). For panels (b) and (c), mean of replicate reactions (n = 2) is shown with error bars representing standard deviation. All experiments were performed on at least two independent occasions with similar results.

Source data

Extended Data Fig. 5 Bioactivity of an unstable ClpP inhibitor.

(a) RT–PCR quantification of clpP3 transcripts in the heterologous producer S. coelicolor M1154. Complementation of cac16-17 is provided by the plasmid pIJ-ncac16-17. Mean with error bars showing standard deviation is plotted (n = 3) for three independent cultures. Significance is tested against pCGW with **p = 0.0008 or *p = 0.0392 by two-sided Kruskal-Wallis with Dunn’s multiple comparison test. (b) Extracted ion chromatograms of n-butanolic extracts from S. coelicolor M1154 strains carrying the cac cluster with cac8 deleted, as indicated. Complementation of cac8 is provided by the plasmid pIJ10257-cac8. Results are representative of three independent fermentations. (c) Quantification of results shown in (b) for biological triplicate fermentations (n = 3) with error bars showing standard deviation. (d) S. coelicolor pIJGUS-pClpP3 pSET152 streaked on media containing X-gluc tested against pure azabicyclenes and bohemamines. 40 μg compound is spotted on each disk. NP indicates the bohemamine-type compound NP25302. Cr indicates crude n-butanolic extract of either S. coelicolor M1154 pCGW-cac-LHK (azabicyclene) or S. sp. NBRC110035 (bohemamine) fermentations, where 10 μL is applied to each disk. An agar plug inoculated with S. coelicolor M1154 pCGW-cac-LHK is used as a positive control. (e) S. coelicolor pIJGUS-pClpP3 pSET152 co-streaked with the bohemamine producing organism Streptomyces sp. NBRC110035 or S. coelicolor M1154 pCGW (negative control). (f) Kirby–Bauer growth inhibition assays with agar plugs inoculated with S. coelicolor M1154 strains, as indicted on the left. Indicator strains labelled along the top are environmental Streptomyces spp. (g) Time course of clipibicyclene production using S. coelicolor pIJGUS-pClpP indicator assay. 2.5 μL of each sample is spotted as indicated. All experiments were repeated on at least two independent occasions with similar results.

Source data

Extended Data Fig. 6 Reconstitution of ClpP in vitro.

(a) In vitro processing of S. cattleya ClpPs was determined by intact protein MS after incubation alone or with cognate ClpPs, as listed. Incubations were carried out in the presence or absence of ADEP to determine its effect. (b) The activity of a variety of substrates and the effects of activator peptides were measured. % activity is calculated based on rate of the reaction, normalized to that with Suc-LLVY-AMC substrate and no activator peptide. (c) Thermal shift assays of S. cattleya ClpPs in the presence or absence of ADEP. Numbers indicate melting temperature (°C). Assays were performed in technical duplicate with representative curve shown. For panels (a) and (b) means of technical triplicate reactions are reported. All experiments were performed on two independent occasions with similar results.

Extended Data Fig. 7 Behaviour of clipibicyclene in culture supernatant.

(a) Extracted ion chromatogram shows that clipibicyclene (m/z = 347.1605) is removed upon incubation with ClpPec. 85 μM enzyme was incubated with dichloromethane extract of S. coelicolor M1154 pCGW-cac-LHK prior to LC–MS analysis. (b) Stability of clipibicyclene over the course of zero to three days in water or various medias and at the temperatures indicated was assessed by LC–MS quantification.

Extended Data Fig. 8 Structural details of the ClpPec:clipibicyclene axial pore.

(a) Cartoon representation of the asymmetric unit which contains two complete ClpPec tetradecamers. (b) Left, cartoon representation of the axial pore of a ClpPec:clipibicyclene heptamer. The grey circle highlights the unmodelled N-terminal regions of the complex. Protein chains are labelled alphabetically. Right, detailed structure of the N-terminus of chain A (residues 2–20) depicted in stick representation. The 2mFo-DFc is shown in black mesh and contoured at 1σ. (c) Superposition of the ClpP:clipibicyclene tetradecamer (left) and the monomer (right) with apo ClpP, coloured teal and white, respectively. The apo ClpPec model was obtained from the PDB (1TYF).

Extended Data Fig. 9 Structures of the clipibicyclene adduct in from each chain.

The clipibicyclene adduct is depicted in stick representation and is coloured by atom (C, white; N, blue; O, red). The adducts were defined by a carbamoyl linkage between the Oɣ of Ser97 and the nitrogen of the azetine ring (Fig. 6a). As expected, the seven-membered ring of clipibicyclene is no longer intact. Instead, it is replaced by an imide moiety connecting the azetine portion of the adduct to its aliphatic tail. The 2mFo-DFc map is shown in black mesh and contoured at 0.9 σ and the Fo-Fc omit map is coloured green mesh and contoured at 3 σ. The chain to which each adduct is linked is labelled alphabetically. We use the adduct from chain A as the representative conformer to describe the ClpPec:clipibicyclene complex.

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Source Data Fig. 4

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Source Data Extended Data Fig. 1

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Culp, E.J., Sychantha, D., Hobson, C. et al. ClpP inhibitors are produced by a widespread family of bacterial gene clusters. Nat Microbiol 7, 451–462 (2022). https://doi.org/10.1038/s41564-022-01073-4

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