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Antimicrobial peptides (AMPs) are ubiquitous and display broad-spectrum antimicrobial activity that can control bacterial colonization of surfaces. Ceragenins are small-molecule mimics of AMPs and have several advantages over AMPs, including cost of manufacture and stability. A ceragenin, CSA-120, modified with an acrylamide group was directly incorporated into fluoropolymer coatings as a means of inhibiting bacterial biofilm formation. The ceragenin-containing coatings displayed improved performance. By conjugating a copper chelating group to the ceragenin, chelation of 64Cu by the conjugate was effective and provided a stable complex that allowed in vivo imaging. This conjugate may provide a means of identifying infection sites in patients presenting general signs of infection without localized symptoms. A combination nanoparticle comprised of a maghemite core for enhanced T2 MRI contrast diagnostics, a colloidal silver shell acting as an antimicrobial and therapeutic vehicle, and a ceragenin (CSA- 124) surfactant providing microbial adhesion was synthesized and characterized by multiple methods. Silver nanoparticles conjugated with ceragenin, CSA-124, as a potential Gram-positiveselective antimicrobial were synthesized and termed as CSA-SNPs. Herein, CSA-SNPs are characterized using multiple methods and the antimicrobial properties are determined through minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) and time-kill study. Prostanoids are a natural subclass of eicosanoids generated mainly from metabolic oxidation of arachidonic acid. Cyclopentenone prostaglandins (cyPGs) contain a highly reactive α,β-unsaturated carbonyl group in their cyclopentenone ring and possess three main potentially therapeutic properties: anti-inflammatory, antiproliferative and antiviral. We designed and synthesized EC and its derivatives in reducing secretion of pro-inflammatory cytokines IL-6 and IL-12. Mucosal-Associated Invariant T (MAIT) Cells are unique innate-like T cells and play a key role in host defense against bacterial and fungal infection as well as in human autoimmune diseases. The MAIT cells are activated through T-cell receptor αβ chain (TCR-αβ) binding with the MR1-ligand, which is vitamin B metabolites presented on MR1. Rribityllumazines, one of important MR1-ligand was synthesized in my study.

The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development. In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4. The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways. Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host. In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.

Thesis (PhD (Viticulture and Oenlogy))--University of Stellenbosch, 2008. / Includes bibliography and list of tables and figures. / ENGLISH ABSTRACT: Over the last decade research has shown the importance of small antimicrobial peptides in the innate immunity of plants. These peptides do not only play a critical role in the multilayered defense systems of plants, but have proven valuable in the engineering of disease resistant food crops towards the ultimate aim of reducing the dependency on chemical fungicides. As the lists of isolated and characterized peptides grew, it became clear that other biological activities, in addition to the antimicrobial capacity, could be linked to some of these peptides; these alternative activities could have important applications in the field of medicine. This has made the defensin encoding genes prime targets for the agricultural and medical biotechnology sectors. To this end we set out to evaluate South African flora for the presence of plant defensin sequences and to isolate plant defensin genes that might be useful in biotechnology applications. Moreover, by isolating and characterizing these novel peptides, also in an in planta environment and in interaction with fungal pathogens, important knowledge will be gained of the biological role and importance of the peptides in the plant body. The plant host targets were South Africa Brassicaceae species including indigenous species, as well as Vitis vinifera, as the most important fruit crop in the world and since no defensins have been isolated from this economically important crop plant. The Brassicaceae family has been shown to be abundant in defensin peptides and several of the best characterized peptides with potent activity have been isolated from this family. Based on initial activity screens conducted on selected South African Brassicaceae spp. we concluded that these spp. contain promising antifungal peptide activities, warranting further efforts to isolate the genes and encoding peptides and to characterize them further. The preliminary activity screens used a peptide-enrichment isolation strategy that favored the isolation of basic, heat-stable peptides; these properties are characteristic