Antimicrobial activity of the selected peptides
The antimicrobial activity of the selected peptides was assessed by their ability to sterilize a culture of bacteria or fungi in rich growth media such that no growth occurred in a 24 hour incubation. We used Gram positive bacteria (Staphylococcus aureus
), Gram negative bacteria (Escherichia coli
and Pseudomonas aeruginosa
) and a fungus (Cryptococcus neoformans
). The chemical antibiotic ampicillin and the antimicrobial peptide indolicidin from bovine neutrophils17
served as positive controls for the microbes and gave the expected minimal inhibitory concentrations (MICs) (). The perfringolysin O membrane spanning domain (PMSD) which is an anionic membrane spanning beta-hairpin peptide in the context of a large protein toxin18,19
but does not cause leakage as a free peptide (unpublished observation) served as a negative control. The data shown in indicates that all of the selected peptides have potent antimicrobial activity against all of the organisms. We refer to the minimum effective concentration as a minimum sterilizing concentration (MSC) rather than a minimum inhibitory concentration (MIC) because wells with no growth after 24 hours showed few, if any, colony forming units (CFU) when spread on nutrient agar plates compared to 108
CFU/ml in peptide free wells. In fact, sterilized wells remained sterile indefinitely on a bench top at room temperature, thus the microbes are being killed rather than just inhibited. We recently tested these peptides against other microorganisms including the Gram positive bacterium Bacillus anthracis and the fungus Candida albicans and we find the same low micromolar sterilizing activity. The low micromolar, sterilizing antimicrobial activity of these peptides is comparable to the natural antimicrobial peptide indolicidin () and to other natural antimicrobial peptides20,21
. Therefore, the peptides that we selected from the simple combinatorial library using an in vitro
liposome leakage assay are potent, broad-spectrum antimicrobial agents.
Time-course of antimicrobial activity
To help elucidate the killing mechanism, we varied the pre-incubation time and order of addition of peptide, microbes and growth media. The results (not shown) reveal only small changes, if any, in bacterial MSC values for pre-incubation times ranging from the standard three hours to just a few seconds. In fact, even reverse addition (growth media added to cells first, followed immediately by peptide) did not change MSC values substantially. We conclude from these experiments that the lethal step for bacteria occurs immediately and renders the cells incapable of recovering after only a few moments of contact with peptide. This result is consistent with the membrane permeabilization experiments described below which show immediate effects of these peptides on the integrity on microbial membranes.
The fungus Cryptococcus required more than one hour of pre-incubation for complete sterilization, suggesting a different mode of action. Addition of rich growth medium to fungi that had been preincubated with peptides for intermediate times resulted in a reduction in growth of C. neoformans after overnight incubation, but not sterilization. Consistent with these observations, we show below that these peptides permeabilize fungal membranes much more slowly than bacterial membranes.
Selective biological activity
To distinguish selective antimicrobial activity from non-selective lytic activity, we measured the toxicity and lytic activity of the selected peptides using erythrocytes and mammalian cultured cells. In we show the hemolytic activity of the selected peptides against sheep and human erythrocytes. Buffer served as a negative control and 100% hemolysis of cells was achieved by suspending the cells in distilled water for 1 hr. The non-specific lytic peptide melittin gave 100% hemolysis at 5 μM or above. The negative control peptide, PMSD, gave <5% hemolysis at 15 μM. The selected peptides have little hemolytic activity against the more robust sheep erythrocytes. Some have activity against human erythrocytes, but only at the highest concentration. The hemolytic activity is comparable to that caused by the natural antimicrobial peptide indolicidin. Similarly, the membrane active peptides were tested for cytotoxicity against living mammalian cells lines. Cell viability was measured using the MTT assay, which measures the activity of mitochondrial reductases (). The selected peptides have little toxic effect on the mammalian HEK293T and NIH3T3 cells even at 15 μM, which is well above the MSC levels. The non-selective lytic peptide melittin is always 100% lethal at 5 or 15 μM. Among the selected peptides, the rank order of the slight MTT cytotoxicity is similar to the rank order of slight hemolytic activity.
