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Keywords:

  • antimicrobials;
  • cytotoxicity;
  • HUVEC;
  • mitochondria;
  • pluronic acid;
  • Pseudomonas aeruginosa

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  CSA-13 is an antimicrobial cationic steroid with some toxicity against eukaryotic cells. The purpose of this work was to test whether pluronic acid F-127 could interfere with the toxicity of CSA-13 on human umbilical vein endothelial (HUVEC) without modifying its bactericidal activity against Pseudomonas aeruginosa.

Methods and Results:  The addition of pluronic acid F-127 slightly decreased the number of dead cells after exposure to CSA-13. Pluronic acid F-127 blocked the permeabilizing effect of CSA-13 on the plasma membrane of HUVEC (uptake of ethidium bromide, release of lactate dehydrogenase) without modifying its toxic effect on their mitochondrial function (MTT test, uptake of tetramethyl rhodamine ethyl ester).

Conclusion:  Pluronic acid F-127 decreased the toxicity of CSA-13 against eukaryotic cells without completely protecting them from mitochondrial damage at high concentrations of the drug.

Significance and Impact of the Study:  This work establishes that studies on the toxic effects of synthetic antimicrobials on eukaryotic cells should not only focus on the permeability of the plasma membrane but also on the integrity of the mitochondria.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cystic fibrosis (CF) is a congenital exocrinopathology provoked by the mutation of a chloride channel, the cystic fibrosis transmembrane regulator expressed by exocrine glands. The mutation of this channel greatly impairs its activity and the movements of ions in exocrine cells. This accounts for the high viscosity of the pancreatic or bronchial secretions which provokes either obstructions of the pancreatic secretory ducts and pancreatic insufficiency or bronchiolar obstructions and lobar atelectasis (Di Sant’Agnese 1956). The disease leads to the premature death of the patients after recurrent and intractable infections of the upper airways.

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacteria especially present in water and soil. It is also very often responsible for opportunistic infections in immunocompromised patients, and this pathogen is the predominant cause of fatal pulmonary infections in patients with CF (Gomez and Prince 2007). Ps. aeruginosa interacts with the highly viscous mucus of these patients and adheres to epithelial cells of their respiratory airways. This initial adhesion is followed by the secretion of exopolysaccharides. The formation of this organic matrix is at the basis of a complex tridimensional structure, the biofilm (Høiby et al. 2010). The transfer of bacteria from planktonic conditions to a biofilm is coupled to a change in gene regulation and expression. This switch contributes to the increased resistance of these bacteria to antibiotherapy (Folsom et al. 2010).

The tracheal and bronchial epithelia are constantly exposed to environmental pathogens. Colonization of the upper airways by these micro-organisms is prevented by innate mechanisms of defences. Antimicrobial peptides, which are part of the innate immune system, play a role in this prevention (Seil et al. 2010). These peptides are overexpressed in CF lungs (Chen et al. 2004) and are potentially able to prevent infections by Ps. aeruginosa (Bals et al. 1999). They are sensitive to the proteases secreted by Ps. aeruginosa. They also form a complex with different components of the biofilm like DNA (Bucki et al. 2007a) or glycosaminoglycans which inhibit their antimicrobial activity (Benincasa et al. 2009; Bergsson et al. 2009).

Considering the very interesting potentialities of the antimicrobial peptides as bactericidal drugs (Bucki et al. 2007a), attempts were made to develop alternatives to these peptides which would be resistant to the proteases and which would not be inhibited by the components of the bacterial biofilms. Ceragenins were synthesized by the addition of several aminoalkyl groups to cholic acid (Li et al. 1999). These compounds, like antimicrobial peptides, are facially amphiphiles and are active against both Gram-positive and Gram-negative bacteria (Schmidt et al. 2001). Contrary to the antimicrobial peptides, they are resistant to proteases and are not inactivated by components of the biofilms (Bucki et al. 2007b). They can sensitize Ps. aeruginosa within biofilms to antibiotics (Nagant et al. 2010), and we have recently shown that a ceragenin like CSA-13 can prevent the initial phase of the biofilm formation (Nagant et al. 2011).