features of plant antimicrobial peptides. These peptide fractions showed strong antifungal activities against the test organisms. A PCR-amplification strategy was subsequently designed and implemented, leading to the isolation of 14 novel defensin peptide encoding genes from four South African Brassicaceae spp., including the indigenous South African species Heliophila coronopifolia. Amino acid sequence analysis of these peptides revealed that they are diverse in amino acid composition and share only 42% homology at amino acid level. This divergence in amino acid composition is important for the identification of new biological activities within closely related plant defensins. Single amino acid changes have been contributed with the divergent biological activities observed in closely related plant defensin peptides. Phylogenetic analysis conducted on the deduced amino acid sequences revealed that all the new defensins share a close relationship to other Brassicaceae members of the plant defensin superfamily and was furthest removed from the defensins isolated from the families Solanaceae and Poaceae. Classification analysis of these peptides showed that they belong to subgroup A3 of the defensin superfamily. A putative defensin sequence was also isolated from V. vinifera cultivar, Pinotage, and termed Vv-AMP1. Genetic characterization showed that only a single gene copy of this peptide is present within the V. vinifera genome, situated on chromosome 1. Genetic characterization of this peptide encoding gene within the Vitis genus showed that this gene has stayed conserved throughout the divergent evolution of the Vitis genus. Expression studies of Vv-AMP1 revealed that this gene is expressed in a tissue specific and developmentally regulated manner, being only expressed in grape berries and only at the onset of vèraison. Induction of Vv-AMP1 in grapevine leaf material could never be achieved through the external application of hormones, osmotic stress, wounding, or pathogen infection by Botrytis cinerea. Deduced amino acid analysis showed that Vv-AMP1 encoded for a 77 amino acid peptide consisting of a 30 amino acid signal peptide and a 47 amino acid mature peptide, with putative antifungal activity. The Vv-AMP1 peptide grouped with the subclass B type defensins, which have been documented to have both antifungal and antibacterial activities. The Vv-AMP1 signal peptide directed the green fluorescent protein (GFP) reporter gene to the apoplastic regions in cells with high levels of accumulation in the vascular tissue and the guard cells of the stomata. Recombinant Vv-AMP1 peptide was successfully purified from a bacterial host and shown to have a size of 5.495 kDa. Recombinant Vv-AMP1 showed strong antifungal activity at low concentrations against a broad spectrum of fungal pathogens, which included Verticillium dahliae (IC50 of 1.8 μg mL-1) and the necrotrophic pathogen Botrytis cinerea (IC50 of 12-13 μg mL-1). Antifungal activity of Vv-AMP1 did not induce morphological changes in fungal hyphae, but its activity was associated with induced membrane permeabilization in treated hyphae. Vv-AMP1 was successfully introduced into Nicotiana tabacum as confirmed by Southern blot analysis and 20 individual lines were generated. Genetic characterization confirmed the integration and expression of the gene in the heterologous tobacco environment. The peptide was under control of its native signal sequence which has been shown to direct its product to the apoplastic regions of cells. The transgenic lines were analyzed to determine the presence and activity of the grapevine defensin peptide. Western blot analyses of partially purified plant extracts detected a signal of the expected size in both the untransformed control and the transgenic lines. Comprehensive analysis of EST databases identified three highly homologous sequences from tobacco that probably caused the background signal in the control. These crude protein extracts were able to inhibit the growth of V. dahliae in vitro when tested in a microtiter plate assay, but the inhibition could not be conclusively linked to the presence of the transgenic peptide, since non-expressing transgenic lines, included as controls, also showed inhibition. Similar results were obtained with infection studies, clearly showing that despite successful integration and expression of the transgene, the peptides was either not functional in the heterologous environment, or perhaps unstable under the particular regulatory conditions. This peptide belongs to a subclass of peptides known for associated activities that might activate tight control by plant hosts if threshold levels are reached. These aspects need further investigation, specifically since it is in stark contrast to previous results obtained with defensins from a different subclass. This study has also yielded significant other related resources that would