Fig 1.
Structures of selected lipophosphonoxins DR5047, DR5026.
Table 1.
Antibacterial activity of lipophosphonoxins DR5047, DR5026.
Fig 2.
The effect of LPPOs on the biosynthesis of selected macromolecules.
In all panels, DR5026 is shown with black circles, DR5047 grey circles, and control (no compound added) empty circles. The red symbols depict the effect of a known inhibitor. The amount of the radiolabeled material incorporated at the time of inhibitor addition (shown with arrows) was set as 1. A. The effect on RNA synthesis. Rif, rifampicin. B. The effect on protein synthesis. Cm, chloramphenicol. C. The effect on DNA synthesis. D. The effect on lipid synthesis. Cer, cerulenin. E. The effect on cell wall synthesis. Amp, ampicillin. The experiments were conducted in three biological replicates. Representative experiments are shown. The error was below 10%.
Fig 3.
TEM pictures of B. subtilis cells.
0.25% phosphotungstic acid at pH 7.3 was used for staining. A. Untreated. B. Treated with 10 mg/L of DR5026 for 15 min. C. Treated with 10 mg/L of DR5026 for 30 min. D. Treated with 20 mg/L of DR5026 for 15 min. E. Treated with 20 mg/L of DR5026 for 30 min. The scale bars in the right-hand corners of the pictures represent 500 nm.
Fig 4.
Localization of DR5026 in B. subtilis cells.
A. A scheme of the experiment. SN, supernatant; P, pellet. B. HPLC data of supernatant after cell sedimentation, SN1. C. HPLC analysis of cell debris and remaining non-lysed cells, P2. D. HPLC analysis of cell cytoplasm, SN3. E. HPLC analysis of the plasma membrane fraction, P3. The dotted red line: DR5026 treated cells; the blue line: mock-treated cells. The arrows indicate where DR5026 eluted from the column. The identity of DR5026 was confirmed by MS detection.
Fig 5.
Leakage of contents for LUVs of different lipid composition measured by the release of CF (increase in fluorescence intensity).
The maximum CF release was induced by 0.1% (v/v) Triton X-100. Total phospholipid concentration was 60 μM and temperature was kept at 25°C. LPPO concentration was 0.8 mg/L.
Fig 6.
Conductance of DR5047 (A) and DR5026 (B) single pores measured in 1M KCl, 10 mM Tris, pH 7.4 at membrane potential of 45 mV. The histograms of different conductance states were fitted with Gaussian functions. C. Representative single channel recordings of DR5047 and DR5026 in planar lipid membranes.
Fig 7.
Permeabilization of cytoplasmic membrane induced by DR5026 and DR5047.
At time 0 s permeabilizing agents were added to the cells at final concentrations of 10 mg/L or 10 μM for LPPOs and melittin, respectively. Increase in fluorescence intensity signifies that the membrane impermeant dye PI entered the cells.
Fig 8.
Interaction of fluorescently labeled LPPO DR5823 with model membrane.
(A) Excitation (solid line) and emission (dotted line) spectra of DR5823 in buffer (in green) and liposomes (in red). (B) Incorporation of DR5823 to the liposome membrane.
Fig 9.
A model of the interaction of DR5026 with a bacterial membrane.
The final state of MD simulation after 55 ns shows DR5026 molecules penetrated into the phospholipid bilayer. The nitrogen atoms from the iminosugar modules of DR5026 are highlighted as blue spheres. Phosphorus atoms of the phospholipid bilayer (PB) are depicted as yellow/red spheres. For clarity, almost all atoms of the PB are hidden.
Table 2.
Calculated LogD values for DR-5047 and DR-5026.
Fig 10.
B. subtilis 168 develops resistance against rifampicin (rif) but not against DR5026.
B. subtilis was incubated with subcytotoxic concentrations of rif (starting at 0.01 mg/L; MIC 0.06 mg/L) and DR5026 (0.5 mg/L MIC ~ 3 mg/L) and grown for 24 h. Then, aliquots of the cultures were transferred to new tubes with fresh medium and a two-fold increased concentration of the active compound. A binary representation is shown; 1 indicates growth of the cells, i. e. their resistance to the respective compound; 0 represents lack of growth—the cells were sensitive to the compound and no resistant cell appeared within the time frame of the experiment. The experiment was conducted in three biological replicates with the same results.