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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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Related in: MedlinePlus

Conductance.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.
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pone.0145918.g006: Conductance.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.

Mentions: To investigate in detail the permeabilizing effect of LPPOs we tested them for pore-forming activity by conductance measurements in planar lipid membranes (black lipid membranes, BLM) formed from asolectin. When LPPOs were added, abrupt stepwise variations in membrane current were observed, indicative of opening and closing of individual pores. The pore conductance histograms and the representative current traces are shown in Fig 6. Consistent with the faster CF leakage rate from liposomes induced by DR5026, this LPPO formed most frequently pores of higher conductance than DR5047. The most frequent single pore conductance step observed for DR5026 was 310 ± 128 pS. The variance of pore conductance was rather broad for DR5047, and could be described by three conductance distributions. The most frequent conductance step resulted in 70 ± 37 pS; however, conductance states of 270 ± 79 pS and 510 ± 77 pS were observed as well. The sizes of pores formed by both LPPOs are within the range of other well-characterized pore-forming agents. Under comparable experimental conditions (1 M salt solution) the LPPOs’ major conductance units were larger than those induced by gramicidin A that forms ca. 20 pS channels, which are ~0.4 nm wide [42, 43]. On the other hand, a more potent pore-former such as alamethicin forms ca. 600 pS channels of ~2 nm in diameter.


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)

Conductance.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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145918.g006: Conductance.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.
Mentions: To investigate in detail the permeabilizing effect of LPPOs we tested them for pore-forming activity by conductance measurements in planar lipid membranes (black lipid membranes, BLM) formed from asolectin. When LPPOs were added, abrupt stepwise variations in membrane current were observed, indicative of opening and closing of individual pores. The pore conductance histograms and the representative current traces are shown in Fig 6. Consistent with the faster CF leakage rate from liposomes induced by DR5026, this LPPO formed most frequently pores of higher conductance than DR5047. The most frequent single pore conductance step observed for DR5026 was 310 ± 128 pS. The variance of pore conductance was rather broad for DR5047, and could be described by three conductance distributions. The most frequent conductance step resulted in 70 ± 37 pS; however, conductance states of 270 ± 79 pS and 510 ± 77 pS were observed as well. The sizes of pores formed by both LPPOs are within the range of other well-characterized pore-forming agents. Under comparable experimental conditions (1 M salt solution) the LPPOs’ major conductance units were larger than those induced by gramicidin A that forms ca. 20 pS channels, which are ~0.4 nm wide [42, 43]. On the other hand, a more potent pore-former such as alamethicin forms ca. 600 pS channels of ~2 nm in diameter.

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