(LPS), thereby increasing the permeability of the outer membrane (Hancock et al. 1991). Streptomycin, genta- mycin, and polymixin B have been shown to bind ...
Springer-Verlag 1997
Parasitol Res (1997) 83: 198 – 202
ORIGINAL PAPER
Mohammad Maarouf · Franc¸oise Lawrence Spencer Brown · Malka Robert-Gero
Biochemical alterations in paromomycin-treated Leishmania donovani promastigotes
Received: 13 June 1996 / Accepted: 11 September 1996
Abstract Paromomycin is used for the treatment of leishmaniasis in humans, but little is known about its mechanism of action. Investigating the effect of this antibiotic on promastigotes of Leishmania donovani, we showed that inhibition of the multiplication of these parasites could be related to its effect on RNA synthesis and to modifications of membranous polar lipids and membrane fluidity, leading to altered membrane permeability.
Introduction Leishmaniases are parasitic diseases caused by different species of Leishmania, protozoa transmitted by hematophagous biting insectes, the sandflies. The reservoir hosts are humans and domestic or wild animals. Four main clinical forms are known in humans: visceral, cutaneous, mucocutaneous, and diffuse cutaneous leishmaniasis. Leishmaniasis, found on 4 continents, is endemic in 21 countries in the New World and in 61 in the Old World. The annual incidence is estimated to about 600,000 new officially reported cases, the global prevalence is 12 million cases, and the population at risk totals approximately 350 million (WHO 1995). Treatment of leishmaniasis with sodium stibogluconate, pentamidine, or amphotericin B is expensive and potentially toxic, and the administration of these drugs
may require a prolonged stay in hospital. Paromomycin (PR) is an aminocyclitol aminoglycoside antibiotic that is currently used for treatment of cutaneous and visceral leishmaniasis (El-On et al. 1992; Scott et al. 1992). We are interested in the mechanism of action of this antibiotic in Leishmania (Maarouf et al. 1995). In some protozoa these types of antibiotic are known for their inhibitory effect on protein synthesis (Edlind 1989, 1991). It has also been reported that polycationic and aminoglycoside compounds bind to membrane lipids; gentamycin and polymixin B interact with the Mg2+binding sites on Escherichia coli lipopolysaccharides (LPS), thereby increasing the permeability of the outer membrane (Hancock et al. 1991). Streptomycin, gentamycin, and polymixin B have been shown to bind to LPS and to lipid A of Pseudomonas aeruginosa (Moore et al. 1986). The aim of this work was to study the effect of PR on macromolecular synthesis and on the membrane properties of Leishmania. Herein we report that RNA synthesis is inhibited first by this antibiotic, followed by protein synthesis. Furthermore, the antibiotic induces alterations in the uptake of macromolecular precursors. Our use of fluorescent probes to study membrane fluidity and balance between polar and apolar lipids indicates that PR increases the amount of membranous polar lipids, leading to decreased membrane fluidity.
Materials and methods M.Maarouf1 · F. Lawrence · M. Robert-Gero (&) Institut de Chimie des Substances Naturelles, C.N.R.S., F-91198 Gif-sur-Yvette Cedex, France Tel.: 33 (1) 69-82-30-37; Fax: 33 (1) 69-82-72-47 S. Brown Service de Cytome´trie, Institut des Sciences Ve´ge´tales, Avenue de la Terrasse, Gif-sur-Yvette, France 1 Present address: University of Damascus, Faculty of Pharmacy, Department of Microbiology, Damascus, Syria
Materials The radioactive precursors [methyl-3H]-thymidine (20 Ci/mmol), [5, 6-3H]-uridine (39.70 Ci/mmol), [35S]-L -methionine (1140 Ci/ mmol), and [4, 5-3H]-L -leucine (60 Ci/mmol) were obtained from DuPont NEN (France); PR, chloramphenicol, and cycloheximide came from Sigma (France). Neomycin was kindly provided by Dr. S. Gero (ICSN-CNRS, Gif-sur-Yvette, France), and all compounds used were of the highest purity available. The fluorescent probes 1-(4-trimethylammonium)-6-phenylhexa-1,3,5-triene p-toluene sulfonate (TMA-DPH) and Nile red were purchased from Molecular Probes (Eugene, ore. USA).
199 Parasites Leishmania donovani strain MHOM/ IN/ 80/ DD8, originating from the strain collection of the WHO reference center in the London School of Hygiene and Tropical Medicine (UK), was provided by Dr. D.A. Evans. Promastigotes were routinely grown at 26°C in semidefined RPMI 1640 medium containing 2 mM glutamine, 25 mM HEPES (pH 7.4), and 10% heat-inactivated fetal calf serum (Bioproducts Boehringer, France). PR was dissolved in sterilized water prior to its addition to the cultures.
