1 Institut für Medizinische Mikrobiologie und Immunologie der Universität Bonn, D-53105 Bonn, Germany; 2 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100 Israel
Received 22 September 2003; returned 14 October 2003; revised 19 November 2003; accepted 20 November 2003
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Abstract |
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Methods: MIC determinations and chequerboard titrations followed established procedures. Mode of action studies included killing kinetics and a series of experiments designed to characterize the impact of the diastereomeric peptides on bacterial membranes.
Results: The tested diastereomers displayed high antimicrobial and broad spectrum activity with amphipathic-2D being the most active peptide. Synergic activities were observed with individual strains. Mode of action studies clearly demonstrated that the cytoplasmic membrane is a primary target for the peptides and that membrane disruption constitutes a significant bactericidal activity for the major fraction of a bacterial population. However, depending on the indicator strain, the results also suggest that additional molecular events contribute to the overall activity.
Keywords: synergic activities, membrane permeabilization, intracellular targets
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Introduction |
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The increasing resistance of bacteria to conventional antibiotics encouraged strong efforts to develop antimicrobial agents with new mechanisms of action. Recently, a variety of de novo designed AMPs have been synthesized combining optimized characteristics such as high positive charge, amphipathicity and -helicity. Unfortunately, high antimicrobial activity was frequently accompanied by relatively high haemolytic activity.8 Furthermore, all L-amino acid amphipathic peptides are sensitive to degradation and clearance by serum components. In recent studies, D-amino acids were incorporated into cytolytic peptides and although the resulting diastereomers simultaneously lost their
-helical structure and haemolytic activity, they retained high antibacterial activity, thus providing a basis for designing novel peptide antibiotics composed of D- and L-amino acids that are selective to microorganisms.11,12 The diastereomers were composed of leucine and lysine (15 amino acids), one-third of which were D-amino acids.13 These peptides had been evaluated with regard to their activity against standard laboratory strains, their haemolytic activity, their structural features and mode of action on model membranes, demonstrating their high potential for the development of new target antibiotics.
In this paper, we describe the in vitro activities of these diastereomers, on their own and in combination with conventional antibiotics, against a set of clinical isolates. To study the hypothesis that permeabilization of the cytoplasmic membrane is responsible for killing, we correlated cell viability and various cytoplasmic membrane functions under identical experimental conditions. Our data strongly suggest that membrane perturbation represents a major event for killing; however, additional lethal or sublethal events clearly contribute to killing of individual strains.
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Materials and methods |
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Eighteen clinical isolates from distinct patients as well as two standard laboratory strains were included in this study: Staphylococcus simulans 22; Micrococcus luteus; Enterococcus faecium (two strains, I-11305b and I-11054); Staphylococcus haemolyticus, coagulase-negative (CoNS; I-10925); Staphylococcus epidermidis, methicillin-resistant (MRSE; LT1324); Staphylococcus aureus, methicillin-susceptible (two strains, 5185 and I-11574); Staphylococcus aureus, methicillin-resistant (MRSA; two strains, LT1338 and LT1334); Citrobacter freundii (I-11090); Klebsiella pneumoniae (I-10910); Escherichia coli (two strains, I-11276b and O-19592); Stenotrophomonas maltophila (two strains, O-16451 and I-10717); Pseudomonas aeruginosa (two strains, 4991 and I-10968); Candida albicans (two strains, I-11301 and I-11134). The strains were maintained on MuellerHinton (MH) agar or on blood agar.
Growth media
All strains were subcultured weekly on blood agar (Becton Dickinson, Erembodegem, Belgium) or MuellerHinton (MH; Oxoid, Basingstoke, UK) agar. For the efflux experiments, PYG medium containing 0.2% Bacto-Peptone (Difco, MI, USA), 0.8% glucose (Merck, Darmstadt, Germany), 0.4% yeast extract (Oxoid), 2 mM potassium phosphate (Merck) (pH 7) was used.
