Structure, dynamics and mapping of membrane-binding residues of micelle-bound antimicrobial peptides by natural abundance 13C NMR spectroscopy

AbstractWorldwide bacterial resistance to traditional antibiotics has drawn much research attention to naturally occurring antimicrobial peptides (AMPs) owing to their potential as alternative antimicrobials. Structural studies of AMPs are essential for an in-depth understanding of their activity, mechanism of action, and in guiding peptide design. Two-dimensional solution proton NMR spectroscopy has been the major tool. In this article, we describe the applications of natural abundance 13C NMR spectroscopy that provides complementary information to 2D 1H NMR. The correlation of 13Cα secondary shifts with both 3D structure and heteronuclear 15N NOE values indicates that natural abundance carbon chemical shifts are useful probes for backbone structure and dynamics of membrane peptides. Using human LL-37-derived peptides (GF-17, KR-12, and RI-10), as well as amphibian antimicrobial and anticancer peptide aurein 1.2 and its analog LLAA, as models, we show that the cross peak intensity plots of 2D 1H–13Cα HSQC spectra versus residue number present a wave-like pattern (HSQC wave) where key hydrophobic residues of micelle-bound peptides are located in the troughs with weaker intensities, probably due to fast exchange between the free and bound forms. In all the cases, the identification of aromatic phenylalanines as a key membrane-binding residue is consistent with previous intermolecular Phe-lipid NOE observations. Furthermore, mutation of one of the key hydrophobic residues of KR-12 to Ala significantly reduced the antibacterial activity of the peptide mutants. These results illustrate that natural abundance heteronuclear-correlated NMR spectroscopy can be utilized to probe backbone structure and dynamics, and perhaps to map key membrane-binding residues of peptides in complex with micelles. 1H–13Cα HSQC wave, along with other NMR waves such as dipolar wave and chemical shift wave, offers novel insights into peptide–membrane interactions from different angles

Design, Development, and Characterization of Novel Antimicrobial Peptides for Pharmaceutical Applications

Candida species are the fourth leading cause of nosocomial infection. The increased incidence of drug-resistant Candida species has emphasized the need for new antifungal drugs. Histatin 5 is a naturally occurring human salivary antifungal peptide and the first line of defense against infections of the oral cavity. This research has focused on understanding the activity of histatin 5, and subsequently designing novel peptides that may serve as models for the further development of therapeutics to treat fungal infection. This objective has been achieved in three steps: studying the structural requirement of histatin 5 involved in antifungal activity, the identification of a short peptide sequence, referred to as KM motif, important for fungicidal activity, and finally, the development of a novel antifungal peptide with potent activity. In the initial phase of this work it was demonstrated that reversing the sequence of histatin 5 C-16 peptide to create a retro peptide did not interfere with the fungicidal activity or secondary structure of the peptide. This suggested that the spatial arrangement of amino acid residues was more relevant for fungicidal activity than the actual peptide sequence. In the second phase of the work, we identified and characterized a five amino acid sequence, termed the KM motif, within histatin 5 that maintained fungicidal properties. Although this short peptide was less active than histatin 5, the data suggested it was killing fungi via a mechanism similar to histatin 5. In the final phase, a novel antimicrobial peptide, termed KM-12, was generated containing two KM motifs dimerized via disulfide bonds. The activity of KM-12 on C. albicans was approximately fifteen times more potent than the monomeric peptide and ten times more active than the native histatin 5. KM-12 was shown to have antifungal activity with several Candida species, including fluconazole resistant species. In conclusion, KM-12 is promising antifungal peptide that will serve as a lead candidate for the development of antifungals peptide for pharmaceutical applications

Études spectroscopiques du mécanisme d'action de peptides synthétiques à potentiel antimicrobien