Determination of growth-inhibitory concentrations Of a culture of promastigotes (106 cells/ml), 1 ml was seeded in 24well Nunclon plates and incubated at 26°C for 3 days in the absence or presence of various concentrations of PR. The colorimetric MTT dye-reduction assay (Microculture tetrazolium test; Mossman 1983) was used to measure cell proliferation. Growth inhibition was calculated with respect to the optical density (OD570 nm) measured with untreated control cells.
PR effect on DNA, RNA, and protein syntheses Parasites were seeded as described above in 24-well Nunclon plates. Promastigotes of L. donovani were treated with 150 lM PR for various periods. Assays were carried out in duplicate using untreated cells as a control. Cells were incubated at 26°C. In all, 5 lCi of an appropriate labeled precursor was added to the cultures for 10 min at the end of the treatment. The uptake and the incorporation of radioactive molecules were measured after fixation and permeabilization of the cells by incubation with 5% trichloroacetic acid (TCA) for 20 min at 4°C. Centrifugation at 8,000 g for 5 min permitted the separation of the supernatant (TCA-soluble fraction) from the pellet (TCA-insoluble fraction). Protein concentrations were determined on the insoluble fraction after hydrolysis with 1 M NaOH for 1 h at 80°C by the dye-binding method of Bradford (1976) using bovine serum albumin as the standard. The radioactivity incorporated into the insoluble fraction is expressed in counts per minute per milligram of protein.
with PBS to a cellular density of 107 promastigotes ml–1 and then stained. For some experiments carried out to check the reversibility of the PR effect, promastigotes treated for 72 h were centrifuged, resuspended in medium without PR for 24 h, and prepared as described above. All experiments were conducted at 22°C under incandescent light using glassware rather than plastic. Determination of membrane fluidity by flow cytometry Promastigotes treated or not treated with PR were prepared as described above, stained by 2 lM TMA-DPH for 2 min, and then analyzed by flow cytometry. Membrane fluidity was assessed by measurement of the fluorescence anisotropy (r) of TMA-DPH (Fox and Delohery 1987; Gantet et al. 1990) following its insertion into the plasma membrane of promastigotes. The EPICS flow cytometer (Coulter, Hialeah, Fla.) had a 100lm nozzole and standard optic such that each cell was exposed and measured for about 1.5 ls, i.e., 2 orders of magnitude above the fluorescence lifetime of these probes. The argon laser (SpectraPhysics 2025–05) with vertically polarized output was set at 351– 364 nm and 50 mW. The signals were processed on an analog card linked to a 1,024-channel ADC to derive the blue emission anisotropy (r) as described by Rodes et al (1994). The forward-angle light scatter (FALS) was used to gate the analysis upon intact cells. The mean r value was calculated on 10,000 cells. Determination of lipids Promastigotes treated or not treated with PR were prepared as described for fluidity measurements, stained by 100 ng ml–1 Nile red for 5 min, and then individually analyzed by flow cytometry. The excitation wavelength of the same argon laser was set at 488 nm and 400 mW. The fluorescent emission was filtered through a 515-nm long-pass filter (interference and absorbance) and split with a 590-nm dichroic mirror, sending the red component through a 610-nm long-pass filter to one photomultiplier and the yellow component through a 560-nm short-pass fillter to another photomultiplier. Cellular fluorescence in the absence of dye was negligible. In each analysis, 10,000 promastigotes were studied. The parameter of FALS and the intensity of red (IRF) and yellow fluorescence (IYF) were measured.
PR effect on cytoplasmic and mitochondrial protein syntheses Of a promastigote culture in the exponential growth phase (107cells/ml), 1 ml was seeded in each well of 24-well Nunclon plates. A first treatment for 3 h with 10 mM PR was performed, followed by a second one for 10 min with either 7 mM chloramphenicol or 70 lM cycloheximide. In parallel, cells were treated with the same concentration of either chloramphenicol or cycloheximide alone. Labeled methionine (100 lCi/ml) was always added for the last 10 min of the treatment. The incorporation of methionine was determined as described above. Fluorescent probes TMA-DPH was prepared as a 2 mM stock solution in dimethylformamide and was stored at 4°C. An intermediate dilution of 50 lM was prepared daily from the 2 mM stock with phosphatebuffered saline (PBS), agitated vigorously for 10 min to eliminate the solvent, and used for staining. Nile red was prepared at 100 lg ml)1 (0.3 lM) in acetone and was conserved at 4°C in a dark environment.