Ceragenins not only interact with the bacterial membranes but at higher concentrations also permeabilize the plasma membrane of eukaryotic. This toxic effect limits their clinical use (Nagant et al. 2010). Attempts were made to attenuate the deleterious effects of ceragenins. The haemolytic activity of the ceragenin CSA-13 was greatly reduced when the erythrocytes were exposed to the drug in the presence of pluronic acid F-127 (Leszczyńska et al. 2011).

The protocol used by Leszczynska and collaborators only tested the permeabilizing properties of CSA-13 at the plasma membrane level. Ceragenins which disrupt the integrity of the bacterial membranes and which are derived from bile acids might be deleterious to mitochondria (Voronina et al. 2004). Pluronic acid F-127 itself might also affect cellular metabolism at the mitochondrial level (Alakhova et al. 2010). It thus seemed mandatory to reconsider the effect of the combination of pluronic acid F-127 plus CSA-13 on nucleated cells with aerobic as well as anaerobic metabolism. In this study, we examined the interaction between pluronic acid F-127 and CSA-13 on Ps. aeruginosa and on human umbilical vein endothelial cells (HUVEC). Our results show that pluronic acid F-127 decreased the sensitivity of bacteria to CSA-13. The surfactant also inhibited the deleterious effect of CSA-13 on the plasma membrane of HUVEC. Exposure of eukaryotic cells to CSA-13 dissipated their mitochondrial membrane potential in the absence or in the presence of pluronic acid F-127. We thus conclude that CSA-13 not only affects the integrity of the plasma membrane of eukaryotic cells but has also toxic effects on their mitochondria. Pluronic acid F-127 can only relieve part of these deleterious effects and cannot thus be proposed as a dispersing agent neutralizing the toxicity of the cationic steroid.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Measurement of the uptake of propidium iodide (PI) by Pseudomonas aeruginosa

The permeability of the bacteria was estimated using PI (López-Amorós et al. 1995). Bacteria of the Ps. aeruginosa ATCC 15692 PAO1 strain were incubated at 37°C overnight in cation-adjusted Mueller–Hinton broth (CAMHB) under constant agitation. Cells were washed three times and resuspended in 1 m mol l−1 potassium phosphate buffer (PPB; pH7) to a final OD600nm of 0·100 ± 0·005. Various concentrations of CSA-13 alone or with 10% pluronic acid F-127 were diluted in PPB, and 100 μl of these solutions was plated in a 96-well microplate. One hundred microlitres of the bacterial suspension containing 12 μ mol l−1 PI (Invitrogen, Groningen, The Netherlands) was added to each well. A control (100 μl of the bacterial suspension containing 12 μ mol l−1 PI and 100 μl of PPB) and a blank (200 μl of PPB containing 6 μ mol l−1 PI) were included in the experiment. The fluorescence was monitored for 1 h in a microplate reader (Synergy HT; BioTek, Winooski, VT, USA) at excitation and emission wavelengths of 540 and 590 nm, respectively.

Measure of the viability of Pseudomonas aeruginosa

An overnight growth culture of PA01 at 37°C in CAMHB was adjusted to a final OD600nm of 0·100 ± 0·005. One hundred microlitres of the bacterial suspension was added in the wells of a 96-well microtitre plate. The cells were grown at 37°C for 3 and 24 h in the presence of 100 μl of increasing concentrations of CSA-13 and in the absence or in the presence of 5% pluronic acid F-127. At the end of the incubation, the bacterial suspensions were diluted 10²- to 106-fold. One millilitre of each dilution was plated in duplicate on a Tripticase soy agar (TSA) plate and the number of colonies was counted after an incubation at 32°C for 48 h. Results were expressed as colony-forming units (CFU) ml−1.

Measure of the uptake of ethidium bromide by human umbilical vein endothelial cells

Human umbilical vein endothelial cell cells were cultured to confluence in endothelial cell growth medium in a T75 flask at 37°C in a humidified atmosphere of 5% CO2. The cells were detached using 10% trypsin-EDTA. They were plated overnight in 96-well black-walled plates (96F Nunclon Delta Black Microwell SI; VWR, Leuven, Belgium) and maintained in culture medium for a minimum of 12 h. Ten minutes prior to the measurement of the fluorescence, the media was removed and ethidium bromide was added to the wells at a 10 mg l−1 final concentration. The plates were incubated at 37°C in the microplate reader. The light emitted at 590 nm after excitation at 540 nm was measured every 15 s. The basal rate of uptake of ethidium bromide was measured for 5 min. The cells were then exposed to various concentrations of CSA-13 alone or in the presence of 3% pluronic acid F-127. The measurement in the presence of the drug was performed for 50 min.

