José María Saugar

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.

Activity of Cecropin A-Melittin Hybrid Peptides against Colistin-Resistant Clinical Strains of Acinetobacter baumannii: Molecular Basis for the Differential Mechanisms of Action

ABSTRACT Acinetobacter baumannii has successfully developed resistance against all common antibiotics, including colistin (polymyxin E), the last universally active drug against this pathogen. The possible widespread distribution of colistin-resistant A. baumannii strains may create an alarming clinical situation. In a previous work, we reported differences in lethal mechanisms between polymyxin B (PXB) and the cecropin A-melittin (CA-M) hybrid peptide CA(1-8)M(1-18) (KWKLFKKIGIGAVLKVLTTGLPALIS-NH2) on colistin-susceptible strains (J. M. Saugar, T. Alarcón, S. López-Hernández, M. López-Brea, D. Andreu, and L. Rivas, Antimicrob. Agents Chemother. 46:875-878, 2002). We now demonstrate that CA(1-8)M(1-18) and three short analogues, namely CA(1-7)M(2-9) (KWKLFKKIGAVLKVL-NH2), its Nα-octanoyl derivative (Oct-KWKLFKKIGAVLKVL-NH2), and CA(1-7)M(5-9) (KWKLLKKIGAVLKVL-NH2) are active against two colistin-resistant clinical strains. In vitro, resistance to colistin sulfate was targeted to the outer membrane, as spheroplasts were equally lysed by a given peptide, regardless of their respective level of colistin resistance. The CA-M hybrids were more efficient than colistin in displacing lipopolysaccharide-bound dansyl-polymyxin B from colistin-resistant but not from colistin-susceptible strains. Similar improved performance of the CA-M hybrids in permeation of the inner membrane was observed, regardless of the resistance pattern of the strain. These results argue in favor of a possible use of CA-M peptides, and by extension other antimicrobial peptides with similar features, as alternative chemotherapy in colistin-resistant Acinetobacter infections.

Activities of Polymyxin B and Cecropin A-Melittin Peptide CA(1-8)M(1-18) against a Multiresistant Strain of Acinetobacter baumannii

ABSTRACT Polymyxin B (PXB) and the cecropin A-melittin hybrid CA(1-8)M(1-18) (KWKLFKKIGIGAVLKVLTTGLPALIS-NH2) were compared for antibiotic activity on reference and multiresistant Acinetobacter baumannii strains. Significant differences for both peptides were observed on their inner membrane interaction and inhibition by environmental factors, supporting the use of CA(1-8)M(1-18) as a potential alternative to PXB against Acinetobacter.

Fungus-Elicited Metabolites from Plants as an Enriched Source for New Leishmanicidal Agents: Antifungal Phenyl-Phenalenone Phytoalexins from the Banana Plant (Musa acuminata) Target Mitochondria of Leishmania donovani Promastigotes

ABSTRACT Two antifungal phenyl-phenalenone phytoalexins isolated from the banana plant (Musa acuminata) elicited with the fungus Fusarium oxysporum, together with a methoxy derivative of one of them and two epoxide precursors of their chemical synthesis, were tested for leishmanicidal activity on Leishmania donovani promastigotes and L. infantum amastigotes. Drugs inhibited proliferation of both forms of the parasite with a 50% lethal concentration range between 10.3 and 68.7 μg/ml. Their lethal mechanism was found linked to the respiratory chain by a systematic approach, including electron microscopy, measurement of the oxygen consumption rate on digitonin-permeabilized promastigotes, and enzymatic assays on a mitochondrial enriched fraction. Whereas the whole set of compounds inhibited the activity of fumarate reductase in the mitochondrial fraction (50% effective concentration [EC50] between 33.3 and 78.8 μg/ml) and on purified enzyme (EC50 = 53.3 to 115 μg/ml), inhibition for succinate dehydrogenase was only observed for the two phytoalexins with the highest leishmanicidal activity: anigorufone and its natural analogue 2-methoxy-9-phenyl-phenalen-1-one (EC50 = 33.5 and 59.6 μg/ml, respectively). These results provided a new structural motif, phenyl-phenalenone, as a new lead for leishmanicidal activity, and support the use of plant extracts enriched in antifungal phytoalexins, synthesized under fungal challenge, as a more rational and effective strategy to screen for new plant leishmanicidal drugs.

Identification of new leishmanicidal peptide lead structures by automated real-time monitoring of changes in intracellular ATP.

Leishmanicidal drugs interacting stoichiometrically with parasite plasma membrane lipids, thus promoting permeability, have raised significant expectations for Leishmania chemotherapy due to their nil or very low induction of resistance. Inherent in this process is a decrease in intracellular ATP, either wasted by ionic pumps to restore membrane potential or directly leaked through larger membrane lesions caused by the drug. We have adapted a luminescence method for fast automated real-time monitoring of this process, using Leishmania donovani promastigotes transfected with a cytoplasmic luciferase form, previously tested for anti-mitochondrial drugs. The system was first assayed against a set of well-known membrane-active drugs [amphotericin B, nystatin, cecropin A-melittin peptide CA(1-8)M(1-18)], plus two ionophoric polyethers (narasin and salinomycin) not previously tested on Leishmania, then used to screen seven new cecropin A-melittin hybrid peptides. All membrane-active compounds showed a good correlation between inhibition of luminescence and leishmanicidal activity. Induction of membrane permeability was demonstrated by dissipation of membrane potential, SYTOX trade mark Green influx and membrane damage assessed by electron microscopy, except for the polyethers, where ATP decrease was due to inhibition of its mitochondrial synthesis. Five of the test peptides showed an ED50 around 1 microM on promastigotes. These peptides, with equal or better activity than 26-residue-long CA(1-8)M(1-18), are the shortest leishmanicidal peptides described so far, and validate our luminescence assay as a fast and cheap screening tool for membrane-active compounds.