Results and discussion Effect of PR on parasite multiplication The doubling time of untreated, exponentially growing promastigotes was 9 h. PR inhibited the growth in a time- and concentration-dependent manner. This antibiotic was most efficient when added to the cultures near the end of the logarithmic phase. The 50% growth-inhibitory concentration (IC50 value) after 72 h of contact was 120 ± 20 lM for the strain studied. Neomycin, a very close structural analog of PR, had only a weak effect on these parasites, as 400 lM of this antibiotic decreased the viability by only 20% under the same conditions.
Treatment of cells for flow cytometry
Effect of PR on macromolecular biosynthesis and protein profile
Promastigotes (20–30 × 106 ml–1) were treated for various periods with 200 lM PR or 400 lM neomycin in 50-ml Nunclon flasks containing 5 ml of culture. Control and treated cells were diluted
As shown in Table 1, PR affected the uptake and incorporation of the precursors. It very rapidly (up to 3 h)
200 Table 1 Effect of 150 lM PR on the uptake and incorporation of radioactive precursors into macromolecules of promastigotes of Leishmania donovani DD8 Results (mean values for 3 independent
experiments) are expressed as percentages with respect to untreated cultures (NM Not measured, Incorp. incorporation)
Treatment
Thymidine
Uridine
time(h)
Uptake
Incorp.
Uptake
Incorp.
Uptake
Incorp.
1 3 6 24 48
115 152 NM NM NM
121 113 NM NM NM
94 77 62 NM NM
79 66 7 NM NM
110 NM 75 51 49
90 NM 95 45 30
stimulated both thymidine uptake into the cells and its further incorporation into DNA. Under the same experimental conditions, uridine uptake and incorporation were inhibited, the latter being more severely affected (93% inhibition after 6 h). The effect on protein synthesis was slower, leucine uptake being decreased after 6 h of contact, but a significant effect on the incorporation was observed at 48 h. This late inhibition of protein synthesis may have been a consequence of the inhibition of RNA synthesis. Analysis of the protein profile of PR-treated promastigotes revealed overexpression of the two main heat-shock proteins (HSP) of 70 and 83 kDa (data not shown). Cycloheximide and chloramphenicol are known to inhibit specifically cytoplasmic and mitochondrial protein synthesis, respectively. The combined addition of PR with either cycloheximide or chloramphenicol increased the inhibition of protein synthesis, indicating that PR affects both types (Table 2). These results are in agreement with our previous finding that PR interferes with the dissociation of mitochondrial and cytoplasmic ribosomes (Maarouf et al. 1995). Decreased transcription and translation induced by PR have been reported in Escherichia coli (Jime´rez 1976), Tetrahymena thermophila (Eustice and Wilhelm 1984), and wheat germ (Wilhelm et al. 1978). Effect of PR on membrane fluidity The above-mentioned changes in the uptake properties of PR-treated cells suggest that PR also alters some membrane properties. Analysis by flow cytometry showed that in exponentially growing promastigotes, PR
Table 3 Effect of PR on the membrane fluidity of promastigotes as assessed by emission anisotropy r of TMA-DPH Treatment, reversibility testing, and cell staining were carried out as described in Materials and methods. Data are given as r values and represent mean values ± SD for 3 experiments
Phase of growth
Leucine
Table 2 Effect of PR on mitochondrial and cytoplasmic protein syntheses in L. donovani. Cells were treated as described in Materials and methods. Results are expressed as the percentage of inhibition of methionine incorporation in treated cells with respect to untreated controls. Data represent mean values ± SD for 3 independent experiments Conditions
Promastigotes
Control PR Cycloheximide Chloramphenicol PR + cycloheximide PR + chloramphenicol
100 47.0 51.5 43.0 78.5 76.0
Stationary
1.0 3.5 1.0 1.5 1.0
at 200 lM affected the membrane slowly, as a significant increase in the r value could be measured only from 24 h onward. The antibiotic raised the r value by 45% after 72 h, indicating a rigidification of the membrane (Table 3). When the 72-h-treated promastigotes were washed and recultured in PR-free medium for 24 h, the r value remained high, indicating a rather stable alteration (Table 3). This stability indicates that the effect of this positively charged antibiotic was not due to a simple binding to negatively charged phospholipids. Although steady-state fluorescence anisotropy is only partly dependent on membrane fluidity, these results reveal a rigidifying effect of PR on membranes of treated promastigotes. Neomycin at 400 lM did not modify membrane fluidity (Table 3), although fluorescence microscopy showed an accumulation of TMA-DPH in the outer membrane of the treated cells. As stationary-phase promastigotes conserve their physiologic integrity only briefly (24–48 h), we used a
