Antibiotic acyldepsipeptides activate ClpP peptidase to degrade the cell division protein FtsZ - PubMed (original) (raw)

Antibiotic acyldepsipeptides activate ClpP peptidase to degrade the cell division protein FtsZ

Peter Sass et al. Proc Natl Acad Sci U S A. 2011.

Abstract

The worldwide spread of antibiotic-resistant bacteria has lent urgency to the search for antibiotics with new modes of action that are devoid of preexisting cross-resistances. We previously described a unique class of acyldepsipeptides (ADEPs) that exerts prominent antibacterial activity against Gram-positive pathogens including streptococci, enterococci, as well as multidrug-resistant Staphylococcus aureus. Here, we report that ADEP prevents cell division in Gram-positive bacteria and induces strong filamentation of rod-shaped Bacillus subtilis and swelling of coccoid S. aureus and Streptococcus pneumoniae. It emerged that ADEP treatment inhibits septum formation at the stage of Z-ring assembly, and that central cell division proteins delocalize from midcell positions. Using in vivo and in vitro studies, we show that the inhibition of Z-ring formation is a consequence of the proteolytic degradation of the essential cell division protein FtsZ. ADEP switches the bacterial ClpP peptidase from a regulated to an uncontrolled protease, and it turned out that FtsZ is particularly prone to degradation by the ADEP-ClpP complex. By preventing cell division, ADEP inhibits a vital cellular process of bacteria that is not targeted by any therapeutically applied antibiotic so far. Their unique multifaceted mechanism of action and antibacterial potency makes them promising lead structures for future antibiotic development.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Effects of ADEPs on the growth of Gram-positive bacteria. ADEP treatment results in strong filamentation of B. subtilis 168 (A) as well as swelling of S. aureus HG001 (C) and S. pneumoniae G9A (E) compared with untreated control cells (B, D, and F, respectively), indicating cell division inhibition. Cells were treated for 4–5 h with inhibitory ADEP concentrations (A, 0.25 μg/mL ADEP2; C, 1 μg/mL ADEP2; and E, 0.8 μg/mL ADEP1). (Scale bars, 5 μm.) Accordingly, growth of ADEP-treated B. subtilis 168 (G) at 0.39 μg/mL ADEP1 (twice the MIC) increases along with the control, whereas the colony forming units (cfu) stagnate (H). At higher concentrations (1.56 μg/mL ADEP1, eight times the MIC), ADEP1 exhibits bactericidal effects, indicated by cell growth inhibition and the concomitant drop in cfu. Growth curves were confirmed by three independent experiments and values of a representative experiment are depicted. MIC, minimal inhibitory concentration.

Fig. 2.

Fig. 2.

ADEP inhibits septum formation in B. subtilis and S. aureus. Fluorescence images show B. subtilis 168 and S. aureus HG001 cells costained with FM5-95 membrane dye and DAPI nucleoid dye after ADEP treatment. (A) B. subtilis 168, control; (B) B. subtilis 168, plus 0.25 μg/mL ADEP2; (C) B. subtilis 168 ΔclpP (strain QB4916), plus 0.25 μg/mL ADEP2; (D) S. aureus HG001, control; and (E) S. aureus HG001, plus 1.0 μg/mL ADEP2. After 60 min of ADEP treatment, septum formation is strictly inhibited in both wild-type species, whereas septa were normally formed in the ADEP-treated clpP deletion background, demonstrating an essential role of ClpP for ADEP activity. (Scale bars, 5 μm.)

Fig. 3.

Fig. 3.

ADEP inhibits Z-ring formation in B. subtilis. Fluorescence images show the localization of GFP-tagged FtsZ of B. subtilis 168 strain 2020 during exponential growth in the absence or presence of 0.25 μg/mL ADEP2. The fluorescence images shown are overlays of GFP (green) and FM5-95 (red) channels. (A) In untreated cells, FtsZ condenses at midcell to form the Z-ring. (B) After 20 min of ADEP treatment, cells uniformly showed delocalization of GFP–FtsZ along with a significant loss of Z-ring formation. (C and D) Lack of Z-rings and resulting filamentous growth after prolonged ADEP treatment. (Scale bars, 5 μm.)

Fig. 4.

Fig. 4.

ADEP triggers the delocalization of PBP2 in S. aureus. In correspondence to the results shown for B. subtilis (Fig. 3), the fluorescence images of GFP-tagged PBP2 of S. aureus strain RNpPBP2-31 show the delocalization of GFP–PBP2 upon ADEP treatment (1 μg/mL), which is most probably because of the inhibition of septum formation at the stage of Z-ring assembly. (Scale bars, 2.5 μm.)

Fig. 5.

Fig. 5.

ADEP induces the ClpP-dependent degradation of FtsZ in bacterial cells. ADEP treatment of exponentially growing WT cells of (A) B. subtilis 168 and (B) S. aureus HG001 resulted in a decreased abundance of FtsZ over time, compared with the untreated control. Immunodetection of FtsZ or DivIVA was performed using specific anti-FtsZ or anti-DivIVA antibodies, respectively, as indicated in the margin. DivIVA served as a housekeeping protein control. (C) Immunodetection of FtsZ in untreated or ADEP-treated B. subtilis strain QB4916 (Δ_clpP_). (D) ADEP-induced degradation of FtsZ after 60 min in B. subtilis strain BJK474 (Δ_spx_ Δ_clpXCE_) and the respective control strains B. subtilis strain BJK424 (Δ_spx_) and B. subtilis PY79 (WT).

Fig. 6.

Fig. 6.

ADEP–ClpP degrades FtsZ and αβ-tubulin in vitro. (A) Time-dependent degradation of purified FtsZ by the ADEP–ClpP complex in vitro. (B and C) Mass spectra of in vitro FtsZ degradation assays containing purified, native FtsZ protein (∼39.8 kDa) and ClpP–6HIS protein (∼22.8 kDa) after incubation for 60 min at 37 °C (B) in the absence or (C) in the presence of 10 μg/mL ADEP2. Compared with the control assay, the mass spectrum of the ADEP2-supplemented degradation assay was characterized by the loss of the FtsZ peak and the concomitant appearance of several peaks in the low-molecular range (<4 kDa). (D) Time-dependent degradation of purified αβ-tubulin by the ADEP–ClpP complex in vitro. Immunodetection of αβ-tubulin was performed using anti–α-tubulin or anti–β-tubulin antibodies as indicated. Both α- and β-tubulin are targets for ADEP–ClpP-dependent degradation; however, β-tubulin is degraded faster than α-tubulin for yet unknown reasons.

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