Claire Lacombe

Different membrane behaviour and cellular uptake of three basic arginine-rich peptides.

Cell penetrating peptides (CPPs) are peptides displaying the ability to cross cell membranes and transport cargo molecules inside cells. Several uptake mechanisms (endocytic or direct translocation through the membrane) are being considered, but the interaction between the CPP and the cell membrane is certainly a preliminary key point to the entry of the peptide into the cell. In this study, we used three basic peptides: RL9 (RRLLRRLRR-NH(2)), RW9 (RRWWRRWRR-NH(2)) and R9 (RRRRRRRRR-NH(2)). While RW9 and R9 were internalised into wild type Chinese Hamster Ovary cells (CHO) and glycosaminoglycan-deficient CHO cells, at 4°C and 37°C, RL9 was not internalised into CHO cells. To better understand the differences between RW9, R9 and RL9 in terms of uptake, we studied the interaction of these peptides with model lipid membranes. The effect of the three peptides on the thermotropic phase behaviour of a zwitterionic lipid (DMPC) and an anionic lipid (DMPG) was investigated with differential scanning calorimetry (DSC). The presence of negative charges on the lipid headgroups appeared to be essential to trigger the peptide/lipid interaction. RW9 and R9 disturbed the main phase transition of DMPG, whereas RL9 did not induce significant effects. Isothermal titration calorimetry (ITC) allowed us to study the binding of these peptides to large unilamellar vesicles (LUVs). RW9 and R9 proved to have about ten fold more affinity for DSPG LUVs than RL9. With circular dichroism (CD) and NMR spectroscopy, the secondary structure of RL9, RW9 and R9 in aqueous buffer or lipid/detergent conditions was investigated. Additionally, we tested the antimicrobial activity of these peptides against Escherichia coli and Staphylococcus aureus, as CPPs and antimicrobial peptides are known to share several common characteristics. Only RW9 was found to be mildly bacteriostatic against E. coli. These studies helped us to get a better understanding as to why R9 and RW9 are able to cross the cell membrane while RL9 remains bound to the surface without entering the cell.

Structural requirements for antimicrobial versus chemoattractant activities for dermaseptin S9

Dermaseptin S9 (Drs S9), GLRSKIWLWVLLMIWQESNKFKKM, isolated from frog skin, does not resemble any of the cationic and amphipathic antimicrobial peptides identified to date, having a highly hydrophobic core sequence flanked at either side by cationic termini. Previous studies [Lequin O, Ladram A, Chabbert A, Bruston F, Convert O, Vanhoye D, Chassaing G, Nicolas P & Amiche M (2006) Biochemistry45, 468–480] demonstrated that this peptide adopted a non‐amphipathic α‐helical conformation in trifluoroethanol/water mixtures, but was highly aggregated in aqueous solutions and in the presence of sodium dodecyl sulfate micelles. Circular dichroism, FTIR and attenuated total reflectance FTIR spectroscopies, combined with a surface plasmon resonance study, show that Drs S9 forms stable and ordered β‐sheet aggregates in aqueous buffers or when bound to anionic or zwitterionic phospholipid vesicles. These structures slowly assembled into amyloid‐like fibrils in aqueous environments via spherical intermediates, as revealed by electron microscopy and Congo red staining. Drs S9 induced the directional migration of neutrophils, T lymphocytes and monocytes. Interestingly, the antimicrobial and chemotactic activities of Drs S9 are modulated by its amyloid‐like properties. Whereas spherical oligomers of Drs S9 exhibit antimicrobial activity, the soluble, weakly self‐associated forms of Drs S9 act on human leukocytes to promote chemotaxis and/or immunological response activation in the same range of concentration as amyloidogenic peptides Aβ(1–42), the most fibrillogenic isoform of amyloid beta peptides, and the prion peptide PrP(106–126).

Peptide secretion in the cutaneous glands of South American tree frog Phyllomedusa bicolor: an ultrastructural study

The development of the dermal glands of the arboreal frog Phyllomedusa bicolor was investigated by immunocytochemistry and electron microscopy. The 3 types of glands (mucous, lipid and serous) differed in size and secretory activity. The mucous and serous glands were apparent in the tadpole skin, whereas the lipid glands developed later in ontogenesis. The peptide antibiotics dermaseptins and the D-amino acid-containing peptide opioids dermorphins and deltorphins are abundant in the skin secretions of P. bicolor. Although these peptides differ in their structure and activity they are derived from precursors that have very similar preproregions. We used an antibody to the common preproregion of preprodermaseptins and preprodeltorphins and immunofluorescence analysis to show that only the serous glands are specifically involved in the biosynthesis and secretion of dermaseptins and deltorphins. Scanning and transmission electron microscopy revealed that the serous glands of P bicolor have morphological features, especially the secretory granules, which differ from those of the glands in Xenopus laevis skin.

