Luis Rivas

Animal antimicrobial peptides: An overview

Antibiotic peptides are a key component of the innate immune systems of most multicellular organisms. Despite broad divergences in sequence and taxonomy, most antibiotic peptides share a common mechanism of action, i.e., membrane permeabilization of the pathogen. This review provides a general introduction to the subject, with emphasis on aspects such as structural types, post-translational modifications, mode of action or mechanisms of resistance. Some of these questions are treated in depth in other reviews in this issue. The review also discusses the role of antimicrobial peptides in nature, including several pathological conditions, as well as recent accounts of their application at the preclinical level.

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.

Impaired Virulence and In Vivo Fitness of Colistin-Resistant Acinetobacter baumannii

Acinetobacter baumannii (American Type Culture Collection strain 19606) acquires mutations in the pmrB gene during the in vitro development of resistance to colistin. The colistin-resistant strain has lower affinity for colistin, reduced in vivo fitness (competition index, .016), and decreased virulence, both in terms of mortality (0% lethal dose, 6.9 vs 4.9 log colony-forming units) and survival in a mouse model of peritoneal sepsis. These results may explain the low incidence and dissemination of colistin resistance in A. baumannii in clinical settings.

N-Terminal Fatty Acid Substitution Increases the Leishmanicidal Activity of CA(1-7)M(2-9), a Cecropin-Melittin Hybrid Peptide

ABSTRACT In order to improve the leishmanicidal activity of the synthetic cecropin A-melittin hybrid peptide CA(1-7)M(2-9) (KWKLFKKIGAVLKVL-NH2), a systematic study of its acylation with saturated linear fatty acids was carried out. Acylation of the Nɛ-7 lysine residue led to a drastic decrease in leishmanicidal activity, whereas acylation at lysine 1, in either the α or the ɛ NH2 group, increased up to 3 times the activity of the peptide against promastigotes and increased up to 15 times the activity of the peptide against amastigotes. Leishmanicidal activity increased with the length of the fatty acid chain, reaching a maximum for the lauroyl analogue (12 carbons). According to the fast kinetics, dissipation of membrane potential, and parasite membrane permeability to the nucleic acid binding probe SYTOX green, the lethal mechanism was directly related to plasma membrane permeabilization.

The cost of resistance to colistin in Acinetobacter baumannii: a proteomic perspective

Colistin resistance in Acinetobacter baumannii, a pathogen of clinical concern, was induced in the susceptible strain ATCC 19606 by growth under increasing pressure of the antibiotic, the only drug universally active against multi‐resistant clinical strains. In 2‐D difference gel electrophoresis (DIGE) experiments, 35 proteins with differences in expression between both phenotypes were identified, most of them appearing as down regulated in the colistin‐resistant strain. These include outer membrane (OM) proteins, chaperones, protein biosynthesis factors, and metabolic enzymes, all suggesting substantial loss of biological fitness in the resistant phenotype, as substantiated by complementary experiments in the absence of colistin. Results shed light on the scarcity of widespread clinical outbreaks for resistant phenotypes.

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.

Miltefosine (Hexadecylphosphocholine) Inhibits Cytochrome c Oxidase in Leishmania donovani Promastigotes

ABSTRACT Miltefosine (hexadecylphosphocholine [HePC]) is currently on trial as a first-choice, orally active drug for the treatment of visceral leishmaniasis when resistance to organic pentavalent antimonials becomes epidemic. However, data on the targets involved in its leishmanicidal mechanism have, until now, been only fragmentary. We have carried out a systematic study of the alterations induced on the bioenergetic metabolism of Leishmania donovani promastigotes by HePC. Overnight incubation with HePC caused a significant decline in the intracellular ATP levels of the parasites, together with a reduction in the oxygen consumption rate and mitochondrial depolarization, while the integrity of the plasma membrane remained undamaged. In a further step, the effects of HePC on the respiratory chain were addressed in digitonized parasites. The inhibition of the oxygen consumption rate caused by HePC was not reverted either with the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone or with tetramethyl-p-phenylenediamine plus ascorbate, which feeds the electron transport chain at the level of cytochrome c. These results suggest that cytochrome c oxidase is a likely target in the complex leishmanicidal mechanism of HePC. This was further confirmed from the finding that this enzyme was specifically inhibited in a dose-dependent manner by HePC, but not the cytochrome c reductase, ruling out an unspecific effect of HePC on the respiratory chain.

The Dendritic Cell Receptor DC-SIGN Discriminates among Species and Life Cycle Forms of Leishmania

Infection of dendritic cells by the human protozoal parasite Leishmania is part of its survival strategy. The dendritic cell receptors for Leishmania have not been established and might differ in their interactions among Leishmania species and infective stages. We present evidence that the surface C-type lectin DC-SIGN (CD 209) is a receptor for promastigote and amastigote infective stages from both visceral (Leishmania infantum) and New World cutaneous (Leishmania pifanoi) Leishmania species, but not for Leishmania major metacyclic promastigotes, an Old World species causing cutaneous leishmaniasis. Leishmania binding to DC-SIGN was found to be independent of lipophosphoglycan, the major glycoconjugate of the promastigote plasma membrane. Our findings emphasize the relevance of DC-SIGN in Leishmania-dendritic cell interactions, an essential link between innate and Leishmania-specific adaptive immune responses, and suggest that DC-SIGN might be a therapeutic target for both visceral and cutaneous leishmaniasis

A cutaneous gene therapy approach to treat infection through keratinocyte-targeted overexpression of antimicrobial peptides

Infection represents a major associated problem in severely burned patients, as it causes skin graft failure and increases the risk of mortality. Topical and systemic antibiotic treatment is limited by the appearance of resistant bacterial strains. Antimicrobial peptides (AMPs) are gene‐ encoded “natural antibiotics” that form part of the innate mechanism of defense and may be active against such antibiotic‐resistant microorganisms. Several microbicidal peptides are expressed in human skin under inflammatory conditions, and their function is not only limited to microbial killing but also influences tissue repair and adaptive immunity. Protein delivery through cutaneous gene therapy is a promising therapeutic tool for both skin and nonskin diseases. Here we present a gene transfer approach aimed at delivering antimicrobial peptides from keratinocytes. Adenoviral vectors encoding antimicrobial peptide genes were used to infect human keratinocytes growing either on plastic or as part of cultured skin equivalents. Inhibition of bacterial growth occurred both in conditioned media and in direct contact with AMPs gene‐ transduced keratinocytes. In addition, we showed cooperative effects after transfer of combinations of genes encoding for AMPs with structural differences. Combined cutaneous tissue engineering in conjunction with (microbicidal) gene therapy emerges as a tailored therapeutic approach that is useful for wound coverage and, in this case, concomitantly combating infection.

Safety and Efficacy of Antimicrobial Peptides against Naturally Acquired Leishmaniasis

ABSTRACT Leishmaniases, which are important causes of morbidity and mortality in humans and dogs, are extremely difficult to treat. Antimicrobial peptides are rarely used as alternative treatments for naturally acquired parasitic diseases. Here we report that the acylated synthetic antimicrobial peptide Oct-CA(1-7)M(2-9) is safe and effective for treating naturally acquired canine leishmaniasis.