Limits...
Insights into the Mechanism of Action of Bactericidal Lipophosphonoxins.

Panova N, Zborníková E, Šimák O, Pohl R, Kolář M, Bogdanová K, Večeřová R, Seydlová G, Fišer R, Hadravová R, Šanderová H, Vítovská D, Šiková M, Látal T, Lovecká P, Barvík I, Krásný L, Rejman D - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences v.v.i., Flemingovo nám. 2, 166 10 Prague 6, Czech Republic.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

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%.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4696656&req=5

pone.0145918.g002: 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%.

Mentions: We grew B. subtilis to mid-exponential phase and then added a radiolabeled precursor for the biosynthesis of the macromolecule of interest (e. g. 3H uridine for RNA; for details see Mat&Met). We then distributed the culture into several aliquots, one of which was allowed to grow without the addition of any compound, and to the others we added one or the other LPPO or an antibiotic known to inhibit the synthesis of the particular macromolecule (e. g. rifampicin that binds to RNAP and stops transcription of DNA into RNA;) [41]. We subsequently withdrew aliquots at time points and determined the amount of the radioactive label being incorporated into the macromolecule. As can be seen in Fig 2, the two LPPOs did not significantly affect the biosynthesis of any of the macromolecules while known inhibitors were able to do so.


Insights into the Mechanism of Action of Bactericidal Lipophosphonoxins.

Panova N, Zborníková E, Šimák O, Pohl R, Kolář M, Bogdanová K, Večeřová R, Seydlová G, Fišer R, Hadravová R, Šanderová H, Vítovská D, Šiková M, Látal T, Lovecká P, Barvík I, Krásný L, Rejman D - PLoS ONE (2015)

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%.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696656&req=5

pone.0145918.g002: 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%.
Mentions: We grew B. subtilis to mid-exponential phase and then added a radiolabeled precursor for the biosynthesis of the macromolecule of interest (e. g. 3H uridine for RNA; for details see Mat&Met). We then distributed the culture into several aliquots, one of which was allowed to grow without the addition of any compound, and to the others we added one or the other LPPO or an antibiotic known to inhibit the synthesis of the particular macromolecule (e. g. rifampicin that binds to RNAP and stops transcription of DNA into RNA;) [41]. We subsequently withdrew aliquots at time points and determined the amount of the radioactive label being incorporated into the macromolecule. As can be seen in Fig 2, the two LPPOs did not significantly affect the biosynthesis of any of the macromolecules while known inhibitors were able to do so.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences v.v.i., Flemingovo nám. 2, 166 10 Prague 6, Czech Republic.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus