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.

Histatin 5 (Hst5) is a human salivary antimicrobial peptide that targets fungal mitochondria. In the human parasitic protozoa Leishmania, the mitochondrial ATP production is essential, as it lacks the bioenergetic switch between glycolysis and oxidative phosphorylation described in some yeasts. On these premises, Hst5 activity was assayed on both stages of its life cycle, promastigotes and amastigotes (LC ₅₀ =7.3 and 14.4 μ,M, respectively). In a further step, its lethal mechanism was studied. The main conclusions drawn were as follows: 1 ) Hst5 causes limited and temporary damage to the plasma membrane of the parasites, as assessed by electron microscopy, depolarization, and entrance of the vital dye SYTOX Green; 2 ) Hst5 translocates into the cytoplasm of Leishmania in an achiral receptor‐independent manner with accumulation into the mitochondrion, as shown by confocal microscopy;and 3) Hst5 produces a bioenergetic collapse of the parasite, caused essentially by the decrease of mitochondrial ATP synthesis through inhibition of F ₁ F ₀ ‐ATPase, with subsequent fast ATP exhaustion. By using the Hst5 enantiomer, it was found that the key steps of its lethal mechanism involved no chiral recognition. Hst5 thus constitutes the first leishmanicidal peptide with a defined nonstereospecific intracellular target. The prospects of its development, by its own or as a carrier molecule for other leishmanicidal molecules, into a novel anti‐Leishmania drug with a preferential subcellular accumulation are discussed.— Luque‐ Ortega, J. R., van't Hof, W., Veerman, E. C. I., Saugar, J. M., Rivas, L. The human antimicrobial peptide histatin 5 is a cell‐penetrating peptide targeting mitochondrial ATP synthesis in Leishmania. FASEB J. 22, 1817–1828 (2008)

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-NH ₂ ) 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-NH ₂ ), its N ^α -octanoyl derivative (Oct-KWKLFKKIGAVLKVL-NH ₂ ), and CA(1-7)M(5-9) (KWKLLKKIGAVLKVL-NH ₂ ) 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.

Giardia duodenalis and Cryptosporidium spp. are enteric protozoan causing gastrointestinal illness in humans and animals. Giardiasis and cryptosporidiosis are not formally considered as neglected tropical diseases, but belong to the group of poverty-related infectious diseases that impair the development and socio-economic potential of infected individuals in developing countries. We report here the prevalence and genetic diversity of G. duodenalis and Cryptosporidium spp. in children attending rural primary schools in the Bahir Dar district of the Amhara Region, Ethiopia. Stool samples were collected from 393 children and analysed by molecular methods. G. duodenalis was detected by real-time PCR, and the assemblages and sub-assemblages were determined by multilocus sequence-based genotyping of the glutamate dehydrogenase and β-giardin genes of the parasite. Detection and identification of Cryptosporidium species was carried out by sequencing of a partial fragment of the small-subunit ribosomal RNA gene. The PCR-based prevalences of G. duodenalis and Cryptosporidium spp. were 55.0% (216/393) and 4.6% (18/393), respectively. A total of 78 G. duodenalis isolates were successfully characterized, revealing the presence of sub-assemblages AII (10.3%), BIII (28.2%), and BIV (32.0%). Discordant typing results AII/AIII and BIII/BIV were identified in 7.7% and 15.4% of the isolates, respectively. An additional five (6.4%) isolates were assigned to assemblage B. No mixed infections of assemblages A+B were found. Extensive genetic variation at the nucleotide level was observed within assemblage B (but no within assemblage A), resulting in the identification of a large number of sub-types. Cryptosporidium diversity was demonstrated by the occurrence of C. hominis, C. parvum, and C. viatorum in the population under study. Our data suggest an epidemiological scenario with an elevated transmission intensity of a wide range of G. duodenalis genetic variants. Importantly, the elevated degree of genetic diversity observed within assemblage B is consistent with the occurrence of intra-assemblage recombination in G. duodenalis.