Over-activation of a nonessential bacterial protease DegP as an antibiotic strategy

Abstract

Rising antibiotic resistance urgently begs for novel targets and strategies for antibiotic discovery. Here, we report that over-activation of the periplasmic DegP protease, a member of the highly conserved HtrA family, can be a viable strategy for antibiotic development. We demonstrate that tripodal peptidyl compounds that mimic DegP-activating lipoprotein variants allosterically activate DegP and inhibit the growth of an Escherichia coli strain with a permeable outer membrane in a DegP-dependent fashion. Interestingly, these compounds inhibit bacterial growth at a temperature at which DegP is not essential for cell viability, mainly by over-proteolysis of newly synthesized proteins. Co-crystal structures show that the peptidyl arms of the compounds bind to the substrate-binding sites of DegP. Overall, our results represent an intriguing example of killing bacteria by activating a non-essential enzyme, and thus expand the scope of antibiotic targets beyond the traditional essential proteins or pathways.

Fig. 1. Design of the DegP-activating compounds mimicking Lpp^+Leu.

a Crystal structure of the Lpp trimer (PDB code, 1EQ7; cartoon presentation) with a close-up view of the C-terminal region. All Tyr56 (cyan sticks) and Arg57 (magenta sticks) residues roughly sit in the same plane. Distances from the center to the Tyr56-Cα and Arg57-Cα are 5.0 Å and 7.3 Å, respectively. b Synthesis of the tripodal peptidyl compounds, TMB_(Cys_peptide). Peptides are attached to the benzyl positions of 1,3,5-trimethylbenzene (TMB) via the N-terminal cysteine. Distance from the center to the cysteine-Cα in the extended tripodal compound is about 6.7 Å.

Fig. 2. Tripodal peptidyl compounds activate the DegP protease.

Data are presented as dot plots with mean ±1 SD (n = 3 independent experiments). a, b Activation effects of compounds were monitored by measuring cleavage rates of the reporter peptide (100 μM) by DegP (1 μM for TMB_CKYRKL and TMB_CYRKL (a); 10 μM for Lpp^+Leu, TMB_CRKL, and TMB_CKL (b)) with increasing concentrations of Lpp^+Leu or tripodal compounds. The insets show close-up views. c Effect of alanine substitution on each position of the pentapeptide (CYRKL) for DegP activation. d DegP activation effects of TMB_CYYKI or its variants with an alanine substitution were monitored as described in Fig. 2a. e Degradation of denatured OmpA (2 μM) by DegP (5 or 0 μM) was monitored by SDS-PAGE in the absence or presence of TMB_CYYKI (10 μM).

Fig. 3. Tripodal compounds inhibit bacterial growth in a DegP-dependent fashion.

Data are presented as dot plots with mean ±1 SD (n = 3 independent experiments). a Growth of the imp4213 cells in the presence of TMB_CYYKI or TMB_CYRKL. Overnight cultures in LB broth were diluted 100-fold in the presence of different concentrations of TMB_CYYKI or TMB_CYRKL. Growth was monitored by OD₆₀₀ after 12 h at 30 °C. b Time-kill curves of the imp4213 strain with TMB_CYYKI, TMB_CYRKL or no compound. Cells at log phase (OD₆₀₀ = 0.01) were incubated at 30 °C in the absence or presence of TMB_CYYKI (160 µM) or TMB_CYRKL (640 µM). Cells at different times were taken out to determine colony forming units (CFUs). c Time-kill curves of the imp4213 ∆degP strain with TMB_CYYKI, TMB_CYRKL, or no compound at 30 °C. d, e Time-kill curves of the imp4213 ∆degP strain that has a plasmid expressing either DegP^WT (d) or DegP^S210A (e) in the absence or presence of arabinose (Ara) at 30 °C or 42 °C. f Time-kill curves of the imp4213 ∆degP strain containing a DegP^WT- or DegP^S210A-plasmid in the presence of TMB_CYYKI (2x MIC) at 30 °C. g, h Time-kill curves of the imp4213 strain at 30 °C in the presence of TMB_CYYKI (160 µM), chloramphenicol (20 µg/ml), rifampicin (0.016 μg/ml), tetracycline (2 μg/ml), or both an antibiotic and TMB_CYYKI. Those of the cells at stationary phase in the absence or presence of TMB_CYYKI are also shown.

Fig. 4. TMB_CYYKI induces an elongated morphology.

a–c Representative micrographs of E. coli W3110 (wild-type), ∆degP, or degP^R207P/Y444A strains (a), the imp4213 strain in the absence or presence of TMB_CYYKI (160 µM; b), or the imp4213 ∆degP strain in the absence or presence of TMB_CYYKI (160 µM; c), at 30 °C or 42 °C after 4 h of incubation from OD₆₀₀ = 0.1. d, e Quantification of cell length (n = 152–221) of the wild-type, ∆degP, or degP^R207P/Y444A strains at 42 °C (d), or the imp4213 strain at 30 °C in the absence or presence of TMB_CYYKI (160 µM; e). Data are presented as dot plots with mean ±1 SD (n = 3 independent experiments).

Fig. 5. Co-crystal structures reveal that the tripodal compounds mainly bind to substrate-binding sites of DegP.

a Superposition of three trimers from the previously reported dodecameric structure (PDB code, 3OTP) and our structures with TMB_CYRKL or TMB_CYYKI. b Pentapeptides in DegP^S210A•TMB_CYYKI are shown in spheres (rainbow colors from the blue N-terminus to the red C-terminus) on the two substrate-binding sites of DegP. Three DegP monomers are colored differently in surface presentation. Two insets show close-ups of peptapeptides (yellow sticks with electron density maps) and nearby DegP residues (orange sticks) at the PDZ1 pocket (above) and the active-site region (below) of DegP.

Fig. 6. The compound with three arms has the best activity and properties different from those with one or two arms.

a Structures of compounds with three, two, or one peptidyl arm(s) (TMB_CYYKI, DMB_CYYKI, or MMB_CYYKI, respectively). b Activation effects of TMB_CYYKI, DMB_CYYKI, and MMB_CYYKI were monitored by measuring cleavage rates of the reporter peptide (100 µM) by DegP (1 µM) with increasing concentrations of compounds. Data are presented as dot plots with mean ± 1 SD (n = 3 independent experiments). c–g Isothermal titration calorimetry (ITC) experiments of TMB_CYYKI (c), 18–58 (d), DMB_CYYKI (e, g for 0–2 and 0–0.4 molar ratio, respectively), and MMB_CYYKI (f) with DegP^S210A. Data were fitted to the single binding site model. h DegP assembly was monitored by measuring fluorescence resonance energy transfer (FRET) of the donor- and acceptor-labeled DegP variants (0.1 M total) with increasing concentrations of 18–58, TMB_CYYKI, DMB_CYYKI, or MMB_CYYKI. Data were fitted to the Hill equation. Data are presented as dot plots with mean ± 1 SD (n = 3 independent experiments).

Fig. 7. A model for bacterial killing by DegP over-activation.

DegP mainly degrades misfolded proteins, but its over-activation by the tripodal compounds triggers excessive degradation of newly synthesized proteins and leads to bacterial death.