Antimicrobial lipopeptide tridecaptin A1 selectively binds to Gram-negative lipid II

Abstract

Tridecaptin A₁ (TriA₁) is a nonribosomal lipopeptide with selective antimicrobial activity against Gram-negative bacteria. Here we show that TriA₁ exerts its bactericidal effect by binding to the bacterial cell-wall precursor lipid II on the inner membrane, disrupting the proton motive force. Biochemical and biophysical assays show that binding to the Gram-negative variant of lipid II is required for membrane disruption and that only the proton gradient is dispersed. The NMR solution structure of TriA₁ in dodecylphosphocholine micelles with lipid II has been determined, and molecular modeling was used to provide a structural model of the TriA₁-lipid II complex. These results suggest that TriA₁ kills Gram-negative bacteria by a mechanism of action using a lipid-II-binding motif.

Keywords: antibiotic; lipid II; membrane pore; peptide; peptidoglycan.

Fig. 1.

Structures of the tridecaptin analogs TriA₁ and Oct-TriA₁.

Fig. 2.

(A) Bacterial growth kinetics. Optical densities of E. coli cells exposed to 2× MIC of ampicillin (8 μg/mL), chloramphenicol (32 μg/mL), tridecaptin A₁ (6.25 μg/mL), and polymyxin B (4 μg/mL). Tridecaptin A₁ reduces cell density after 20 min of exposure. (B) Time-kill assays. E. coli cells were treated with 10× MIC of each antibiotic (50 μM), and the number of viable cells was determined at different time points. Tridecaptin A₁ kills cells more slowly than polymyxin B.

Fig. 3.

Inner-membrane assays. (A) Membrane depolarization assay. Addition of TriA₁ to DiBAC₄-treated E. coli cells decreases fluorescence by dilution, but no membrane depolarization is observed. Polymyxin B leads to expected fluorescence increase as the inner membrane is depolarized. (B) Membrane disruption assay. Addition of TriA₁ to SYTOX Green-treated E. coli cells does not cause immediate pore formation. Triton X-100 causes rapid inner-membrane lysis. (C) Disruption of proton motive force detected as fluorescence decrease from BCECF-AM–treated E. coli cells. Glucose increases the proton motive force, increasing the cytoplasmic pH and increasing fluorescence. Valinomycin disperses the electrochemical gradient, and addition of TriA₁ rapidly decreases fluorescence. Subsequent addition of nigericin further decreases fluorescence at a faster rate than TriA₁.

Fig. 4.

(A) Structure of Gram-negative and Gram-positive lipid II. (B) ITC of TriA₁ + Gram-negative lipid II. (C) Spot-on-lawn assay with E. coli cells. (Left) TriA₁ (50 μM). (Middle) 1:1 TriA₁ (100 μM):G⁺LII (100 μM). (Right) 1:1 TriA₁ (100 μM):G⁻LII (100 μM). TriA₁ is active, and premixing with Gram-positive lipid II (G+LII) slightly reduces the zone of inhibition. Premixing with Gram-negative lipid II (G-LII) abolishes activity.

Fig. 5.

(A) In vitro assay measures formation of small proton pores. BCECF is encapsulated in LUVs with an internal pH of 8, and the external buffer is pH 6. Pore formation results in a proton gradient and decrease in fluorescence. (B) BCECF LUVs with no lipid II, 1 mol% Gram-negative lipid II, or 1 mol% Gram-positive lipid II are treated with 1.8 μM Oct-TriA₁. Gram-negative lipid II significantly accelerates pore formation.

Fig. 6.

Total synthesis of (Z,Z)-farnesyl Gram-negative lipid II (1). (A) TMSOTf, 4 Å MS, CH₂Cl₂, rt, 18 h, 61%. (B) (i) ZnCl₂, AcOH/Ac₂O, rt, 24 h (ii) Zn, THF/AcOH/Ac₂O, rt, 24 h, 63% (two steps). (C) (i) H₂, Pd/C, MeOH, rt, 3 h, (ii) (ⁱPr)₂NP(OBn)₂, tetrazole, CH₂Cl₂, rt, 2 h, (iii) 30% H₂O₂/THF, −78 °C, 2 h, 84% (three steps). (D) DBU, CH₂Cl₂, rt, 0.5h, quant. (E) Tetrapeptide 5, TFA/CH₂Cl₂, 2h; HATU, DIPEA, DMF, rt, 24 h, 78%. (F) (i) H₂, Pd/C, MeOH, rt, 2.5 h, (ii) CDI-activated (Z,Z)-farnesyl phosphate, DMF, rt, 4d, (iii) NaOH, H₂O/dioxane, 37 °C, 2 h, 25% (three steps).

Fig. 7.

(A) NMR solution structure of TriA₁ in DPC micelles containing Gram-negative lipid II. Orange: hydrophobic residues; purple: d-Dab8; and cyan: other residues. (B) Lipid II analog 1 docked into TriA₁. Hydrophobic residues interact with the lipid II terpene tail, and the pentapeptide occupies the binding pocket. (C) Modeled interaction shows H-bonding between d-Dab8 and DAP3.

Fig. 8.

Resistance study with E. coli cells.