Insights into the Mechanism of Action of Bactericidal Lipophosphonoxins.

The advantages offered by established antibiotics in the treatment of infectious diseases are endangered due to the increase in the number of antibiotic-resistant bacterial strains. This leads to a need for new antibacterial compounds. Recently, we discovered a series of compounds termed lipophosphonoxins (LPPOs) that exhibit selective cytotoxicity towards Gram-positive bacteria that include pathogens and resistant strains. For further development of these compounds, it was necessary to identify the mechanism of their action and characterize their interaction with eukaryotic cells/organisms in more detail. Here, we show that at their bactericidal concentrations LPPOs localize to the plasmatic membrane in bacteria but not in eukaryotes. In an in vitro system we demonstrate that LPPOs create pores in the membrane. This provides an explanation of their action in vivo where they cause serious damage of the cellular membrane, efflux of the cytosol, and cell disintegration. Further, we show that (i) LPPOs are not genotoxic as determined by the Ames test, (ii) do not cross a monolayer of Caco-2 cells, suggesting they are unable of transepithelial transport, (iii) are well tolerated by living mice when administered orally but not peritoneally, and (iv) are stable at low pH, indicating they could survive the acidic environment in the stomach. Finally, using one of the most potent LPPOs, we attempted and failed to select resistant strains against this compound while we were able to readily select resistant strains against a known antibiotic, rifampicin. In summary, LPPOs represent a new class of compounds with a potential for development as antibacterial agents for topical applications and perhaps also for treatment of gastrointestinal infections

Quantitative Conformational Analysis of Functionally Important Electrostatic Interactions in the Intrinsically Disordered Region of Delta Subunit of Bacterial RNA Polymerase

International audienceElectrostatic interactions play important roles in the functional mechanisms exploited by intrinsically disordered proteins (IDPs). The atomic resolution description of long-range and local structural propensities that can both be crucial for the function of highly charged IDPs presents significant experimental challenges. Here, we investigate the conformational behavior of the δ subunit of RNA polymerase from Bacillus subtilis whose unfolded domain is highly charged, with 7 positively charged amino acids followed by 51 acidic amino acids. Using a specifically designed analytical strategy, we identify transient contacts between the two regions using a combination of NMR paramagnetic relaxation enhancements, residual dipolar couplings (RDCs), chemical shifts, and small-angle scattering. This strategy allows the resolution of long-range and local ensemble averaged structural contributions to the experimental RDCs, and reveals that the negatively charged segment folds back onto the positively charged strand, compacting the conformational sampling of the protein while remaining highly flexible in solution. Mutation of the positively charged region abrogates the long-range contact, leaving the disordered domain in an extended conformation, possibly due to local repulsion of like-charges along the chain. Remarkably, in vitro studies show that this mutation also has a significant effect on transcription activity, and results in diminished cell fitness of the mutated bacteria in vivo. This study highlights the importance of accurately describing electrostatic interactions for understanding the functional mechanisms of IDPs

Conductance.

<p>Conductance of <b>DR5047</b> (<b>A</b>) and <b>DR5026</b> (<b>B</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. <b>C</b>. Representative single channel recordings of <b>DR5047</b> and <b>DR5026</b> in planar lipid membranes.</p

The effect of LPPOs on the biosynthesis of selected macromolecules.

<p>In all panels, <b>DR5026</b> is shown with black circles, <b>DR5047</b> 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. <b>A.</b> The effect on RNA synthesis. Rif, rifampicin. <b>B.</b> The effect on protein synthesis. Cm, chloramphenicol. <b>C</b>. The effect on DNA synthesis. <b>D</b>. The effect on lipid synthesis. Cer, cerulenin. <b>E</b>. 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%.</p

Structures of selected lipophosphonoxins DR5047, DR5026.

<p>Structures of selected lipophosphonoxins DR5047, DR5026.</p

A model of the interaction of DR5026 with a bacterial membrane.

<p>The final state of MD simulation after 55 ns shows <b>DR5026</b> molecules penetrated into the phospholipid bilayer. The nitrogen atoms from the iminosugar modules of <b>DR5026</b> 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.</p

Localization of DR5026 in <i>B</i>. <i>subtilis</i> cells.

<p><b>A</b>. A scheme of the experiment. SN, supernatant; P, pellet. <b>B.</b> HPLC data of supernatant after cell sedimentation, SN1. <b>C.</b> HPLC analysis of cell debris and remaining non-lysed cells, P2. <b>D.</b> HPLC analysis of cell cytoplasm, SN3. <b>E.</b> HPLC analysis of the plasma membrane fraction, P3. The dotted red line: <b>DR5026</b> treated cells; the blue line: mock-treated cells. The arrows indicate where <b>DR5026</b> eluted from the column. The identity of <b>DR5026</b> was confirmed by MS detection.</p

TEM pictures of <i>B</i>. <i>subtilis</i> cells.

<p>0.25% phosphotungstic acid at pH 7.3 was used for staining. <b>A.</b> Untreated. <b>B</b>. Treated with 10 mg/L of <b>DR5026</b> for 15 min. <b>C</b>. Treated with 10 mg/L of <b>DR5026</b> for 30 min. <b>D</b>. Treated with 20 mg/L of <b>DR5026</b> for 15 min. <b>E.</b> Treated with 20 mg/L of <b>DR5026</b> for 30 min. The scale bars in the right-hand corners of the pictures represent 500 nm.</p

<i>B</i>. <i>subtilis</i> 168 develops resistance against rifampicin (rif) but not against DR5026.

<p><i>B</i>. <i>subtilis</i> was incubated with subcytotoxic concentrations of rif (starting at 0.01 mg/L; MIC 0.06 mg/L) and <b>DR5026</b> (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.</p

Permeabilization of cytoplasmic membrane induced by DR5026 and DR5047.

<p>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.</p