Hemolytic and cytotoxic activity
In some of these experiments, we also used a random pooled peptide stock from the original library to compare selected peptides to the average library peptide. We pooled about 200 randomly chosen peptides from the library and used this peptide pool to evaluate the potential of the library overall prior to selection, on permeabilization in living microorganisms and mammalian cells. In we show that the random pooled peptides cause slight toxicity which is at the same level as the selected antimicrobial peptides.
Biological membrane permeabilization
In order to correlate the selective killing of microbes by these peptides to their selection using an in vitro liposome leakage assay, we tested whether killing of bacterial cells is due to membrane permeabilization. To study the effect of these peptides on the membranes of living microbial cells, we used the fluorescence of SYTOX Green, a membrane impermeant, DNA binding dye. Membrane permeabilization allows entry of the dye, as indicated by an increase in fluorescence. In SYTOX Green fluorescence is monitored after addition of 5 μM peptide to E. coli cells suspended in PBS buffer. Treatment with melittin shows an accelerating activity in which 50% of the maximum SYTOX Green uptake occurred by about 15 minutes and complete SYTOX Green leakage occurred by 25 min. The selected peptides all showed significant SYTOX Green influx. The control AMP indolicidin causes only a very small amount of dye influx, suggesting a different mechanism of antimicrobial activity. The antibiotic ampicillin has no effect in the influx of SYTOX Green into the cells. The selected peptides have a direct effect on the living bacterial inner cell membrane.
Mitochondrial membrane permeabilization
To help elucidate the mechanism of action of these peptides in cells and to establish their versatility, we repeated the SYTOX Green experiments using cell suspensions under three different conditions. Cells re-suspended in PBS (above) will be metabolically dormant due to the lack of nutrients, but they may still have a residual transmembrane electrochemical potential and ATP store. Cells supplemented with growth media (LTM) will have metabolic activity, including a transmembrane potential, and cells treated with 40 μM valinomycin will have fully dissipated transmembrane potential and no ATP stores. We observed little difference in the SYTOX Green influx under these different conditions, as shown in , suggesting that the peptide-membrane interactions and membrane disruption are independent of the metabolic state of the cells. The negative-inside electrochemical potential of bacterial plasma membranes has been suggested to be an important factor in AMP activity and selectivity for microbes22,23
. The data presented here, however, shows that these peptides cause the rapid and extensive permeabilization of microbial membranes independent of the membrane potential.
In we compare membrane permeabilization of other classes of microbes. Consistent with their broad-spectrum activity, the selected peptides permeabilize the membranes of the bacterium S. aureus. Against the fungus C. neoformans the peptides behave somewhat differently. Although all of the selected peptides are potently fungicidal (), some show little leakage of SYTOX Green into the fungal cells compared to the lytic peptide melittin which completely permeabilizes the fungal cells in just a few minutes. This is probably related to the observation, above, that sterilization of C. neoformans requires more than one hour, compared to only seconds for the bacteria. Similar sterilizing antifungal activity and slow activity is observed for these peptides against other fungal species (RR and WCW unpublished results). This observation suggests that the mode of antifungal activity is different from the mechanism of action in bacteria. The mechanism of antifungal activity is under further study.
We also measured the permeabilizing effect of these peptides on living mammalian cells, HEK293T and NIH293T (not shown). Treatment of these cells with 5 μM peptide as in caused no detectable influx of SYTOX Green into cells in 40 minutes. Mean SYTOX Green fluorescence after 40 minutes was -6 ± 8% (SD) of the buffer and melittin controls. Melittin at 5 μM concentration was used as a positive control and it caused maximum dye uptake by the cells in less than 10 minutes. The observation that the selected peptides have little or no permeabilizing effect on mammalian cells is consistent with their lack of hemolytic or cytotoxic activity ().
