Temporins: Small Antimicrobial Peptides with Leishmanicidal Activity*

Leishmaniasis encompasses a wide range of infections caused by the human parasitic protozoan species belonging to the Leishmania genus. It appears frequently as an opportunistic disease, especially in virus-infected immunodepressed people. Similarly to other pathogens, parasites became resistant to most of the first-line drugs. Therefore, there is an urgent need to develop antiparasitic agents with new modes of action. Gene-encoded antimicrobial peptides are promising candidates, but so far only a few of them have shown anti-protozoa activities. Here we found that temporins A and B, 13-amino acid antimicrobial peptides secreted from the skin of the European red frog Rana temporaria, display anti-Leishmania activity at micromolar concentrations, with no cytolytic activity against human erythrocytes. To the best of our knowledge, temporins represent the shortest natural peptides having the highest leishmanicidal activity and the lowest number of positively charged amino acids (a single lysine/arginine) and maintain biological function in serum. Their lethal mechanism involves plasma membrane permeation based on the following data. (i) They induce a rapid collapse of the plasma membrane potential. (ii) They induce the influx of the vital dye SYTOX™ Green. (iii) They reduce intracellular ATP levels. (iv) They severely damage the membrane of the parasite, as shown by transmission electron microscopy. Besides giving us basic important information, the unique properties of temporins, as well as their membranolytic effect, which should make it difficult for the pathogen to develop resistance, suggest them as potential candidates for the future design of antiparasitic drugs with a new mode of action.


INTRODUCTION
Leishmaniasis includes a wide variety of clinical symptoms caused by infection with different species of the genus Leishmania, a human protozoan parasite with a worldwide incidence of 12-14 million people affected. The disease is widespread mostly in tropical and subtropical countries (1). Leishmania is a digenetic parasite with two major stages, differing widely in their antigenic pattern, metabolism, and plasma membrane composition (2)  At present, the only available treatment is based on chemotherapy with organic pentavalent antimonials as the first -line drugs (3,4). However, their efficacy is rapidly eroding due to the increasing appearance of resistant clinical isolates (5,6) and the severe side-effects associated with the treatment (7). Despite the availability of alternative drugs, such as amphotericin B or miltefosine, there is a real need for the development of new agents with new modes of action 4 charge and a potential to adopt an amphipathic α-helix and/or β-sheet structures upon their interaction with membranes. To date, there is a compelling evidence that a common step in the microbial killing mechanism is their electrostatic interaction with the negatively charged cell membrane followed by its permeation/disruption (13,14).
During the last few years, a considerable number of studies have been carried out with AMPs on bacteria (15,16) , fungi (17, 18), viruses (19,20) and tumor cells (21,22) , in attempting to understand the parameters responsible for their activity. Nevertheless, reports on the activity and the mode of action of AMPs toward protozoan and metazoan parasites are very scarce (23). For Leishmania, these include dermaseptins (24) and SPYY (25), both isolated from the skin of frogs, gomesin, from the hemocytes of the tarantula spider, Acanthoscurria gomesiana (26), and indolicidin (27) , from granules of bovine neutrophils. In 1998, Diaz-Achirica and coworkers (28) and more recently Chicharro (29) and Luque-Ortega and colleagues (30) investigated the leishmanicidal activity of highly positively charged cecropin A-melittin hybrid peptides. Their data suggested that the killing of the parasite was strongly correlated with plasma membrane permeabilization. Conversely, authophagic cell death has been described for indolicidin -treated parasites (27).
Here we report the biological function and the mode of action of temporin A (FLPLIGRVLSGIL-NH 2 ) and temporin B (LLPIVGNLLKSLL-NH 2 ), both isolated from the frog Rana temporaria, against Leishmania promastigotes and amastigotes. Temporins are short (13-residues) peptides, with an amidated C-terminus and with only one positively-charged amino acid. They were first identified in R. temporaria skin secretions (31) and further detected in several North American Rana species, such as R. clamitans (32), R. luteiventris (33), R. pipiens (33), and R. grylio (34). Temporins, together with indolicidin, are among the smallest by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 5 AMPs isolated so far from animal sources. However, in contrast with indolicidin, temporins are non-hemolytic (35,36) . Previous studies indicated that these molecules are active mainly against Gram-positive bacteria, Candida albicans, and some human tumor cell lines (12). Our data show that temporins A and B have leishmanicidal activity at concentrations that are not toxic to human red blood cells. In addition, in contrast t o most AMPs, temporins preserve biological function in serum. Furthermore, studies on their mode of action suggest that they act directly on the membrane of the parasite and destroy its integrity; therefore, it should make it difficult for the pathogen to develop resistance. Besides providing important basic information, the small size of temporins, their destructive mode of action and ability to maintain activity in serum, suggest them as attractive templates for further development of new antiparasitic drugs.
(i) Inhibition of 3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction to insoluble formazan by mitochondrial reductases was used as a short-term viability parameter of the parasites, and assayed immediately after peptide incubation (28). To the 100-µl aliquot of parasite suspension, 1 ml of Hanks-Glc was added to slow down peptide activity. The parasites were then collected by centrifugation, resuspended in 100 µl of a 0.5 mg/ml MTT solution in Hanks -Glc, transferred into a 96-well culture microplate, and incubated for 2 h at 26 °C or 32 °C, for promastigotes or amastigotes, respectively. The reduced formazan was solubilized by the addition of an equal volume of 10% (w/v) sodium dodecyl sulfate, incubated overnight at 26 ºC and measured in a 450 Bio-Rad Microplate Reader equipped with a 595-nm filter.
(ii) Measuring the inhibition of parasite proliferation. To measure inhibition of promastigotes proliferation, the 10-µl aliquot was added to 100 µl of RPMI medium, devoid of phenol red, and supplemented with 10% HIFCS. The surviving parasites were allowed to proliferate for 72 h; then, 100 µl of MTT solution (1 mg/ml in Hanks -Glc) was added and its reduction was measured as described above. For amastigotes, the inhibition of proliferation was performed in M199 growth medium. After 5 days, the cells were centrifuged, washed with Hanks, and resuspended in 0.5 mg/ml MTT solution. All assays were performed in triplicate and the 8 experiments were repeated twice. The results were normalized to those of the corresponding controls in the absence of the peptide.
Modification of Bioenergetic Parameters−We carried out two different assays. (ii) In-vivo monitoring of intracellular ATP variation. The variation of intracellular ATP level was monitored as previously described (30). Briefly, promastigotes transfected with the pX63NEO-3Luc expression vector were incubated for 15 min at 26 °C with 25 µM DMNPEluciferin, a free membrane-permeable caged luciferase substrate. Changes in luminescence were measured in the Fluorostat Galaxy microplate reader fitted with luminescence optics, with readings averaged every 10 s. Peptides were added once the luminescence reached a plateau.
This point was considered as time zero and its luminescence value was taken as 100%. The decrease in luminescence was monitored continuously for 60 min. All assays were performed in triplicate and the luminescence values were normalized to those of the corresponding controls in the absence of the peptide.
Permeation of the Plasma Membrane of the Promastigotes and Amastigotes−We adapted the procedure described by Thevissen and coworkers (42) to assess the permeability of Leishmania membrane. Briefly, the parasites were incubated with 1 µM SYTOX TM Green in Hanks-Glc for 15 min in the darkness. After peptide addition, the increase in fluorescence, due to the binding of the dye to intracellular DNA, was measured in the same microplate reader described above, using 485 nm and 520 nm filters for excitation and emission wavelengths, respectively.
Maximal membrane permeation was defined as that obtained after the addition of 0.1% Triton X-100 (29).
Other Methods −Hydrophobicity was calculated according to the normalized scale of Eisenberg (43). LC 50 (the concentration of peptide required to inhibit half of the maximum MTT reduction) and its 95% confidence limits were determined by the Litchfield and Wilcoxon procedure using the PHARM/PCS Version 4 software package for PCs. The number of experiments analyzed was indicated in the legend of each figure.

RESULTS
Leishmania Killing by Temporins A and B−Temporins were tested for their activity and plausible mode of action on both promastigote and amastigote stages of Leishmania. The ability of a drug to kill the mammalian intracellular stage of the parasite is crucial for its potential to be developed as a therapeutic agent. Note that this form has been shown to be more resistant to other cationic AMPs than the corresponding form present in the insect host (29).  Table I for comparison. These results indicate that whereas the antibacterial potency of temporin A was approximately 2-fold higher than that of temporin B, both peptides were similarly active toward the promastigotes, and at least 5-fold more efficient than cecropin A, a potent 36-residue AMP (44). Interestingly, we found that, in contrast to most natural AMPs (40), temporins maintained high activity in serum (Table I). Furthermore, temporins inhibited MTT reduction of the metacyclic promastigotes (Fig. 1B), the circulating form of the parasite in the blood of an infected mammal for about 24 h before becoming amastigote, to a slightly lower extent than that found for the procyclic promastigotes (Fig.1A).

The Influence of External Parameters on the Activity of Temporins on Leishmania
Promastigotes−Τo better understand the interaction of these peptides with the surface of the parasite, we carried out a systematic variation of the conditions used in the short-term standard assay for determining the ability of temporins to inhibit MTT reduction.
(i) To study the role of electrostatic interactions, we tested temporins in the absence and presence of 50 µg/ml heparin, a strongly anionic polysaccharide. Temporin A, at 15 µM, caused 90.8 ± 2.3% inhibition of MTT reduction in the absence of heparin (Fig. 1A), whereas after a 15-min incubation with heparin, prior to the addition of the peptide to the promastigotes, the inhibition was 78.4 ± 3.2%. This effect was clearly weaker than that obtained for other peptides having a higher cationic character such as CA(1-8)M(1-18) (28). In another assay, temporin A was assayed at 10 µM for its ability to inhibit the MTT reduction using two isosmotic incubation media: the standard Hanks medium (140 mM NaCl) and the same medium but containing 280 mM D-sorbitol instead of NaCl. Only a small difference in the activity was detected under the two conditions: the percentage of inhibition increased from 59.4 ± 1.7 in the standard assay, to 66.7 ± 2.9 in D -sorbitol medium.
(ii) Temperature is a main factor that affects membrane fluidity. We noticed that the leishmanicidal activity of temporins, tested at 15 µM peptide concentration, decreased from 90.8 To determine whether the promastigote membrane is a potential target for temporins, we performed a peptide-induced membrane depolarization assay using the membrane potential-sensitive dye bisoxonol. Both temporins caused rapid dissipation 12 of the membrane potential in a concentration-dependent manner (Fig. 2). In the case of temporin A, which displayed a slightly stronger effect than temporin B, the depolarization induced at 20 µM, a concentration that inhibited more than 97% of the parasite proliferation, was identical to that found for CA(1-8)M(1-18) when tested at 2.5 µM. Under these conditions, the hybrid peptide killed all the parasites through a membrane-permeabilization mechanism (28). Thus, our findings suggest a similar mode of action for temporins.
The data revealed also that the membrane perturbation induced by temporins leads to a strong collapse of the bioenergetic metabolism of the parasite. This was monitored by the fast and dose-dependent decrease of luminescence in parasites expressing a cytoplasmic luciferase, when incubated with DMNPE-luciferin (Fig. 3). This means that the intracellular level of ATP diminishes concomitantly with the decrease in luminescence, since the intracellular ATP concentration is the limiting step for the bioluminescence reaction in 3-Luc promastigotes (30,46). Also under these conditions, temporin B displayed a slightly lower efficacy compared with temporin A at the same peptide concentration.
To get a better insight into the extent of membrane damage caused by temporins, we measured fluorimetrically the influx of SYTOX TM Green (MW = 900) into the parasites, as its fluorescence is enhanced when bound to intracellular nucleic acids. The influx of the dye was prevented in intact membranes but not in those parasites having lesions with a size large enough to allow the entrance of the dye (42). Here, the effect caused by temporin A was significantly higher than that for temporin B (Fig. 4, panels A and B). We further investigated whether the mode of action of temporins against Leishmania amastigotes was similar to that found for promastigotes. To this end, we tested the peptide ability to induce the permeation of the amastigote membrane by using three assays and found inhibits promastigotes proliferation at a micromolar range higher than that for temporins.
However, the LC 50 of indolicidin is much lower than that of temporins, i.e. 3.5 x 10 -5 µM and ~8.5 µM, respectively (27). Furthermore, here we demonstrate that temporins function at physiological salt concentrations and in 33% human serum, whereas many AMPs are inactive in these conditions. In addition, they preserve their activity on the mammalian stage of the parasite.
Preliminary experiments have also shown that both temporins are devoid of cytotoxic effects on murine macrophages (RAW 264,7 line), which are capable to act as host cells for Leishmania , at the maximal concentration tested (80 µM). In addition, treatment of the infected macrophages with 15 µM temporin B also resulted in a lethal effect on the parasites, as the macrophage population remained apparently unharmed and fully viable whereas the ratio amastigote:macrophage, assessed by Giemsa staining, was reduced by a half (data not shown).
Previous studies reported that short cecropin-melittin hybrid peptides possess potent leishmanicidal activity (30) . Nevertheless, in contrast with temporins, they are high positively charged molecules (4 basic amino acids).
Our data envisage that the lethal concentrations of temporins against Leishmania are similar to those found against Candida (12). However, they are higher than those obtained against Gram-positive bacteria, but much lower than the values obtained against Gram-negative ones (31,49). This suggests that the outer membrane of Gram-negative bacteria, which is composed predominantly of hydrophobic and negatively charged moieties, namely lipopolysaccharides (LPS), acts as a barrier against temporins. The finding that E. coli strains deficient in the number of LPS or with a reduced amount of sugar residues of their LPS chain are more sensitive to these peptides (47) supports this notion.
