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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

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.
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pone.0145918.g003: 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.

Mentions: As LPPOs did not target the biosynthesis of any of the tested macromolecules, we decided to use electron microscopy to gain insights into the potential mode of function of these compounds. We treated the cells with LPPOs or water and withdrew aliquots after 15 and 30 min, respectively. The cells were then used for electron microscopy assays. Transmission electron microscopy (TEM) with negative staining technique was used to examine the B. subtilis cell morphology before and after the treatment with LPPOs. Fig 3 shows that the addition of either LPPO resulted in a major damage to the integrity of the cell. The cells appeared to be leaking the intracellular material to the outside, and after the longer incubation with LPPOs the cells even completely disintegrated. The leaking and disintegration were suggestive of an effect on the cytoplasmic membrane.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145918.g003: 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.
Mentions: As LPPOs did not target the biosynthesis of any of the tested macromolecules, we decided to use electron microscopy to gain insights into the potential mode of function of these compounds. We treated the cells with LPPOs or water and withdrew aliquots after 15 and 30 min, respectively. The cells were then used for electron microscopy assays. Transmission electron microscopy (TEM) with negative staining technique was used to examine the B. subtilis cell morphology before and after the treatment with LPPOs. Fig 3 shows that the addition of either LPPO resulted in a major damage to the integrity of the cell. The cells appeared to be leaking the intracellular material to the outside, and after the longer incubation with LPPOs the cells even completely disintegrated. The leaking and disintegration were suggestive of an effect on the cytoplasmic membrane.

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