MIC determinations
The determination of MICs was carried out in microtitre plate assays. Since cationic peptides bind to polystyrene, 96-well polypropylene plates (Life Technologies) were used throughout.14 Binding became particularly obvious at low MICs, e.g. below 1 µM, and was further reduced by coating with bovine serum albumin (BSA). A series of two-fold dilutions in MH broth was prepared from a stock solution of the respective peptide (16 µM). The indicator strains were grown to an optical density (600 nm) of 1.0 in half-concentrated MH broth and diluted 1:105 with the same medium. Then 100 µL of this suspension was mixed with 100 µL of the peptide dilution in the well of a microtitre plate. After incubation for 18 h at 37°C, the MIC was read as the lowest concentration of antimicrobial agent resulting in the complete inhibition of visible growth and results given are mean values of three independent determinations.
Chequerboard titrations
Synergic activities were assayed by chequerboard titrations with half-concentrated MH broth. Fractional inhibitory concentration (FIC) indices were calculated as follows: [(A)/MICA] + [(B)/MICB] = FICA + FICB = FIC index, where MICA and MICB are the MICs of drugs A and B when used alone, and (A) and (B) are the MICs of drugs A and B when used in combination. The interaction was defined as synergic if the FIC index was 0.5 and antagonistic if the FIC index was >4.0; no interaction was observed if the FIC index was >0.54.0.15
Killing kinetics
All strains were grown overnight in half-concentrated MH broth and diluted in fresh medium to an optical density of 0.1. Peptides were added in concentrations corresponding to 1, 2 and 4 times the MIC as determined after 18 h. The viable count was monitored up to 22 h. Aliquots were taken at defined intervals, diluted in 10 mM potassium phosphate buffer, and 100 µL of the dilutions were plated in triplicate on MH agar. The plates were incubated at 37°C and the colony forming units (cfu) were counted after 24 h. Since we expected an inoculum effect on the killing kinetics, we carried out MIC determinations not only with the standard inoculum of 105 cfu/mL, but also with 108 cfu/mL and did not see significant differences between the MIC values, particularly when BSA coated microtitre plates were used.
Efflux of radioactively labelled glutamate
The influence of peptide amphipathic-2D on the uptake and retention of glutamate was investigated as described previously with some modifications.16 Briefly, strains were cultured in PYG medium at 37°C to an absorbance of 1.0 at 600 nm. Centrifuged cells were resuspended 1:3 in fresh medium, which was supplemented with 100 µg of chloramphenicol per mL to prevent glutamate incorporation. After 15 min of pre-incubation, radiolabelled L-[3H]glutamate (42 Ci/mMol) was added (final concentration 10 µCi/mL), and the culture was immediately divided into two parts. One aliquot was transferred into a flask that already contained amphipathic-2D (10 times the MIC for the respective strain) to tests its effect on the uptake of glutamate; the other part was run as control. After 30 min of incubation, the control was further subdivided into two aliquots, one of which received amphipathic-2D (10 times the MIC) to follow the effect on pre-accumulated amino acids. Samples of 100 µL were filtered through cellulose acetate filters (pore size, 0.2 µm; Schleicher & Schüll, Dassel, Germany) and washed twice with 5 mL of 200 mM potassium phosphate buffer (pH 7) containing 100 µM unlabelled glutamate. Filters were dried and transferred to counting vials filled with scintillation fluid (Quickszint 100; Zinsser, Frankfurt, Germany). The radioactivity was measured in a beta-counter (1900CA; Packard, Downers Grove, IL, USA).
Estimation of the membrane potential
Cells were grown in PYG at 37°C to an absorbance of 1 at 600 nm, centrifuged and resuspended 1:3 in fresh medium. To monitor the membrane potential, 1 µCi/mL of [3H]tetraphenylphosphonium bromide (TPP+; 26 Ci/mMol) was added. TPP+ is a lipophilic cation which diffuses across the bacterial membrane in response to a trans-negative . The culture was treated with amphipathic-2D (10 times the MIC for the respective strain) and samples were filtered and washed as described above. Counts were corrected for unspecific binding of [3H]TPP+ by subtracting the radioactivity of 10% butanol-treated cell aliquots. For calculation of the membrane potential (
), TPP+ concentrations were applied to the Nernst equation [
= (2.3 x R x T/F) x log (TPP+in/TPP+out)]. A mean
was calculated from a minimum of two independent determinations.