Release of lactate dehydrogenase (LDH)

The permeabilization of the plasma membrane of HUVEC was estimated by measuring the release of LDH in the extracellular medium. After incubation of the cells for 12 h in a 96-well plate, the medium was replaced and various concentrations of CSA-13 (in the presence or absence of 3% pluronic acid F-127) were added to each well. The plate was incubated at 37°C for 10 min. At the end of the incubation, the plate was centrifuged at 1200 rev min−1 pm for 10 min and 30 μl of the supernatant was transferred to a new clear walled 96-well plate. The LDH activity was measured as previously described (Métioui et al. 1994). A NADH solution (TRIS 56 m mol l−1, EGTA 5·6 m mol l−1 and 170 μ mol l−1β-NADH) was added to each well. The assay was started by the addition of 1·22 m mol l−1 (final concentration) pyruvate to the wells. The activity of LDH was assayed at 30°C for 20 min. The absorbance was measured at 340 nm. Blank samples were obtained using the supernatant of cells not treated with CSA-13 and pluronic acid F-127. Total LDH activity was measured after the lysis of HUVEC by sonication for 15 s at a 15 μm amplitude (Soniprep 150; MSE, London, UK). For each assay, the results were obtained by calculating the rate of decrease of the absorbance provoked by the oxidation of NADH. Each value was then corrected by subtracting the blank values, and the release of LDH was expressed as percentage of the total cellular content.

Tetrazolium test (MTT test)

The toxicity of CSA-13 on eukaryotic cells was estimated by measuring the ability of eukaryotic cells to reduce the yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to purple formazan. This reduction is catalysed by oxidoreductases which use electrons from mitochondria-generated NADH as the primary reductants (Mosmann 1983). HUVEC were grown overnight in 96-well plates at 37°C in a CO2 atmosphere. The next day, the incubation medium was discarded and replaced by fresh medium containing various concentrations of CSA-13 with or without 6% pluronic acid F-127. The cells were incubated for 20 min at 37°C, the medium was removed before the addition of 100 μl MTT solution (1 mg ml−1 medium) to the wells. After the incubation of the cells with MTT for 3·5 h, formazan crystals developed in respiring and early apoptotic cells. These crystals were solubilized with dimethylsulphoxide and the absorbance measured at 540 nm with the microplate reader.

Estimation of the mitochondrial membrane potential

The integrity of the mitochondria was studied as described using tetramethylrhodamine ethyl ester (TMRE), a cell-permeable fluorescent probe sequestered by active mitochondria (Bermpohl et al. 2005). HUVEC plated in a 24-well plate were incubated in a phosphate-buffered saline (PBS) solution for 1 h in the presence of various concentrations of CSA-13, with or without 5% pluronic acid F-127. After removal of the medium, the cells were incubated for 1 h in PBS containing 200 n mol l−1 TMRE. At the end of this second incubation, the medium was removed and the cells were washed twice with PBS. One millilitre of a 1% (v/v) Triton X-100 solution was added to the wells to lyse the cells. Two hundred microlitres of the homogenate was transferred to the wells of a black-walled microplate. The fluorescence was measured with the microplate reader (excitation wavelength 544 nm and emission wavelength 590 nm).

Statistical analysis

Results were analysed using a two-way anova followed by a Bonferroni post-test performed for each dose.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Evaluation of the toxicity of CSA-13 on Pseudomonas aeruginosa

The fluorescence of bacteria incubated at 37°C in the absence of CSA-13 and in the presence of PI did not change during 60 min confirming that the bacteria excluded PI (Fig. 1, upper panel). CSA-13, in the 0-10 mg l−1 concentration range, sharply increased the uptake of PI during the first 10 min (Fig. 1, upper panel). The uptake of PI decreased at higher concentrations of the drug. As shown in the middle panel of Fig. 1, the addition of 5% pluronic acid F-127 to the medium had no significant effect on the basal fluorescence of the cells. It did not significantly affect either the response to CSA-13 (Fig. 1, lower panel).