Comparative Study of Two Plasticins: Specificity, Interfacial Behavior, and Bactericidal Activity

A comparative study was designed to evaluate the staphylococcidal efficiency of two sequence-related plasticins from the dermaseptin superfamily we screened previously. Their bactericidal activities against Staphylococcus aureus as well as their chemotactic potential were investigated. The impact of the GraS/GraR two-component system involved in regulating resistance to cationic antimicrobial peptides (CAMPs) was evaluated. Membrane disturbing activity was quantified by membrane depolarization assays using the diS-C3 probe and by membrane integrity assays measuring beta-galactosidase activity with recombinant strain ST1065 reflecting compromised membranes and cytoplasmic leakage. Interactions of plasticins with membrane models composed of either zwitterionic lipids mimicking the S. aureus membrane of CAMP-resistant strains or anionic lipids mimicking the negative charge-depleted membrane of CAMP-sensitive strains were analyzed by jointed Brewster angle microscopy (BAM), polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), and differential scanning calorimetry (DSC) to yield detailed information about the macroscopic interfacial organization, in situ conformation, orientation of the peptides at the lipid-solvent interface, and lipid-phase disturbance. We clearly found evidence of distinct interfacial behaviors of plasticins we linked to the distribution of charges along the peptides and structural interconversion properties at the membrane interface. Our results also suggest that amidation might play a key role in GraS/GraR-mediated CAMP sensing at the bacterial surface.

Intrinsic flexibility and structural adaptability of Plasticins membrane-damaging peptides as a strategy for functional versatility.

The Plasticins are a family of antimicrobial, 23–29-residue Gly-Leu-rich ortholog peptides from the frog skin that have very similar amino acid sequences, hydrophobicities, and amphipathicities but differ markedly in their conformational plasticity and spectrum of activity. The intrinsic flexibility and structural malleability of Plasticins modulate their ability to bind to and disrupt the bilayer membranes of prokaryotic and eukaryotic cells, and/or to reach intracellular targets, therefore, triggering functional versatility. The discussion is opened herein on several examples of other membrane-active peptides, like viral fusion peptides, cell-penetrating peptides, that are able to display antimicrobial activity. Hence, Plasticins could be regarded as models of multipotent membrane-active peptides guided by structural plasticity.

Investigating the role of GXXXG motifs in helical folding and self-association of plasticins, Gly/Leu-rich antimicrobial peptides☆

Plasticins (PTC) are dermaseptin-related antimicrobial peptides characterized by a large number of leucine and glycine residues arranged in GXXXG motifs that are often described to promote helix association within biological membranes. We report the structure and interaction properties of two plasticins, PTC-B1 from Phyllomedusa bicolor and a cationic analog of PTC-DA1 from Pachymedusa dacnicolor, which exhibit membrane-lytic activities on a broad range of microorganisms. Despite a high number of glycine, CD and NMR spectroscopy show that the two plasticins adopt mainly alpha-helical conformations in a wide variety of environments such as trifluoroethanol, detergent micelles and lipid vesicles. In DPC and SDS, plasticins adopt well-defined helices that lie parallel to the micelle surface, all glycine residues being located on the solvent-exposed face. Spectroscopic data and cross-linking experiments indicate that the GXXXG repeats in these amphipathic helices do not provide a strong oligomerization interface, suggesting a different role from GXXXG motifs found in transmembrane helices.

Dermaseptin DA4, although closely related to dermaseptin B2, presents chemotactic and Gram‐negative selective bactericidal activities

Antimicrobial peptides participate in innate host defense by directly eliminating pathogens as a result of their ability to damage the microbial membrane and by providing danger signals that will recruit innate immune cells to the site of infection. Dermaseptin DA4 (DRS‐DA4), a new antimicrobial peptide of the dermaseptin superfamily, was identified based on its chemotactic properties, contrasting with the currently used microbicidal properties assessment. The peptide was isolated and purified by size exclusion HPLC and RP‐HPLC from the skin of the Mexican frog, Pachymedusa dacnicolor. MS and amino acid sequence analyses were consistent with the structure GMWSKIKNAGKAAKAAAKAAGKAALGAVSEAM. CD experiments showed that, unlike most antimicrobial peptides of the dermaseptin superfamily, DRS‐DA4 is not structured in the presence of zwitterionic lipids. DRS‐DA4 is a potent chemoattractant for human leukocytes and is devoid of hemolytic activity; in addition, bactericidal tests and membrane perturbation assays on model membranes and on Escherichia coli and Staphylococcus aureus strains have shown that the antibacterial effects of DRS‐DA4 and permeabilization of the inner membrane are exclusively selective for Gram‐negative bacteria. Interestingly, despite high sequence homology with dermaseptin S4, dermaseptin B2 was not able to induce directional migration of leukocytes, and displayed a broader bactericidal spectrum. A detailed structure–function analysis of closely related peptides with different capabilities, such as DRS‐DA4 and dermaseptin B2, is critical for the design of new molecules with specific attributes to modulate immunity and/or act as microbicidal agents.

ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration

CC chemokine receptor type 2 (CCR2) is a key molecule in inflammatory diseases and is an obvious drug target for the treatment of inflammation. A number of nonpeptidic, competitive CCR2 antagonists have been developed, but none has yet been approved for clinical use. Our aim was to identify a short peptide that showed allosteric antagonism against human and mouse CCR2. On the basis of sequence analysis and 3‐dimensional modeling, we identified an original 7‐d‐amino acid peptidic CCR2 inhibitor that we have called extracellular loop 1 inverso (ECL1i), d(LGTFLKC). In vitro, ECL1i selectively and potently inhibits CC chemokine ligand type 2 (CCL2)‐triggered chemotaxis (IC50, 2 μM) but no other conventional CCL2‐associated events. We used the classic competitive CCR2 antagonist, BMS22 {2‐[(isopropylaminocarbonyl)amino]‐N‐[2‐[[cis‐2‐[[4‐(methylthio)benzoyl] amino]cyclohexyl]amino]‐2‐oxoethyl]‐5‐(trifluoromethyl)benzamide}, as positive control and inhibited CCL2‐dependent chemotaxis with an IC50 of 18 nM. As negative control, we used a peptide with the same composition as ECL1i, but in a different sequence, d(FKLTLCG). In vivo, ECL1i (4 mg/kg) interfered with CCR2‐positive cell recruitment and attenuated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. This study establishes ECL1i as the first allosteric inhibitor of CCR2 with functional selectivity. ECL1i is a promising new agent in therapeutic development, and it may, by its selective effect, increase our understanding of CCR2 signaling pathways and functions.—Auvynet, C., Baudesson de Chanville, C., Hermand, P., Dorgham, K., Piesse, C., Pouchy, C., Carlier, L., Poupel, L., Barthélémy, S., Felouzis, V., Lacombe, C., Sagan, S., Chemtob, S., Quiniou, C., Salomon, B., Deterre, P., Sennlaub, F., Combadière, C. ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2‐dependent migration. FASEB J. 30, 2370–2381 (2016). www.fasebj.org

Pachymodulin, a new functional formyl peptide receptor 2 peptidic ligand isolated from frog skin has Janus-like immunomodulatory capacities.

Recruitment of leukocytes is essential to fight infections or to heal injuries; however, excessive and/or prolonged responses favor the development of major inflammatory pathologies, such as cardiovascular or neurodegenerative diseases. Thus, it is of great interest to seek novel compounds that can regulate leukocyte recruitment depending on the degree of inflammation. We have isolated and characterized, by different chromatographic techniques, mass spectrometry, and Edman sequencing, a new hexapeptide (SSLSKL) from the Mexican frog Pachymedusa dacnicolor, which we named pachymodulin. In vitro, pachymodulin promotes the migration of leukocytes through the binding and activation of the human and mouse N-formyl peptide receptor 2 (huFPR2). In vivo, it exhibits opposite biological activities: under homeostatic conditions, pachymodulin induces the recruitment of leukocytes, whereas under inflammatory conditions, it inhibits this process. Therefore, pachymodulin represents an interesting template in the quest to design new immunomodulatory drugs in the therapy of immune-related diseases.

Peptidoglycan potentiates the membrane disrupting effect of the carboxyamidated form of DMS-DA6, a Gram-positive selective antimicrobial peptide isolated from Pachymedusa dacnicolor skin

The occurrence of nosocomial infections has been on the rise for the past twenty years. Notably, infections caused by the Gram-positive bacteria Staphylococcus aureus represent a major clinical problem, as an increase in antibiotic multi-resistant strains has accompanied this rise. There is thus a crucial need to find and characterize new antibiotics against Gram-positive bacteria, and against antibiotic-resistant strains in general. We identified a new dermaseptin, DMS-DA6, produced by the skin of the Mexican frog Pachymedusa dacnicolor, with specific antibacterial activity against Gram-positive bacteria. This peptide is particularly effective against two multiple drug-resistant strains Enterococcus faecium BM4147 and Staphylococcus aureus DAR5829, and has no hemolytic activity. DMS-DA6 is naturally produced with the C-terminal carboxyl group in either the free or amide forms. By using Gram-positive model membranes and different experimental approaches, we showed that both forms of the peptide adopt an α-helical fold and have the same ability to insert into, and to disorganize a membrane composed of anionic lipids. However, the bactericidal capacity of DMS-DA6-NH2 was consistently more potent than that of DMS-DA6-OH. Remarkably, rather than resulting from the interaction with the negatively charged lipids of the membrane, or from a more stable conformation towards proteolysis, the increased capacity to permeabilize the membrane of Gram-positive bacteria of the carboxyamidated form of DMS-DA6 was found to result from its enhanced ability to interact with peptidoglycan.