Fluorometric assay for membrane potential
Cells were grown in half-concentrated MH broth to an OD600 of 0.5 and incubated for 5 min with 1 µM of the membrane-potential-sensitive fluorescent probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3); Molecular Probes, Leiden, The Netherlands]. Amphipathic-2D was added at a concentration corresponding to 10 times the MIC. Fluorescence was measured at the excitation and emission wavelengths of 492 and 515 nm, respectively.
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Results |
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The primary structures of the diastereomers, all of which were amidated at their C terminus and had a charge of +7, are shown in Table 1. All peptides were derived from an -helical lytic peptide composed of L-lysine and L-leucine (15 amino acids) either by only introducing five D-amino acids or by additional sequence alteration.13 The antibacterial activity of the peptides was tested against a set of fresh clinical isolates including both Gram-positive and Gram-negative species as well as two Candida albicans strains with varying susceptibility patterns. We selected strains with multiple resistances, including those of current clinical concern such as 4-quinolone-resistant Enterobacteriaceae, methicillin-resistant S. aureus, VanA-type vancomycin-resistant enterococci and genera of high natural resistance such as Stenotrophomonas and Pseudomonas. When possible, we included, in addition to two well-characterized laboratory strains, the respective susceptible counterparts. Peptides were tested with a concentration of up to 8 µg/mL corresponding to 4 µM, which is of relevance for the development of a new antibiotic drug.
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Chequerboard titrations were carried out using peptide amphipathic-2D in combination with different conventional antibiotics against selected indicator strains (Table 3). In some cases, amphipathic-2D showed some remarkable synergy with ß-lactams (ampicillin, oxacillin and cefazolin) against some strains. Synergy was also observed for combinations of amphipathic-2D with the DNA gyrase inhibitor ciprofloxacin. All other FIC indices ranged between >0.5 and 4.0 (no interaction); remarkably, no indications of antagonism (FIC of >4.0) were obtained.
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We selected four species of Gram-positive cocci with comparable in vitro susceptibility (MICs of 1 or 2 µM, respectively) and peptide amphipathic-2D for a closer inspection of the killing activity of the diastereomeric peptides. Short-term experiments were carried out to study the immediate impact of the peptides on cells (Figure 1, inserts) and long-term experiments (22 h) for detection of survivors and possible regrowth. The effect of amphipathic-2D was clearly dose-dependent. At 2x MIC, only E. faecium showed a continuous decrease in the number of cfu over 22 h (Figure 1b) whereas with all three staphylococcal strains, survival of subpopulations and subsequent regrowth was observed. At 4x MIC, this was still true for the MRSA strain; complete killing was only achieved with S. simulans 22. With this strain, we also observed the most immediate impact of the peptide on the cells. At time-point zero, i.e. within seconds after addition of the peptide, the number of cfu was reduced by several log and killing proceeded most rapidly for the first 10 min and then slowed down. The enterococcal strain showed similar killing kinetics. With the MRSA strain, there was no immediate impact of the peptide and the number of cfu was only reduced by less than 2 log within 30 min of incubation. The CoNS strain behaved very differently in that, after rapid reduction in cfu in the first 2 min, cells recovered in the broth culture over the next 20 min before the number of cfu decreased again.
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Influence of amphipathic-2D on amino acid accumulation in whole cells
Effects on the integrity of the cytoplasmic membrane should impair energy-dependent transport phenomena. Therefore, we examined the uptake of radioactively labelled glutamate into chloramphenicol-treated Gram-positive bacteria. S. simulans, when pretreated with amphipathic-2D, was unable to carry out active transport of glutamate (Figure 2a). The addition of amphipathic-2D to cells which had accumulated glutamate induced a rapid efflux of the marker within a few minutes. In contrast, the clinical isolates were able to transport glutamate in the presence of amphipathic-2D to some extent. With the enterococcal and the MRSA strain, the uptake was approximately 50% of the untreated control. In contrast, the CoNS strain was hardly impaired (Figure 2c); addition of amphipathic-2D to this strain after accumulation of the glutamate induced rapid efflux; however, the amino acid was completely re-imported within a few minutes. No significant efflux was observed with E. faecium. When compared with the killing kinetics, the results with S. simulans and S. aureus agree well: S. simulans was killed rapidly and, within the time frame of the uptake experiment, there were not enough viable cells left that could carry out glutamate transport. The MRSA strain was killed more slowly and the remaining viable cells could be responsible for the uptake observed. The efflux kinetics were comparable for both strains. Also, the results with the CoNS strain agreed to some extent since in both assays the strain showed short-term recovery from the action of the peptide. In contrast, the enterococcal cells, 99% of which were inactivated within the first 10 min, retained a substantial capacity for transport, indicating that the actual killing may be delayed until plating.