Leishmania promastigotes are surrounded by a glycocalix layer, formed mainly by LPG, a highly anionic molecule bound to the membrane through a glycosylphosphoinositol anchor, which cover more than 40% of the total surface of the parasite (50). We found that temporins A and B do not differ significantly (p>0.1) in their activity against the parental strain of L.
donovani and its R2D2 mutant (data not shown). This is despite the fact that the mutant strain is defective in the biosynthesis of the repetitive units of LPG (37). Conversely, R2D2 is at least two-fold more susceptible to other AMPs, such as CA Interestingly, in contrast with temporins and other AMPs (29,53) , indolicidin, exhibits a consistently reduced activity against parasites defective for LPG biosynthesis, such as the R2D2 mutant (27). However, as reported by Bera and colleagues (27) , since indolicidin produces an autophagic cell death, we cannot exclude the possibility of a receptor -mediated pathway for the induction of autophagy, via LPG. Nevertheless, if the killing mechanism of indolicidin is associated to the interaction with a receptor, this might limit its antiparasitic potency compared with that of temporins.
Overall, our results indicate the Leishmania membrane as being the major target for temporins A and B, based on the following data: (i) the rapid induction of the collapse of the membrane potential as well as the lowering of the intracellular ATP levels and the permeation of the membrane simultaneously with the inhibition of parasite proliferation. All of these findings also correlate with the extent of this inhibition and are in contrast with the transient and slow effects caused by peptides that traverse the membrane and act intracellularly. Since maintenance of the trans-membrane potential depends mainly on the gradient of K + and Na + across the membrane, temporins probably promote plasma membrane permeability to these ions; (ii) the fast kinetics of the drop in the intracellular ATP level and the irreversibility of the process. The hypothesis that the decrease in the intracellular ATP level is due to its release from the parasite is reasonable; indeed, if it happened because of the inhibition of the oxidative phosphorylation, which represents the major source for ATP production in Leishmania (54), we should expect a slower initial kinetic; (iii) the increase in SYTOX TM Green fluorescence, which reflects the membrane perturbation; (iv) the pattern of injured parasites as visualized by transmission electron microscopy. The photographs show damage in the plasma membrane of the parasites in the form of blebs and breakages, as well as a partial depletion in the intracytoplasmic content. A similar effect on the Leishmania morphology was also detected with other membrane-active leishmanicidal peptides, such as dermaseptins (55), cecropin A-melittin hybrids (28)(29)(30) or magainins (56). In addition, temporin-treated parasites were devoid of the extensive cytoplasmic vacuolation as found with indolicidin, which does not affect the integrity of the plasma membrane (27) ; (v) the gap between the concentration values for a short-term effect (MTT reduction assayed after 1h of incubation of the parasites with peptides) and those for inhibition of proliferation, which are significantly lower than those achieved for peptides with an intracellular target (57,58); note also (vi) the previously observed alteration of model membrane structure, by forming transient peptide -phospholipid membrane-spanning pores, as described by the two-states model (12,45,47,48) and (vii) the fact that all-D temporins A and B enantiomers preserved the antibacterial activity of the natural peptide, indicating that chiral targets are not involved (49,59).
Interestingly, although against promastigotes both temporins are almost equally efficient in the inhibition of proliferation, in their capacity to depolarize the membrane potential, and their ability to reduce the intracellular ATP levels, they have distinct effects on the influx of vital dyes such as the SYTOX TM Green. For example, at a given concentration, temporin B was substantially less active than temporin A in promoting the intracellular influx of the dye.
Conversely, a reversal in potency was manifested against amastigotes (Figs. 4C, 4D and 6) and in the calcein-leakage assay from liposomes (47) , where tempor in B was the most active peptide. This suggests that the size of the membrane breakages and the kinetics of their formation differ for the two molecules, and that both phenomena depend on the type of the phospholipid of the target system. Indeed, the calcein-leakage experiments were done with liposomes containing phosphatidylglycerol (PG) and phosphatidylserine (PS) (47) . However, PG is almost absent in the plasma membrane of L. donovani promastigotes (60), whereas PS is mainly located at its cytoplasmic leaflet. Furthermore, promastigotes contain a LPG layer that is absent in amastigotes as well as in the liposomes, and therefore, the ability to traverse this layer is also a crucial parameter and might be different for the two peptides.
In summary, our results demonstrate that temporins A and B are among the smallest highly active natural antiparasitic peptides reported so far that act directly on membranes. This should make it difficult for the parasite to develop resistance. In addition, the small size of temporins and the finding that, in contrast to many natural AMPs, they maintain activity in physiological salt concentration as well as in serum makes them as attractive lead compounds for the future development of antiparasitic drugs with a new mode of action.