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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.

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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.
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pone.0145918.g007: 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.

Mentions: To directly study the permeabilizing activity of LPPOs on live cells we determined their effect on membrane permeability by measuring the influx of propidium iodide (PI) to the bacterial cells. The dye enters only cells with compromised membrane, intercalates into double stranded DNA or RNA, resulting in fluorescence increase. The addition of either tested LPPO at 10 μg/ml induced rapid uptake of PI to B. subtilis, E. faecalis and S. epidermidis cells within several seconds, indicating fast kinetics (Fig 7). Lowering the concentration slowed down the kinetics but the compound remained active (S4 Fig). Finally and importantly, neither LPPO affected the cytoplasmic membrane permeability of mouse macrophages while a known pore former melittin [44] did 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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145918.g007: 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.
Mentions: To directly study the permeabilizing activity of LPPOs on live cells we determined their effect on membrane permeability by measuring the influx of propidium iodide (PI) to the bacterial cells. The dye enters only cells with compromised membrane, intercalates into double stranded DNA or RNA, resulting in fluorescence increase. The addition of either tested LPPO at 10 μg/ml induced rapid uptake of PI to B. subtilis, E. faecalis and S. epidermidis cells within several seconds, indicating fast kinetics (Fig 7). Lowering the concentration slowed down the kinetics but the compound remained active (S4 Fig). Finally and importantly, neither LPPO affected the cytoplasmic membrane permeability of mouse macrophages while a known pore former melittin [44] did 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