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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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B. subtilis 168 develops resistance against rifampicin (rif) but not against DR5026.B. subtilis was incubated with subcytotoxic concentrations of rif (starting at 0.01 mg/L; MIC 0.06 mg/L) and DR5026 (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.
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pone.0145918.g010: B. subtilis 168 develops resistance against rifampicin (rif) but not against DR5026.B. subtilis was incubated with subcytotoxic concentrations of rif (starting at 0.01 mg/L; MIC 0.06 mg/L) and DR5026 (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.

Mentions: Finally we tested the potential for the development of resistance against LPPOs in B. subtilis. Rifampicin was used as a control. We started culturing the cells in the presence of subcytotoxic concentrations of either DR5026 or rifampicin. The cells were incubated for 24 hours after which they were diluted into fresh medium containing a two-fold increased concentration of the respective compound. This scheme was followed for several days. Fig 10 shows that while we were able to obtain cells growing in the presence of 5 mg/L of rifampicin (MIC against wt B. subtilis ~0.06 mg/L), we failed to obtain cells that would grow at concentrations exceeding the MIC of DR5026 (~3 mg/L). Subsequently, the induction of resistance of clinically more relevant species was carried out by repeated passages of E. faecalis and S. agalactiae strains with subinhibitory concentrations of LPPOs. A total of 14 passages were performed, after each the MICs were determined. Similarly to B. subtilis, the MICs remained relatively unchanged (S2 Table).


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)

B. subtilis 168 develops resistance against rifampicin (rif) but not against DR5026.B. subtilis was incubated with subcytotoxic concentrations of rif (starting at 0.01 mg/L; MIC 0.06 mg/L) and DR5026 (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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145918.g010: B. subtilis 168 develops resistance against rifampicin (rif) but not against DR5026.B. subtilis was incubated with subcytotoxic concentrations of rif (starting at 0.01 mg/L; MIC 0.06 mg/L) and DR5026 (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.
Mentions: Finally we tested the potential for the development of resistance against LPPOs in B. subtilis. Rifampicin was used as a control. We started culturing the cells in the presence of subcytotoxic concentrations of either DR5026 or rifampicin. The cells were incubated for 24 hours after which they were diluted into fresh medium containing a two-fold increased concentration of the respective compound. This scheme was followed for several days. Fig 10 shows that while we were able to obtain cells growing in the presence of 5 mg/L of rifampicin (MIC against wt B. subtilis ~0.06 mg/L), we failed to obtain cells that would grow at concentrations exceeding the MIC of DR5026 (~3 mg/L). Subsequently, the induction of resistance of clinically more relevant species was carried out by repeated passages of E. faecalis and S. agalactiae strains with subinhibitory concentrations of LPPOs. A total of 14 passages were performed, after each the MICs were determined. Similarly to B. subtilis, the MICs remained relatively unchanged (S2 Table).

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