image

Figure 1.  Effect of CSA-13 on the uptake of PI by Pseudomonas aeruginosa. Upper and middle panels: The bacteria were incubated for various times in the presence of 6 μ mol l−1 PI, in the presence of increasing concentrations of CSA-13 and in the absence (upper panel) or in the presence (middle panel) of 5% pluronic acid F-127. For the clarity of the graphs, one in every six points has been plotted. Lower panel: The bacteria were incubated for 10 min in the presence of 6 μ mol l−1 PI, in the presence of increasing concentrations of CSA-13 in the absence (closed triangles, full line) or in the presence (open circles, dotted line) of 5% pluronic acid F-127. Results were expressed as arbitrary fluorescence units (AFU) and are the means ± SEM of three independent experiments performed in duplicate. (inline image) Control; (inline image) CSA-13 10 mg l−1; (inline image) CSA-13 50 mg l−1; (inline image) CSA-13 100 mg l−1; (inline image) CSA-13 and (inline image) CSA-13 with pluronic acid F-127.

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Effect of CSA-13 on the viability of Pseudomonas aeruginosa

In a next experiment, the viability of bacteria exposed to various concentrations of CSA-13 was examined. As shown in Fig. 2, the drug dose dependently decreased the viability of Ps. aeruginosa. After an incubation of 3 h, 5 mg l−1 CSA-13 decreased by 99% the number of colony-forming units (CFU), and all the bacteria were killed by a 10-fold higher concentration of CSA-13. Similar results were observed after 24 h except that all the bacteria were killed at a 20 mg l−1 concentration of CSA-13. In the presence of 5% pluronic acid F-127, the antimicrobial property of CSA-13 was maintained but at a slightly lower level. After 3 h, 20 mg l−1 CSA-13 decreased the number of CFU by more than 5·6 and 2·5 log in the absence and in the presence of pluronic acid F-127, respectively (Fig. 2, upper panel). After 24 h, this dose of CSA-13 decreased the number of CFU by more than 7 log and by 3·8 log in the absence or in the presence of pluronic acid F-127 (Fig. 2, lower panel).

image

Figure 2.  Effect of CSA-13 on the viability of Pseudomonas aeruginosa. The bacteria were incubated for three (upper panel) or 24 h (lower panel) in the presence of increasing concentrations of CSA-13 and in the absence (solid line, closed triangles) or in the presence (dotted line, open circles) of 5% pluronic acid F-127. At the end of the incubation, an aliquot of the bacteria was diluted and cultured for 48 h on TSA plates. The colony-forming units (CFU) were counted. After correction for the dilution factors, the number of CFU was plotted as a function of the concentration of CSA-13. Results were expressed as the means and standard deviation of two independent experiments performed in duplicate. (inline image) CSA-13 and (inline image) CSA-13 with pluronic acid F-127.

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Effect of pluronic acid F-127 on the uptake of ethidium bromide by human umbilical vein endothelial cell

In basal conditions, the rate of uptake of ethidium bromide was small and averaged 0·009 ± 0·016 (n = 6) arbitrary fluorescence units (AFU) per second (Fig. 3, upper panel). The addition of 100 mg l−1 CSA-13 increased the rate of the uptake of ethidium bromide to 1·860 ± 0·475 AFU per second (n = 6) after 10 min (Fig. 3, lower panel). Adding 3% pluronic acid F-127 to the medium increased the basal uptake of ethidium bromide (0·527 ± 0·051 (n = 9) AFU per second) (Fig. 3, middle panel). It inhibited the increase of the uptake in response to 50 and 100 mg l−1 CSA-13 (from 1·851 ± 0·480 (n = 6) AFU per second for 100 mg l−1 CSA-13 alone to 0·464 ± 0·114 (n = 7) AFU per second in the presence of CSA-13 and 3% pluronic acid F-127, P < 0·001) (Fig. 3, lower panel).