Peptide effect on membrane potential
The membrane potential sensitive dye diSC3(5) was used to monitor the cytoplasmic membrane depolarization of E. coli cells in the presence of peptides. In we show a typical set of experiments. In each experiment the dye is added to E. coli cells and the fluorescence decreases as the dye accumulates in the membranes and becomes self-quenched. Dissipation of the potential causes an increase in fluorescence due to dye de-quenching. Complete dissipation is given by the valinomycin curve. The potential dissipation caused by the lytic peptide melittin is comparable to that caused by valinomycin, and occurs in less than 30 seconds. Most of the selected peptides affect the membrane potential of cells in less than a minute, although not all to the same extent as melittin and valinomycin. Importantly, this experiment shows that peptide-induced dissipation of the membrane potential can occur in less than one minute, while the SYTOX Green influx requires 10-40 minutes, and often does not even begin for several minutes. This result suggests that a sequence of steps is occurring at the membrane, beginning with depolarization (small ion permeability) and followed by more significant membrane disruption leading to SYTOX Green influx.
Microbial membrane depolarization
The importance of structure to interfacial activity
We have proposed that the rare active peptides selected from the library have the correct balance of solubility, hydrophobicity and amphipathicity (i.e. interfacial activity) such that they can partition into in the membrane-water interface and alter the packing and organization of lipids disruption to cause leakage of contents entrapped in vesicles8,2,15
. In this work, we show that the same peptides also have potent, broad-spectrum antimicrobial activity. However the importance of peptide structure to interfacial activity remains poorly defined for antimicrobial peptides. To examine the importance of secondary structure in membrane disruption, we synthesized analogs of four of the active peptides in which a single D-amino acid was substituted at a position near the C-terminus (). This small change was expected to decrease the peptide secondary structure24
. We compared the structure and activity of these peptides in living cell membranes as well as in liposomes (). In we show the circular dichroism (CD) spectra of the all L peptides and their single D-amino acid variants The all L peptides have β-sheet secondary structure in buffer and in bilayers8
. The D-amino substituted peptides have reduced structure propensity. All four are mostly random coil in buffer. The longer peptides d*VAVR*, d*VAYR* and d*ARVA showed no increase in structure content when 2.5 mM lipid was added. The shorter peptide dVVRG gained significant β-sheet content when bound to membranes.
Properties of D-substituted antimicrobial peptides
Circular dichroism spectroscopy of D-amino acid substituted peptides
Generally, D-substituted peptides had at least slightly lower antimicrobial activity, lower hemolysis and lower cell permeabilizing and vesicle permeabilizing activity as shown in . However the quantitative effect of D-amino acid substitutions on biological activity is mixed, and does not correlate completely with loss or retention of secondary structure. For example, d*VAVR*, although completely unstructured in buffer and in membranes compared to all L*VAVR*, which has significant β-sheet content, retained good antimicrobial activity against E. coli, S. aureus and C. neoformans, while losing most activity against P. aeruginosa. The peptide d*VAYR* retained its activity against S. aureus while losing activity against the other three microorganisms and d*ARVA* lost activity against the three bacteria, but retained activity against the fungus. The one peptide that showed “normal” β-sheet secondary structure in membranes, dVVRG, lost all activity against all four microbes. In all cases, the broad-spectrum antimicrobial activity that is the hallmark of these selected peptides is lost upon a single L to D amino acid substitution; however the pattern of species-specific activity is unpredictable and some potent antimicrobial activity is retained. These results suggest that the coupling between peptide secondary structure and biological or interfacial activity is incomplete. Importantly, by showing that broad-spectrum activity is always lost, even when some species activity is retained, this result supports our hypothesis that the universal broad-spectrum antimicrobial activity of selected peptides is a specific property that is selected for in the liposome-based high throughput screen.