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The potential across the cytoplasmic membrane () of bacterial cells can be estimated by measuring the distribution of a radiolabelled, lipophilic cation such as tetraphenylphosphonium bromide (TPP+). Using this technique, we could show that S. simulans 22 maintained a trans-negative
of approximately 140 mV, which rapidly decreased after addition of amphipathic-2D; however,
did not drop below 100 mV (Figure 3a). The clinical strains initially showed a
of about 100 to 120 mV (Figure 3b and c). E. faecium was only transiently depolarized and
was re-established within about 5 min.
of the CoNS and MRSA strains were hardly affected by the peptide. Clinical strains are known to frequently carry transport proteins of the S. aureus QacA-type which export lipophilic drugs and which had also been reported to use the TPP+ probe as a substrate.17,18 This was recently noticed with a pair of isogenic strains expressing qacA (from plasmid pSK1) and devoid of the plasmid, respectively. In the qacA-expressing mutant,
was underestimated by approximately 50 mV (U. Pag, M. Brown, R. H. Skurray, H.-G. Sahl, unpublished data). The presence of such a multidrug transporter leads to underestimation of
in our clinical strains and could explain the relatively low initial potential level observed. Therefore we also used the fluorescent membrane potential probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] to monitor the effect of amphipathic-2D (Figure 4). Applying this probe, we observed an increase in fluorescence with all strains, being most rapid with S. simulans and rather slower with the MRSA strain. Use of fluorescent probes does not allow any quantification of
or changes in
; however, since the changes in fluorescence were similar with all peptides, the degree of depolarization may be similar to that measured with TPP+ and S. simulans, i.e. approximately 40 mV. Unlike with TPP+, depolarization did not seem to be reversible; however, since DiBAC4(3) is known to traverse depolarized membranes and to subsequently bind to cytoplasmic constituents, its diffusion back across the membrane upon repolarization may be considerably slower than with TPP+ and therefore this dye may not be suitable for detection of transient effects. In contrast, TPP+ hardly binds to cellular components and responds quickly to changes in polarization. Taken together, both methods indicate that amphipathic-2D may impair energized bacterial membranes; however, the effect observed on
seems to be too small to fully explain the rapid killing induced by the peptide.
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Discussion |
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Synergy with conventional antibiotics
Combination studies showed that amphipathic-2D may act synergically with or add positively to the activity of conventional antibiotics such as ß-lactams and fluoroquinolones. However, in most cases, the positive interactions were restricted to a particular test strain and a general rule for a given species or combination could not be detected. The mechanism of the synergic effect remains largely unknown; we observed both amphipathic-2D showing improved activity while the activity of the conventional antibiotic remained unchanged and vice versa; in a few cases, both compounds increased in activity when combined. Thus, through the activity of cell wall biosynthesis inhibitors, the wall architecture may be disturbed and access of amphipathic-2D to the cytoplasmic membrane may be increased, whereas membrane disorganization by the peptide may improve the activity of ß-lactams or help gyrase inhibitors to cross the cytoplasmic membrane. Recently, synergy between cationic peptides (buforin II, magainin II or cecropin P1) and ß-lactam antibiotics was also shown for Gram-negative species such as Stenotrophomonas, Acinetobacter or Pseudomonas.2325 Amphiphilic cationic peptides may also cause degradation of the peptidoglycan and enhance the activity of the ß-lactam antibiotics, by triggering the activity of bacterial murein hydrolases.26 Furthermore, cationic peptides may compete with divalent cations for binding to lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, which results in permeabilization of the outer membrane and promoted access to sites of peptidoglycan synthesis.27 Fosfomycin has been reported to synergize with a number of antibiotics;28,29 however, no synergic effects were observed in combination with amphipathic-2D. In conducting chequerboard titrations, one should recall that such titration assays can only reflect the effectiveness of a combination at a single time point (18 h incubation), but cannot provide information on transient interactions occurring throughout the incubation time.30
Mode of action studies
It is widely accepted that the primary target of cationic AMPs is the cytoplasmic membrane; however, the exact mechanism by which these peptides kill bacteria is still not fully understood. Recent studies on the interaction of the diastereomers with model membranes suggested that the antimicrobial activity is based on membrane perturbation as described by the carpet mechanism.13 The antibiotic peptides are proposed to bind to the surface of the membrane, cover the membrane in a carpet-like manner and disturb its barrier function.12 To test this model with living microbes, we evaluated data from killing curves, efflux experiments and membrane potential, all obtained under identical experimental conditions with various Gram-positive cocci. Treatment of S. simulans 22, a standard laboratory strain which had been used frequently to study the mode of action of antibiotic peptides,16,31,32 yielded a fairly defined picture. We observed rapid killing, significant membrane depolarization and immediate cessation of the uptake as well as rapid efflux of radiolabelled amino acids. Such results are typically seen with membrane permeating agents and are consistent with the data described by Papo et al.13 for spheroplasts of Gram-negative bacteria and intact Gram-positive bacteria.