image

Figure 3.  Effect of CSA-13 on the uptake of ethidium bromide by human umbilical vein endothelial cell (HUVEC). Upper and middle panels: HUVEC were incubated at 37°C in the presence of 10 mg l−1 ethidium bromide in control conditions (upper panel) or in the presence of 3% pluronic acid F-127 (middle panel). Five minutes after the start of the measurement of the fluorescence, the cells were exposed to various concentrations of CSA-13 and the fluorescence was measured for 50 min. For the clarity of the graphs, one every six points has been plotted. The results were expressed as arbitrary fluorescence units (AFU). Lower panel: HUVEC were incubated as described above. The rate of ethidium uptake was measured between 5 min (just before the addition of CSA-13) and 15 min (i.e. 10 min after the addition of CSA-13). The increase in the rate of ethidium bromide uptake (AFU per second) was plotted as a function of the concentration of CSA-13. Results were the means ± SEM of at least six experiments. ***, P < 0·001. (inline image) Control; (•inline image)CSA-13 20 mg l−1 (inline image) CSA-13 30 mg l−1; (inline image) CSA-13 50 mg l−1; (inline image) CSA-13 100 mg l−1; (inline image) CSA-13; and (inline image) CSA-13 with pluronic acid F-127.

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Effect of pluronic acid F-127 on the release of lactate dehydrogenase by human umbilical vein endothelial cell

Lactate dehydrogenase is a cytosolic enzyme with a tetrameric quaternary structure. It has a molecular weight close to 140 kDa and is thus unable to cross the plasma membrane. Its presence in the extracellular fluid is a consequence of the permeabilization of the membrane. As shown in Fig. 4, concentrations of CSA-13 higher than 5 mg/L dose dependently increased the release of LDH. At a 100 mg l−1 CSA-13 concentration, half of the cellular LDH was released in the medium (52 ± 4%, n = 12) within 10 min. In the presence of 3% pluronic acid F-127, only concentrations of CSA-13 higher than 30 mg l−1 increased the release of LDH. Pluronic acid F-127 reduced the release of LDH provoked by 100 mg l−1 CSA-13 from 52 ± 4% (n = 12) to 21 ± 2% (n = 9), P < 0·001.

image

Figure 4.  Effect of CSA-13 on the release of lactate dehydrogenase (LDH) by human umbilical vein endothelial cell (HUVEC). HUVEC were incubated at 37°C for 10 min in the presence of increasing concentrations of CSA-13 in the absence (closed triangles, solid line) or in the presence (open circles, dotted line) of 3% pluronic acid F-127. The LDH released in the medium at the end of the incubation was assayed as described in Materials and Methods. Results were expressed as per cent of the maximal LDH activity in the cells. They are the means ± SEM of at least three experiments. **, P < 0·01; ***, P < 0·001. (inline image) CSA-13 and (inline image) CSA-13 with pluronic acid F-127.

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Effect of pluronic acid F-127 on the MTT test

As shown in Fig. 5, 30 mg l−1 CSA-13 decreased the absorbance of the reduced formazan to 59 ± 5% of the absorbance measured in control conditions (n = 6). The absorbance further dropped at higher concentrations of the cationic steroid and reached 25 ± 3% (n = 11) at 100 mg l−1 CSA-13. The addition of 6% pluronic acid F-127 to the medium had no effect by itself on the absorbance of the reduced formozan (from 0·328 ± 0·046, n = 10 in the absence to 0·321 ± 0·007, n = 12 in the presence of pluronic acid F-127). It slightly but significantly displaced the dose–response curve for CSA-13 to higher concentrations without affecting the maximal effect of the drug (Fig. 5).

image

Figure 5.  Effect of CSA-13 on the MTT test in human umbilical vein endothelial cell (HUVEC). HUVEC were incubated at 37°C for 20 min in the presence of increasing concentrations of CSA-13 in the absence (solid line, closed triangles) or in the presence (dotted line, open circles) of 6% pluronic acid F-127. At the end of the incubation, the incubation medium was removed and a solution of MTT was added to each well. The absorbance measured at 540 nm was used to estimate the formazan produced during the reaction. The results were expressed as the percentage of the absorbance measured in control conditions. They are the means ± SEM of at least three experiments. (inline image) CSA-13 and (inline image) CSA-13 with pluronic acid F-127.