Tableau d'honneur de la Faculté des études supérieures et postdorales, 2016-2017Cette thèse porte sur l’élucidation du mode d’action de peptides synthétiques qui sont des analogues cationiques du peptide modèle 14-mère. À l’instar des peptides antimicrobiens naturels, ces peptides synthétiques induisent des perturbations membranaires et une meilleure connaissance de leur mode d’action s’avère un élément déterminant pour le design de nouveaux peptides synthétiques qui présenteront une activité antibactérienne maximale et une activité hémolytique moindre. Afin de mieux caractériser le rôle de certains déterminants moléculaires sur l’interaction membranaire, nous avons sélectionné deux catégories de peptides, soit des analogues qui ont une structure secondaire en hélice α et qui sont non sélectifs à l’égard des membranes mimétiques de bactérie et des analogues qui forment des feuillets β intermoléculaires et qui sont sélectifs à l’endroit des membranes mimétiques de bactérie. Les études réalisées ont fait intervenir plusieurs techniques spectroscopiques comme la spectroscopie infrarouge, la spectroscopie RMN des solides et la spectroscopie de fluorescence. Tout d’abord, nous avons vérifié le rôle des interactions électrostatiques sur l’interaction membranaire, et ce, en effectuant une étude comparative entre des analogues cationiques et anioniques. Les résultats obtenus démontrent que les peptides anioniques et cationiques ont un impact similaire sur la dynamique des têtes polaires des phospholipides et l’ordre conformationnel des chaînes acyle. Cependant, les tests de relargage ont démontré que seuls les peptides cationiques formant des feuillets β intermoléculaires sont sélectifs alors que les homologues anioniques qui forment des feuillets β intermoléculaires sont inactifs en présence des membranes mimétiques de bactéries et de cellules eucaryotes. Ensuite, pour mieux comprendre le mode de perturbation membranaire des peptides cationiques qui sont non sélectifs et sélectifs, nous avons effectué des études par spectroscopie RMN des solides sur des échantillons de peptides reconstitués dans des bicouches lipidiques mécaniquement orientées entre des lamelles de verre ainsi que échantillons bicellaires. Les résultats ont démontré que les peptides sélectifs perturbaient davantage l’orientation des phospholipides zwitterioniques en comparaison avec les peptides non sélectifs. De plus, les simulations spectrales réalisées sur les spectres expérimentaux suggèrent que les peptides non sélectifs induisent la formation de pores ayant une géométrie toroïdale. En terminant, nous avons étudié la topologie membranaire et la localisation dans la membrane d’un peptide ayant une conformation en hélice α (R5R10) et d’un peptide formant des feuillets β intermoléculaires (R4R11). Les résultats obtenus par RMN de l’azote-15 indiquent que le peptide R5R10 est localisé au niveau de la surface membranaire avec une certaine hétérogénéité dans l’orientation. Cette conclusion est soutenue par des expériences réalisées par spectroscopie infrarouge en mode ATR. Des mesures de distances à partir de la technique REDOR entre les phospholipides et les peptides ont permis de constater que le peptide en hélice α est localisé à proximité du noyau phosphore alors que le peptide en feuillets β agrégés est localisé plus superficiellement au niveau de la surface membranaire. Cette différence de pénétration dans la région interfaciale pourrait expliquer la différence de sélectivité entre le peptide R5R10 (non sélectif) et R4R11 (sélectif). De plus, les tests de relargage de la calcéine ont démontré que les deux peptides induisent une contrainte de courbure positive à la membrane lipidique. Ainsi, l’alliance des résultats suggèrent que le mode de perturbation membranaire des peptides R5R10 et R4R11 est similaire au mécanisme modèle du type sinking-raft.This thesis is related to the determination of the mode of action of synthetic peptides that are cationic derivatives of the model 14-mer peptide. Similarly to the natural antimicrobial peptides, these synthetic peptides induce membrane perturbations and a better understanding of their mechanism of action is of primary importance to design new synthetic derivatives displaying higher antimicrobial and lower hemolytic activities. In order to characterize the role of certain molecular determinants on the membrane interactions, we have focused on two categories of peptides, namely peptides adopting an α-helical conformation which are non-selective towards bacterial mimetic membranes and peptides forming intermolecular β-sheet structures which are selective towards bacterial mimetic membranes. The experiments were carried out with several spectroscopic techniques such as infrared spectroscopy, solid-state NMR spectroscopy and fluorescence spectroscopy. First, we have studied the effect of electrostatic interactions on the membrane interactions by doing a comparative study between cationic and anionic analogs. The results show that both the anionic and the cationic derivatives have a similar impact on the dynamics of the phospholipid polar headgroups and on the conformational order of the acyl chains. However, dye-release experiments have shown that only the cationic derivatives forming intermolecular β-sheet structures are selective whereas their homologous β-sheet aggregated anionic peptides are inactive in the presence of both bacterial and eukaryotic mimetic membranes Then, to better understand the mode of membrane perturbation of non-selective and selective peptides, we have performed solid-state NMR experiments on peptides reconstituted in oriented samples of phospholipids. The results have shown that the selective peptides disrupt even more the orientation of zwitterionic phospholipids than the non selective peptides. In addition, the spectral simulations performed on experimental spectra suggest that the non-selective peptides induce the formation of pores having a toroidal geometry. To conclude, we have studied the membrane topology and the location into the membrane of the α-helical peptide R5R10 and the β-sheet aggregated peptide R4R11. The results obtained using ¹⁵N NMR indicate that the α-helical peptide is located on the membrane surface with a certain degree of heterogeneity. This conclusion is also supported by experiments performed using ATR spectroscopy. Distance measurements between phospholipids and peptides using the REDOR technique indicate that the α-helical peptide is near the phosphate nucleus of phospholipids whereas the β-sheet aggregated peptide is superficially located on the membrane surface. The difference of penetration into the interfacial region could explain the difference of selectivity between the R5R10 peptide (non selective) and the R4R11 peptide (selective). In addition, dye-release experiments have shown that both peptides induce a negative curvature strain to the membrane. Therefore, by taking into account all the results, it seems that the mechanism of action of these peptides is similar to the sinking-raft model mechanism