Role of Positional Hydrophobicity in the Leishmanicidal Activity of Magainin 2

ABSTRACT The emergence of membrane-active antimicrobial peptides as new alternatives against pathogens with multiantibiotic resistance requires the design of better analogues. Among the different physicochemical parameters involved in the optimization of linear antimicrobial peptides, positional hydrophobicity has recently been incorporated. This takes into consideration the concept of the topological distribution of hydrophobic residues throughout the sequence rather than the classical concept of hydrophobicity as a global parameter of the peptide, calculated as the summation of the individual hydrophobicities of the residues. In order to assess the contribution of this parameter to the leishmanicidal mechanisms of magainin 2 analogues, the activities of two of these analogues, MG-H1 (GIKKFLHIIWKFIKAFVGEIMNS) and MG-H2 (IIKKFLHSIWKFGKAFVGEIMNI), which have similar charges, amino acid compositions, and hydrophobicities but different positional hydrophobicities, against Leishmania donovani promastigotes were assayed (T. Tachi, R. F. Epand, R. M. Epand, and K. Matsuzaki, Biochemistry 41:10723-10731, 2002). The activities were compared with that of the parental peptide, F5W-magainin 2 (GIGKWLHSAKKFGKAFVGEIMNS). The three peptides were active at micromolar concentrations, in the order MG-H2 > MG-H1 > F5W-magainin 2. These activities differ from their hemolytic and bactericidal activities. The results demonstrate that positional hydrophobicity, which reflects the presence of short stretches of sequences rich in hydrophobic amino acids, plays an important role in the activities of leishmanicidal peptides.

Thelaziosis in Humans, a Zoonotic Infection, Spain, 2011

After Thelazia callipaeda infection in dogs and cats were reported in Spain, a human case of thelaziosis in this country was reported, suggesting zoonotic transmission. The active reproductive status of this nematode in situ indicates that humans are competent hosts for this parasite.

Synthesis and biological evaluation of fluorescent leishmanicidal analogues of hexadecylphosphocholine (miltefosine) as probes of antiparasite mechanisms.

The leishmanicidal mechanism of miltefosine (hexadecylphosphocholine, MT) is not clearly understood. Valuable insights into its mode of action could be obtained by fluorescence techniques, given suitably emitting analogues. In this regard, the synthesis and biological characterization of two fully competent MT fluorescent analogues is reported here: all-(E)-13-phenyltrideca-6,8,10,12-tetraenylphosphocholine (PTE-MT) and all-(E)-13-phenyltrideca-8,10,12-trien-6-ynylphosphocholine (PTRI-MT). Both compounds show large absorption coefficients and a modest, but usable, fluorescence yield. Their activities were very similar to that of MT and were recognized by the MT uptake system of Leishmania. Their localization in living L. donovani promastigotes by confocal microscopy show a homogeneous intracellular distribution of the fluorescence. The concentration of PTRI-MT within the parasites (ca. 1.7 mM) showed a 100-fold enrichment relative to its external concentration. These results are consistent with a multiple target leishmanicidal mechanism for MT and validate the application of these analogues for pharmacokinetic and diagnostic studies concerning the chemotherapy of leishmaniasis.

Enterocin AS-48 as Evidence for the Use of Bacteriocins as New Leishmanicidal Agents

ABSTRACT We report the feasibility of enterocin AS-48, a circular cationic peptide produced by Enterococcus faecalis, as a new leishmanicidal agent. AS-48 is lethal to Leishmania promastigotes as well as to axenic and intracellular amastigotes at low micromolar concentrations, with scarce cytotoxicity to macrophages. AS-48 induced a fast bioenergetic collapse of L. donovani promastigotes but only a partial permeation of their plasma membrane with limited entrance of vital dyes, even at concentrations beyond its full lethality. Fluoresceinated AS-48 was visualized inside parasites by confocal microscopy and seen to cause mitochondrial depolarization and reactive oxygen species production. Altogether, AS-48 appeared to have a mixed leishmanicidal mechanism that includes both plasma membrane permeabilization and additional intracellular targets, with mitochondrial dysfunctionality being of special relevance. This complex leishmanicidal mechanism of AS-48 persisted even for the killing of intracellular amastigotes, as evidenced by transmission electron microscopy. We demonstrated the potentiality of AS-48 as a new and safe leishmanicidal agent, expanding the growing repertoire of eukaryotic targets for bacteriocins, and our results provide a proof of mechanism for the search of new leishmanicidal bacteriocins, whose diversity constitutes an almost endless source for new structures at moderate production cost and whose safe use on food preservation is well established.

Prevalence and Genotype Identification of Toxoplasma gondii in Wild Animals from Southwestern Spain

Abstract We used PCR to detect Toxoplasma gondii in the principal game species in southwestern Spain. We detected T. gondii in 32.2% of animals tested. Prevalences varied from 14.7% in wild boar (Sus scrofa) to 51.2% in red fox (Vulpes vulpes). The most prevalent genotype was type II (50.0%), followed by type III (20.6%) and type I (5.9%). Mixed infections (11.8%) were detected in wild boar (types I+III) and red fox (types II+III). Polymorphic strains (11.8%) were detected in several species. The high prevalence and the genetic variability shown could have implications for infection of farm animals and humans.