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Effect of pluronic acid F-127 on the mitochondrial membrane potential

The potential of the inner membrane of mitochondria is a major component of the protomotive force responsible for the synthesis of ATP by the mitochondria. TMRE is a dye which accumulates inside intact mitochondria with a negative membrane potential (Scaduto and Grotyohann 1999). When the cells are incubated in the presence of TMRE, the dye accumulates in their mitochondria and increases their fluorescence. The dissipation of the mitochondrial inner membrane potential impairs the uptake of the dye and decreases the cellular fluorescence. As shown in Fig. 6, the fluorescence associated with HUVEC was greatly reduced by a preincubation for 1 h with increasing concentrations of CSA-13. At a 5 and 50 mg l−1 concentration, the drug decreased by, respectively, 60 and 85% the fluorescence associated with the cells (Fig. 6, middle panel). This result confirmed that CSA-13 impaired mitochondrial function. The addition of 5% pluronic acid F-127 to the medium did not significantly affect the basal fluorescence (from 68 612 ± 6504 AFU (n = 8) in the absence to 59 990 ± 2728 AFU (n = 6) in the presence of pluronic acid F-127, P > 0·05) and did not modify the toxic effect of CSA-13 on mitochondria (Fig. 6, middle panel). In a last experiment, the toxic effect of CSA-13 was studied at two different pH’s. As shown in Fig. 6, the dissipation of the potential of the inner mitochondrial membrane by CSA-13 (with or without pluronic acid F-127) was not affected by an incubation of HUVEC at pH 6 (Fig. 6, left panel) or 8 (Fig. 6, right panel) rather than 7.

image

Figure 6.  Effect of CSA-13 on the uptake of tetramethyl rhodamine ethyl ester (TMRE) by human umbilical vein endothelial cell (HUVEC). HUVEC were incubated for 1 h at 37°C in a phosphate buffer at pH 6 (left panel), 7 (middle panel) or 8 (right panel). During this incubation, they were exposed to various concentrations of CSA-13 in the absence (solid lines, closed triangles) or in the presence (dotted lines, open circles) of 5% pluronic acid F-127. After removal of the medium, the cells were incubated for 1 h in the presence of TMRE. At the end of this second incubation, the fluorescence of the cells was estimated as described in Materials and Methods. Results were expressed as percentage of the fluorescence measured in cells incubated for 1 h in control conditions before exposure to TMRE. They are the means ± SEM of five experiments (inline image) CSA-13 and (inline image) CSA-13 with pluronic acid F-127.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In this work, we confirmed that CSA-13 could eradicate high populations of Ps. aeruginosa in planktonic cultures at a concentration (20 mg l−1) with marginal toxicity towards eukaryotic cells. The antimicrobial effect of CSA-13 has been explained by the interaction of the drug with the bacterial membrane. The drug is derived from cholic acid and possesses aminoalkyl groups conferring positive charges to the molecule. It behaves like a facially amphiphile with a hydrophobic side formed by the sterane nucleus and a polar face with the positive charges of the secondary amine groups. First, the drug interacts through its cationic side with the charges of the bacterial membrane and then its hydrophobic surface destroys the membrane (Epand et al. 2007).

The uptake of PI observed in this work (Fig. 1) was secondary to the permeabilization of the membrane by CSA-13, an observation in agreement with the antimicrobial property of the drug. High concentrations of CSA-13 increased the release of haemoglobin from erythrocytes (Leszczyńska et al. 2011), the uptake of ethidium bromide and the release of LDH from HUVEC (Nagant et al. 2010). Our present results further establish that CSA-13 also affected the integrity of mitochondria. In the MTT test, CSA-13 inhibited the reduction of tetrazole by HUVEC, suggesting that the mitochondria of these cells had lost their ability to transfer electrons (Fig. 5). This was confirmed by measuring the uptake of TMRE (Fig. 6). This fluorescent dye accumulates in mitochondria according to the electrical potential across the inner mitochondrial membrane (Scaduto and Grotyohann 1999). CSA-13 decreased the mitochondrial uptake of the dye as reflected by the decrease in the cellular fluorescence after exposure to the drug. This is to our knowledge the first report on an effect of CSA-13 on mitochondria. These results are based on in vitro testing, and it is not clear how interactions of CSA-13 with mitochondria will impact toxicity to eukaryotic cells in vivo. Preliminary results from in vivo experiments indicate that therapeutic concentrations of CSA-13 do not cause local toxicity. Concentrations of CSA-13 necessary to prevent bacterial colonization of medical devices are expected to be well below concentrations found in this work to impact mitochondrial function. Further in vivo experiments will be necessary to determine whether potential adverse effects of CSA-13 on mitochondria decrease the therapeutic window in which this antimicrobial drug can be used.