Membrane-active derivatives of the frog skin peptide Esculentin-1 against relevant human pathogens

Candida albicans represents one of the most prevalent species causing life-threatening fungal infections. Current treatments to defeat Candida albicans have become quite difficult, due to their toxic side effects and the emergence of resistant strains. Antimicrobial peptides (AMPs) are fascinating molecules with a potential role as novel anti-infective agents. However, only a few studies have been performed on their efficacy towards the most virulent hyphal phenotype of this pathogen. The purpose of this work is to evaluate the anti-Candida activity of the N-terminal 1–18 fragment of the frog skin AMP esculentin- 1b, Esc(1–18), under both in vitro and in vivo conditions using Caenorhabditis elegans as a simple host model for microbial infections. Our results demonstrate that Esc(1–18) caused a rapid reduction in the number of viable yeast cells and killing of the hyphal population. Esc(1–18)revealed a membrane perturbing effect which is likely the basis of its mode of action. Esc(1-18) is able (1) to kill both growing stages of Candida; (2) to promote survival of Candida-infected living organisms and (3) to inhibit transition of these fungal cells from the roundish yeast shape to the more dangerous hyphal form at sub-inhibitory concentrations. Pseudomonas aeruginosa is an opportunistic bacterial pathogen that forms sessile communities, named biofilms. The non-motile forms are very difficult to eradicate and are often associated with the establishment of persistent infections, especially in patients with cystic fibrosis. The resistance of P. aeruginosa to conventional antibiotics has become a growing health concern worldwide and has prompted the search for new anti-infective agents with new modes of action. Naturally occurring antimicrobial peptides (AMPs) represent promising future template candidates. Here we report on the potent activity and membrane-perturbing effects of the amphibian AMP esculentin(1-21), on both the free-living and sessile forms of P. aeruginosa, as a possible mechanism for biofilm disruption. Furthermore, the findings that esculentin(1-21) is able to prolong survival of animals in models of sepsis and pulmonary infection indicate that this peptide can be a promising template for the generation of new antibiotic formulations to advance care of infections caused by P. aeruginosa

Synthèse et caractérisation de composés à potentiel antimicrobien à base de peptides et de sulfahydantoïnes inhibitrices de β-lactamases

