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Background Crohn's disease (CD) associated dysbiosis is characterized by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, the chemical nature of the anti-inflammatory compounds has not yet been determined. Methods Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant Lactic Acid Bacteria to prevent DNBS-colitis in mice. Results The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single Microbial Anti-inflammatory Molecule (MAM), a protein of 15 kDa and comprising 53% of nonpolar residues. This last feature prevented the direct characterization of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the NF-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice. Conclusion A 15kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.

Cell-penetrating peptides (CPPs) share the property of cellular internalization. The question of how these peptides reach the cytoplasm of cells is still widely debated. Herein, we have used a mass spectrometry-based method that enables quantification of internalized and membrane-bound peptides. Internalization of the most used CPP was studied at 37°C (endocytosis and translocation) and 4°C (translocation) in wild type and proteoglycandeficient Chinese hamster ovary cells. Both translocation and endocytosis are internalization pathways used by CPP. The choice of one pathway versus the other depends on the peptide sequence (not the number of positive changes), the extracellular peptide concentration, and the membrane components. There is no relationship between the high affinity of these peptides for the cell membrane and their internalization efficacy. Translocation occurs at low extracellular peptide concentration, whereas endocytosis, a saturable and cooperative phenomenon, is activated at higher concentrations. Translocation operates in a narrow time window, which implies a specific lipid/peptide co-import in cells.Cell-penetrating peptides (CPPs) 3 that share the activity of cellular entry are usually short peptides of less than 20 amino acids highly enriched in basic residues. Among them, Antp, Tat-(48 -60), and oligoarginine peptides are the most intensively studied. Despite the wide use of these CPPs as macromolecular delivery devices, the internalization mechanism of these peptides in cells still remains largely controversial. The energy dependence of the internalization mechanism is unique because all endocytotic pathways are inhibited at low temperature. Consequently, at low temperature, internalization likely reflects a direct translocation mechanism. Early studies proposed that Antp enters cells by an energy-independent membrane translocation mechanism (1). This first analysis was then contradicted by other studies that suggested, with the use of inhibitors, the involvement of endocytosis in the cellular internalization of cell-penetrating peptides (2-4).The hypothesis that endocytosis was the only internalization mechanism of CPP resulted from studies examining whether the temperature or the binding to cell-surface glycosaminoglycans (GAGs) were critical for peptide internalization. Most of these interpretations resulted from fluorescence microscopy data. For instance, it was reported that Antp, Tat, and oligoarginine peptides were not efficiently internalized in the Chinese hamster ovary (CHO) mutant pgsA-745 cell line, which does not produce cell-surface heparan sulfate or chondroitin sulfate (5, 6). However, recent data indicates that Tat-mediated transduction occurs in the absence of heparan sulfate and chondroitin sulfate (7). The discrepancies observed between studies may be explained in part by different incubation conditions (peptide/cells ratio and peptide concentration) (8), limits in fluorescence imaging, such as quenching (9), or fluorophore-dependent intracellular trafficking (10), as...

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The third helix of antennapedia homeodomain pAntp-(43 -58) can translocate through cell membrane and has been used as an intracellular vehicle for delivering peptides and oligonucleotides. The conformational and associative behaviour of two peptidic vectors pAntp-(43 -58) and [Pro50]pAntp-(43 -58) has been analyzed by different biophysical methods. pAntp-(43 -58) adopts an amphipathic helical structure in 30 % (by vol.) hexafluoroisopropanol, in perfluoro-tert-butanol and in the presence of SDS micelles.CD spectra indicate that the conformation of [Pro50]pAntp-(43 -58) in contrast to pAntp-(43 -58) is independent of the media used. 'H-NMR spectroscopy in SDS micelles or in perfluoro-tert-butanol allows detection of aggregated peptides probably in a ribbon 2, type conformation. These conformations became the predominant structure when Gln50 was replaced by ProSO. Interproton-distance restraints derived from NOE measurements have been classified in two groups corresponding to two types of structures: a-helix and essentially extended structures. Consecutive CHa(i)lCHa(i+ 1) NOES are only compatible with aggregates. Simulated annealing calculation of dimeric structure agrees with 4 and y/ angles in the p-sheet and y-turn regions. Fluorescence spectroscopy analysis has shown that the indole groups of both peptides penetrate into SDS micelles; both peptides also induce the formation of micelles at very low concentration of SDS (20 pM). Similar interaction was observed with reverse-phase micelles made of bis(2-ethyhexyl) sodium sulfosuccinate and small unilamellar vesicles (SUV) made of a mixture of phosphatidylcholine/phosphatidylserine. 3'P-NMR of vesicles (SUV and large unilamellar vesicles) indicated that the addition of pAntp analogues did not affect the size of phosphatidylcholine/phosphatidylserine vesicles. The addition of pAntp analogues to lipidic dispersions modulates lipid polymorphism in different ways depending on the mixtures of acidic lipids.

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.

The ability of cell-penetrating peptides (CPPs) to cross cell membranes has found numerous applications in the delivery of bioactive compounds to the cytosol of living cells. Their internalization mechanisms have been questioned many times, and after 20 years of intense debate, it is now widely accepted that both energy-dependent and energy-independent mechanisms account for their penetration properties. However, the energy-independent mechanisms, named "direct translocation", occurring without the requirement of the cell internalization machinery, remain to be fully rationalized at the molecular level. Using artificial membrane bilayers, recent progress has been made toward the comprehension of the direct translocation event. This review summarizes our current understanding of the translocation process, starting from the adsorption of the CPP on the membrane to the membrane crossing itself. We describe the different key steps occurring before direct translocation, because each of them can promote and/or hamper translocation of the CPP through the membrane. We then dissect the modification to the membranes induced by the presence of the CPPs. Finally, we focus on the latest studies describing the direct translocation mechanisms. These results provide an important framework within which to design new CPPs and to rationalize an eventual selectivity of CPPs in their penetration ability.