Considering that CSA-13 is a facially amphiphile, we hypothesized that it would behave like a cationic lipid. The non-ionic form of the drug should passively diffuse through the plasma membrane according to its concentration gradient. Once in the cytoplasm, the drug should protonate which would maintain the transmembrane gradient of the non-ionic form of the drug. The pKa values of the various amine groups of CSA-13 are unknown but secondary amines usually dissociate at pH around 10. According to the Henderson-Hasselbach equation, an increase in the extracellular pH from 6 to 8 should increase 100-fold the concentration of the neutral form of the drug (Hallifax and Houston 2006). Our results show that, in spite of the increase in the non-ionic form of CSA-13, the effect of the drug on mitochondria remained unchanged (Fig. 6). This result did not support the model of the diffusion of neutral CSA-13 and suggested that the drug accumulates in the cytosol by a transport mechanism. The drug is derived from cholic acid which crosses the plasma membrane not only by diffusion but also by facilitated and active transporters (Dawson et al. 2009). Some of these transporters are ubiquitous (Ballatori et al. 2009). The synthesis of drugs combining cholic acid and an active molecule has been proposed to facilitate the cellular uptake of these prodrugs by the various transporters of the bile salt (Sievänen 2007). It is thus conceivable that these transporters can also translocate CSA-13 through the plasma membrane. This model is in agreement with recent results of Trainor et al. (2011) who reported that the resistance of Helicobacter pylori to CSA-13 involved HelC (Trainor et al. 2011), an efflux pump able to transport both bile salts and ceragenins. Once inside the cell, the drug might easily interact with the mitochondrial membranes.

Leszczyńska et al. recently reported that adding pluronic acid F-127 to the solution of CSA-13 prevented its haemolytic effect on erythrocytes (Leszczyńska et al. 2011). It did not significantly interfere with its antimicrobial properties. Pluronic acid F-127 is a non-ionic surfactant. It is also a thermoreversible hydrogel which is more soluble at low temperature and which forms a gel at body temperature. It is currently used as a vehicle in pharmaceutical preparations (Escobar-Chávez et al. 2006). Pluronic acid F-127 shifted to the right of the dose–response curve of CSA-13 on the killing of Ps. aeruginosa (Fig. 2). A similar inhibitory trend was reported by Leszczyńska and collaborators (see Fig. 1b in Leszczyńska et al. 2011). The short incubation time used by these authors (1 h) might explain why, according to them, this inhibition was not significant. The selection of the most relevant incubation time depends, in large measure, on the time that the drug is present and available in a living organism. There are no data on the in vivo half-life of CSA-13 but this half-life is likely between 1 h (the incubation time used by Leszczyńska and collaborators) and 24 h (the incubation time necessary to observe a strong inhibition by pluronic acid F-127, see Fig. 2b). Thus, data collected after 3 h (Fig. 2a) were probably more relevant and, at this time, the inhibition exerted by pluronic acid F-127 was significant but rather marginal. In agreement with Leszczyńska et al. (2011), a lower concentration of pluronic acid F-127 (3%) inhibited the permeabilization of the plasma membrane of eukaryotic cells by CSA-13 as estimated by the uptake of ethidium bromide or the release of LDH. But pluronic acid F-127, even at a 6% concentration, did not inhibit the deleterious effect of CSA-13 on mitochondrial function measured by the MTT test and the uptake of TMRE. The discrepancy between the tests measuring plasma membrane integrity and mitochondrial function is reminiscent of the results of Braff et al. (2005). These authors tested the toxicity of LL-37 towards keratinocytes. They reported that, at a 10 μ mol l−1 concentration, the peptide decreased the reduction of tetrazolium (MTT test) without modifying the release of LDH.

In conclusion, our results show that antimicrobial drugs might affect the viability of eukaryotic cells by two mechanisms. First, they increase the permeability of the plasma membrane and the subsequent release of cytosolic components. Second, when they have access to the intracellular compartment, they interfere with the mitochondrial electron transport. Toxicological studies should thus take both mechanisms into consideration.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by grant no. 3.4577.10 from the Fonds National de la Recherche Scientifique (F.N.R.S.) of Belgium. C.N. is a research fellow of the FNRS. The authors wish to thank Asma Hafid for her skilfull technical help during this work.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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