La résistance bactérienne aux antibiotiques est une menace en constante expansion. Les bactéries ne cessent de développer de nouvelles résistances à un rythme parfois plus rapide que notre capacité à développer de nouveaux médicaments pour les combattre. Il est donc important si l'on veut garder la cadence dans ce marathon contre les bactéries de continuer la recherche de nouvelles méthodes pour contrer la résistance aux antibiotiques. La présente étude se divise en deux projets portant chacun sur une approche distincte dans le but de développer de nouveaux antibiotiques. La première partie porte sur l'étude des peptides antimicrobiens comme nouvelle classe d'antibiotiques. Leur mode d'action distinct des antibiotiques sur le marché est prometteur pour un développement de résistance amoindrie. Par contre, leur complexité entraîne une conception de médicament plus ardue. Afin d'aider la compréhension des facteurs influençant l'interaction des peptides avec leur principale cible, les membranes cellulaires, un peptide modèle synthétique simple et neutre ainsi que des analogues chargés positivement furent développés dans notre groupe. Ces peptides, notamment surnommés 14-mère, R4R11 et R5R10, ont d'abord été étudiés par des multiples méthodes spectroscopiques, biophysiques et bio-informatique. Ces études démontrent qu'il est possible de moduler la structure secondaire des peptides par la position des acides aminés cationiques dans la séquence. De plus, la conformation des peptides influence leurs interactions avec les membranes modèles et les cellules où ceux en feuillets β sont plus sélectifs envers les modèles et cellules procaryotes alors que ceux sous forme d'hélice α interagissent indistinctement avec les deux types membranes modèles ou cellules. Il a aussi été observé une différence d'orientation entre les peptides hélicoïdaux neutres et cationiques en présence de différentes membranes lipidiques. Les peptides étudiés ont une plus grande affinité envers les bicouches plus minces et les peptides cationiques ont une plus grande interaction avec les membranes anioniques. Ensuite, le projet s'est poursuivi par l'étude de l'effet de l'ajout d'un groupement ayant une propension à se lier aux membranes procaryotes. Pour ce faire, un ligand bis-dipicolylamine (bis-DPA) a été ajouté à l'extrémité N-terminale des peptides 14-mère, R4R11 et R5R10 afin de former un complexe de Zn(II) in situ avec le ligand bis-DPA (Zn₂•bisDPA). En général, la présence du complexe augmente la sélectivité des peptides par l'entremise d'une interaction plus grande envers les cellules procaryotes et membranes modèles anioniques. La seconde partie vise plutôt la recherche de nouveaux inhibiteurs de β-lactamases visant sur une approche de combinaison de médicaments afin de diminuer la résistance aux antibiotiques β-lactames. Malgré leurs similitudes avec les composés β-lactames et leurs propriétés d'inhibiteurs de protéases, les molécules de la famille des sulfahydantoïnes n'ont pas été investiguées comme inhibiteurs de β-lactamases. Divers analogues ayant comme cœur l'hétérocycle sulfahydantoïne ont été synthétisés à partir d'acides aminés et ont été évalués pour leur pouvoir inhibiteur sur les β-lactamases courantes TEM-1 et TEM-15. De ces analogues, certain ont démontré une inhibition notable des deux β-lactamases avec des valeurs de IC₅₀ entre 130 et 510 μM et des valeurs inférées de Kᵢ entre 32 et 55 μM. Ces résultats indiquent que ce type de composés ont un potentiel intéressant comme futur inhibiteur de β-lactamases.Antibiotic resistance is one of the top worldwide healthcare problems. Bacteria are continuously finding new ways to survive the treatment we develop. To stay ahead in this race, we must accelerate the development of new drugs that counteract bacterial resistance. The present study is divided in two projects each using a separate approach aiming to find new antimicrobial molecules. The first part of this thesis focusses on the study of antimicrobial peptides as a potential new class of antibiotics. Their mode of action is different than commercially available antibiotics and is less prone to induce resistance development. However, the design of new peptides for clinical uses is challenging because of the complexity of these compounds. To have a better understanding of the molecular determinants affecting their interaction with cellular membranes, we have developed a simple synthetic model peptide with a neutral global charge named 14-mer. Multiple analogs were synthesized bearing cationic amino acids at different positions in the sequence. Notably, analogs R4R11 and R5R10, bearing arginine residues at positions 4 and 11, and 5 and 10 respectively, were studied alongside the 14-mer model by various spectroscopic, biophysical and bioinformatics methods. These experiments show that the secondary structure and supramolecular self-assembly can be modulated by the position of the cationic residue in the peptide sequence. The peptide secondary structure affects their interactions with model membranes and living cells. The β-strand peptides tend to interact more selectively toward prokaryotic model membranes and cell whereas α-helical peptides interact indistinctly with both prokaryotic and eukaryotic model membranes and cells. In addition, a difference in peptide orientation has been observed between uncharged and cationic α-helical peptides when they interact with phospholipid membranes. These peptides have generally a higher affinity with thinner bilayers, and cationic peptides possess better interaction with cationic membranes. The investigation was continued by studying the effect of the incorporation of a molecular tag that is known to have a high affinity toward prokaryotic membranes. This was achieved by adding a bis-dipicolylamine (bis-DPA) ligand at the N-terminus of peptides 14-mer, R4R11 and R5R10 to form a Zn(II) complex in situ. The Zn(II) complex tends to increase the selectivity of the studied peptides toward prokaryotic model membranes and cells. The second part of this thesis instead focusses on finding new β-lactamase inhibitors in order to reduce the antibiotic resistance of one of the most versatile antibiotic families, the β-lactams. As potential candidates, the sulfahydantoin family has never been investigated for this application even if they possess similar structure to β-lactam antibiotics and has been shown to inhibit similar enzymes like proteases. To evaluate their potential, we synthesized multiple analogs containing the sulfahydantoin heterocycle starting from amino acids. These analogs were tested as inhibitors of two of the most prevalent β-lactamases, TEM-1 and TEM-15. Out of these compounds, two analogs have shown substantial inhibition with IC₅₀ values between 130 and 510 μM and inferred Kᵢ values between 32 and 55 μM. These results suggest that sulfahydantoin compounds have a good potential for the development of new and improved β-lactamase inhibitors