Conditions of treatment
r 24 h
Logarithmic
± ± ± ± ±
Control PR 200 lM Neomycin 400 lM Control PR 250 lM PR 500 lM
0.144 0.168 0.144 0.145 0.151 0.151
± ± ± ± ± ±
0.020 0.001 0.020 0.008 0.009 0.004
72 h
Reversion
0.144 ± 0.003 0.209 ± 0.036 0.147 ± 0.010 – – –
0.144 ± 0.002 0.196 ± 0.004 0.144 ± 0.002 – – –
201
24-h treatment with high PR concentrations (up to 500 lM). Under these conditions the membrane fluidity of these cells was not significantly affected (Table 3). Effect of PR on lipid metabolism The rigidification of the membrane is probably a consequence of the PR-induced modification of membranous lipid metabolism. A stimulated synthesis (or a stimulated recycling of catabolized lipids) of polar lipids in the membrane could lead to destabilization of the membrane structure and to its consequent rigidification. Nile red is a vital dye that emits a predominantly red fluorescence in polar hydrophobic domains (phospholipids) and a yellow fluorescence in neutral hydrophobic domains (Greenspan et al. 1985). Untreated promastigotes in the logarithmic phase of growth, stained with Nile red and examined by light microscopy, showed that yellow fluorescence was emitted by intracytoplasmic lipidic droplets, whereas red fluorescence was localized in the membrane. In cells treated with 200 lM PR the yellow fluorescence increased by 38% and the red fluorescence increased by 36% after 24 h; after 72 h these increases amounted to 44% and 107%, respectively (Table 4). This indicates a stimulated synthesis of polar lipids localized in the membrane and also a significant increase in the synthesis of intracytoplasmic apolar lipids. After resuspension of 72-h-treated promastigotes in PR-free medium for 24 h, the emission of yellow fluorescence decreased to control values, whereas the effect on polar lipids (red fluorescence) was only partially reversible. In the presence of 400 lM neomycin for 72 h, the synthesis of both lipid types was slightly reduced. Promastigotes in the stationary phase of growth, treated with 250–500 lM PR for 24 h, showed an insignificant increase in red fluorescence (13–15%) and a decrease in yellow fluorescence (11–30%). The FALS value recorded for Nile red-labeled promastigotes treated with 200 lM PR increased by 51% and 130% after 24 and 72 h, respectively. This effect was at least partially reversible in the absence of the antibiotic. Neomycin at 400 lM had no effect on the FALS value after 72 h of treatment (Table 4). This parameter was not modified in stationary-phase promastigotes either. The parameter FALS reflects both the cell volume and the refractive index gradient between the cell surface and its medium (Salzman et al. 1979; Fouchet et al. 1993). Microscopic observations indicated that PRtreated promastigotes were no larger than control ones. We propose that the structural changes induced by increased amounts of polar lipids led to a higher refractive index for the membrane, increasing the FALS value. Furthermore, microscopic examination also showed an increase in the amounts of cytoplasmic apolar lipid droplets. These drops also could have modified the refringence of the cytoplasm and contributed to the increase in FALS. Such drops are considered a sign of toxicity in rat fibroblast cells (Rodes et al. 1994).
Table 4 Effect of PR and neomycin on FALS and on the fluorescence of exponential-phase promastigotes labeled with Nile red. Promastigotes were treated with 200 l M PR for various periods, or with 400 l M neomycin for 72 h. Reversions were carried out for 24 h in PR-free medium on 72-h-treated cells. Measurements were performed as mentioned in Materials and methods. Data represent mean values ± SD for 3 experiments (IRF Intensity of red fluorescence, IYF intensity of yellow fluorescence) Treatment
45 min PR 4 h PR 8 h PR 24 h PR 72 h PR Reversion 24 h 72 h Neomycin
∆% IRF
IYF
FALS
3 23 55 36 107 49 –34
0 13 23 38 44 )15 )28
)1 )1 +1 51 130 52 6
± ± ± ±
10 39 28 8
±9 ± 13 ±9 ±3
± ± ± ±
14 30 9 5
In conclusion, PR affects macromolecular synthesis, leading to growth arrest. Its effect on membrane fluidity and lipid metabolism, provoking altered uptake properties, should also contribute to the leishmanicidal effect of this antibiotic. That neomycin does not alter membrane fluidity indicates that this target is important for the antileishmanial activity of PR, and that the amino group in position 6’’’ of neomycin (which is absent in PR) is involved in this lack of activity
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