However, with the clinical isolates, the picture proved more complicated. Killing kinetics were considerably slower and biphasic. Membrane functions, either measured directly as uptake and retention of amino acids or via the membrane potential, appeared to be only partially or transiently impaired; still, the clinical isolates when subjected to standard MIC determination were of comparable susceptibility. In previous studies, it had also been observed that cationic AMPs may vary greatly in their ability to depolarize the cytoplasmic membrane potential of E. coli and that there is no direct correlation between the ability to permeabilize the membrane and the antimicrobial activity.33
Recently, there has been an increasing number of reports on potential intracellular targets for amphiphilic peptides such as buforin II, indolicidin, cecropin PR-39, HNP-1 and tPMP-13437 and several synthetic peptides.38,39 Membrane permeabilization is thought to provide access to the cytoplasm in addition to contributing to killing. The data presented in our study are in line with such views. The effects on membrane functions produced by 10x MIC in this study could be clearly demonstrated; however, they appear insufficient to explain the killing kinetics observed and the significant variation in the response of the four test strains, three of which are closely related staphylococci. In addition to the individual properties of the cytoplasmic membrane itself that may influence the susceptibility to cationic peptides,39 differences in the accessibility of the membrane caused by variations in the molecular set-up of the cell envelope of individual strains may help explain the differences shown for the various clinical isolates. Moreover, not only intracellular targets and processes may suffer from the action of the peptides. S. simulans with its unique cell wall lytic system, has been shown to be particularly susceptible to cationic agents that replace the lytic enzymes and prematurely induce cell separation and lysis of the septum. This effect significantly contributes to the killing efficiency of the lantibiotics nisin and Pep540,41 and may explain the fast and complete killing of this strain observed with amphipathic-2D. In a more general sense, it had been proposed that depolarization of staphylococcal cells would induce autolysis as a mechanism of programmed cell death42,43 (reviewed by Bayles44). Thus, various degrees of membrane damage and different patterns of additional lethal or sublethal effects produced by the peptides may define the susceptibility of an individual strain and could also explain why results for a given combination of amphipathic-2D with a conventional antibiotic ranged from indifference to synergy within strains of the same species.
The fact that the membrane is a universal target that cannot be changed in its principal set-up, is widely used to explain the broad-spectrum activity of AMPs and their conservation throughout evolution as an effective means for the control of microbes. In light of the additional targets and activities identified recently, it seems justified to broaden this view. The cationic amphiphilic nature of AMPs seems perfectly designed to enable a maximum of interaction, hydrophobic as well as hydrophilic, with cellular constituents. Thus, whenever present in significant concentrations, AMPs could interfere with numerous physiological processes and cause damage at many sites in a cell, which makes development of resistance difficult. Indeed, whereas bacteria exposed to innate immunity factors in the course of evolution have clearly become less susceptible to such peptides,45,46 they did not develop high-level resistance mechanisms as observed with many widely used conventional antibiotics. In that respect, the diastereomers studied here offer a new resource for the development of novel anti-infective agents either to be used on their own or in combination with established agents to restore and boost their activity.
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Acknowledgements |
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Footnotes |
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