Physico-chemical characterization of model bio-membranes and their interaction with potential therapeutic peptides

In the present Ph.D. thesis an extensive structural and functional study on model bio-membranes is presented. The first part of my research has been focused to understand how the lipid composition of the bio-membranes affects their biophysical properties and modulates their interactions with peptides. One of the biological processes involving lipid composition is the interaction between antimicrobial peptides (AMPs) and biological membranes. In fact, the selective interaction of AMPs with prokaryotic cells arises from the difference in the chemical composition between prokaryotic and eukaryotic membranes. Different mechanisms of membrane destruction have been proposed, depending on physico-chemical properties of AMPs and of the target bio-membranes. Among the large number of AMPs present in nature, Myxinidin, from hagfish (Myxine glutinosa L.), is a promising antimicrobial candidate due to its antibacterial activity against different pathogenic Gram negative and Gram positive bacteria. This thesis reports a comparative study of the interaction between Myxinidin and its mutant WMR with two model bio-membranes at different composition and complexity. In particular, in order to understand the role of lipid composition in the peptide-membrane interaction, two different models of bio-membranes have been studied mimicking P. aeruginosa and E. coli cell cytoplasmic membranes. The final goal was to elucidate the effect of amino acid residues substitutions of the peptides and the role of lipid composition on the antibacterial activity of Myxinidin and WMR against these two model bio-membranes.The collected data have allowed to recognize the AMPs specificity for a particular lipid composition and to propose a mechanism of membrane destabilization. In order to study the role of lipid composition in biological processes, another important model bacterial bio-membrane has been studied. In particular the work has been focused on a particular model of bio-membrane representative of Bradyrhizobium BTAi1 Gram negative bacterium, containing an unusual lipopolysaccharide (LPS) in which the lipid A is covalently linked to a hopanoid moiety The aim of this study was to understand the effect of this unique lipid A in modulating the stability and rigidity of the outer membrane of Bradyrhizobium BTAi1 strain. To obtain a wide physico-chemical characterization of the analyzed systems, a combined experimental strategy has been adopted, including spectroscopic and calorimetric techniques such as Circular Dichroism (CD) to study the secondary structure of peptides and its changes in lipid environment; Fluorescence to estimate the microenvironment of the peptides in the vesicles; Dynamic Light Scattering (DLS) to estimate the size and distribution of the liposomes in the absence and in the presence of peptides; (NMR) to obtain information about the conformation of the peptides in membrane environment; Electron Paramagnetic Resonance (EPR) to investigate the dynamics of the lipid hydrophobic tails in the bilayer; Differential Scanning Calorimetry (DSC) to understand the thermotropic behavior of liposomes and the effect of peptides on their phase transition; Isothermal Titration Calorimetry (ITC) to study the energetic of the interaction process between peptides and liposomes

Characterisation of novel antimicrobial peptides from Egyptian scorpion and snake venoms.

Scorpion and snake venoms consist of diverse mixtures of peptides and proteins with varying biological activities and offer an attractive source for the development of novel therapeutics. Smp24 (24 aa) and Smp43 (43 aa) are antimicrobial peptides (AMPs) that were identified from the venom gland of the Egyptian scorpion Scorpio maurus palmatus. These alpha-helical peptides showed potent activity against both Gram positive and Gram negative bacteria with MICs ranging from 4 to 128 ug/ml. Four anti-bacterial peptides were purified using HPLC chromatography from the venom of three different species of Egyptian snakes. The molecular masses of the purified proteins were identified by MALDI-TOF/MS and N-terminal sequences suggest that they are members of the three-finger toxin superfamily. Both SEM and TEM were employed to visualise morphological changes and membrane damage of E. coli and S. aureus in response to different concentrations of Smp peptides at different time intervals. Using DNA microarray, we examined the transcriptomic responses of E. coli to sub-inhibitory doses of Smp24 and Smp43 peptides following 5 hours of incubation. Differentially expressed genes in the presence of peptides or a control antibiotic (Polymyxin B) compared with the absence of peptides were predominantly related to siderophore biosynthesis and transport, as well as more generalised cation transport and oxidative stress responses. The antibacterial effects of Smp peptides were inhibited in the presence of calcium and magnesium ions, but not other cations. Smp peptides offer a promising starting point for the development of new antimicrobial agents and transcriptomic analysis can help identify metabolic processes affected by scorpion venom AMPs which may be beneficial in understanding their mechanism of action