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Abstract
Antimicrobial peptides (AMPs) are promising drugs to kill resistant pathogens. In contrast to bacteria, protozoan parasites, such as Leishmania, were little studied. Therefore, the antiparasitic mechanism of AMPs is still unclear. In this study, we sought to get further insight into this mechanism by focusing our attention on temporin-SHa (SHa), a small broad-spectrum AMP previously shown to be active against Leishmania infantum. To improve activity, we designed analogs of SHa and compared the antibacterial and antiparasitic mechanisms. [K3]SHa emerged as a highly potent compound active against a wide range of bacteria, yeasts/fungi, and trypanosomatids (Leishmania and Trypanosoma), with leishmanicidal intramacrophagic activity and efficiency toward antibiotic-resistant strains of S. aureus and antimony-resistant L. infantum. Multipassage resistance selection demonstrated that temporins-SH, particularly [K3]SHa, are not prone to induce resistance in Escherichia coli. Analysis of the mode of action revealed that bacterial and parasite killing occur through a similar membranolytic mechanism involving rapid membrane permeabilization and depolarization. This was confirmed by high-resolution imaging (atomic force microscopy and field emission gun-scanning electron microscopy). Multiple combined techniques (nuclear magnetic resonance, surface plasmon resonance, differential scanning calorimetry) allowed us to detail peptide-membrane interactions. [K3]SHa was shown to interact selectively with anionic model membranes with a 4-fold higher affinity (KD = 3 x 10−8 M) than SHa. The amphipathic α-helical peptide inserts in-plane in the hydrophobic lipid bilayer and disrupts the acyl chain packing via a detergent-like effect. Interestingly, cellular events, such as mitochondrial membrane depolarization or DNA fragmentation, were observed in L. infantum promastigotes after exposure to SHa and [K3]SHa at concentrations above IC50. Our results indicate that these temporins exert leishmanicidal activity via a primary membranolytic mechanism but can also trigger apoptotis-like death. The many assets demonstrated for [K3]SHa make this small analog an attractive template to develop new antibacterial/antiparasitic drugs.
Highlights
Antimicrobial peptides (AMPs) are a ubiquitous group of natural compounds that play a major role in the innate immune system [1, 2]
The SHa analogs were designed and synthetized by modifying the structural and physicochemical parameters known to control the antimicrobial activity of AMPs, such as net positive charge, hydrophobicity, helicity and amphipathicity
We previously demonstrated by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy that SHa adopts an α-helical conformation in water/2,2,2-trifluoroethanol mixtures and in sodium dodecyl sulfate detergent micelles [15]
Summary
Antimicrobial peptides (AMPs) are a ubiquitous group of natural compounds that play a major role in the innate immune system [1, 2]. All temporins are C-terminally amidated and adopt an amphipathic αhelical structure in apolar media or in membrane mimetic environments [15,16,17] Such structure for a temporin was observed in a media containing bacterial cells [18], using a previous circular dichroism protocol that was used for the first time to study the secondary structure of AMPs (cecropin A and magainin-2) in the presence of E. coli cells [19]. At very high peptide concentrations, the membrane can be disintegrated in a detergent-like manner
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