be instrumental for further possible biotechnology exploitation of some of the novel peptides, but also to provide genetic constructs and plant material that would be invaluable to address fundamentally important questions such as the regulation and mode of action of defensin peptides, specifically in interaction with pathogen hosts. The novel peptides have been transformed to various hosts, including grapevine and these transgenic populations are available to facilitate the next rounds of research into this extremely promising group of antifungal peptides. / AFRIKAANSE OPSOMMING: In die laaste dekade het navorsing die belangrike rol van klein antimikrobiese peptiede in plantweerstandsmeganismes beklemtoon. Hierdie peptiede speel nie alleenlik 'n belangrike rol in die komplekse lae van plantweerstandstelsels nie, maar het ook hulle ekonomiese potensiaal getoon in die manipulering van siekteweerstandbiedendheid in voedselgewasse met die oorkoepelende doel om landbougewasse minder afhanklik van chemiese spuitstowwe te maak. Soos wat die hoeveelheid geïsoleerde en gekarakteriseerde peptiede toeneem, het dit duidelik geword dat ander biologiese aktiwiteite, bykomend tot die antimikrobiese kapasiteit, met sommige van dié peptiede verbind kan word; hierdie alternatiewe aktiwiteite het belangrike toepassing in veral die mediese veld. Dit het die defensin-koderende gene kernteikens vir die landbou- en mediese biotegnologiesektore gemaak. In die studie is daar begin om die Suid-Afrikaanse blommeryk te evalueer vir die teenwoordigheid van plantdefensingene en om dié gene te isoleer wat van ekonomiese belang vir die biotegnologiebedryf kan wees. Deur die in vitro- én in planta karakterisering van die unieke plantdefensinpeptiede word daar gemik daarna om belangrike inligting in te win oor die biologiese rol van die peptiede binne die plantligggaam. Die plantgashere wat geteiken is sluit in die Suid-Afrikaanse Brassicaceae-spesies, insluitende inheemse spesies, asook Vitis vinifera, wat as die belangrikste vrugtegewas ter wêreld beskou word. Die Brassicaceae-familie is welbekend daarvoor dat dit 'n ryk bron van plantdefensinpeptiede is en verskeie van die bes gekarakteriseerde antifungiese defensinpeptiede is van dié familie afkomstig. Aanvanklike aktiwiteitstoetse het getoon dat die Suid-Afrikaanse Brassicaceae-spesies belowende antifungiese aktiwiteit toon, wat die verdere isolering en karakterisering van dié gene en hul peptiedprodukte regverdig. Die aanvanklike aktiwiteitstoetse het 'n selektiewe peptiedverrykingstrategie gevolg wat die isolering van basiese, hittestabiele peptiede bevoordeel het; hierdie eienskappe is baie kenmerkend van plant-antimikrobiese peptiede. Die peptiedfraksies wat met hierdie metode geïsoleer is, het sterk antifungiese aktiwiteit teen die toetsorganismes getoon. Die resultate het gelei tot die ontwikkeling en toepassing van 'n polimerasekettingreaksie-strategie, wat daartoe gelei het dat 14 nuwe defensingene van vier Suid-Afrikaanse Brassicaceae-genera, insluitend die inheemse spesie Heliophila coronopifolia, geïsoleer kon word. Afgeleide aminosuurvolgorde-analises van die nuwe defensinpeptiede het gewys dat hulle slegs 42% homologie het. Hierdie diversiteit in aminosuurvolgorde is belangrik vir die identifisering van nuwe biologiese aktiwitiete binne die groep van verwante peptiede. Navorsing het verder getoon dat enkel-aminosuurverskille bydra tot die diverse spektrum van biologiese aktiwiteite binne 'n groep van verwante defensinpeptiede. Filogenetiese analise van die aminosuurvolgordes het getoon dat al die nuwe defensinpeptiede 'n sterk verwantskap met plantdefensinpeptiede, wat van ander Brassicaceae-spesies geïsoleer is, toon. Daarteenoor het dit die kleinste verwantskap getoon met plantdefensinpeptiede wat van die Solanaceae- en Poaceae-families geïsoleer is. Klassifikasiestudies het bewys dat die nuwe peptiede saam met subgroep A3 van die plantdefensin-superfamilie groepeer. 'n Moontlike plantdefensingeen, genaamd Vv-AMP1, is ook van die V vinifera-kultivar, Pinotage, geïsoleer. Genetiese karakterisering het aangedui dat slegs 'n enkele kopie van die geen in die V. vinifera-genoom teenwoordig en op chromosoom 1 geleë is. Genetiese karakterisering van Vv-AMP1 binne die Vitus-genus het gewys dat die geen binne die genus evolusionêr gekonserveerd is. Uitdrukkingstudies van Vv-AMP1 het verder bewys dat die geen uitgedruk word op 'n weefselspesifieke, ontwikkelingsgekoppelde wyse, naamlik slegs in druiwekorrels en slegs tydens rypwording. Vv-AMP1-uitdrukking kon nooit geïnduseer word in wingerdblare deur die uitwendige toediening van hormone, osmotiese stres, wonding of patogeeninfeksie deur Botrtys cinerea nie. Ontleding van die afgeleide aminosuurvolgorde het gewys dat Vv-AMP1 kodeer vir 'n 77-aminosuurpeptied, wat uit 'n 30-aminosuurseinpeptied en 'n 47-aminosuur-aktiewe peptied met voorspelde antifungiese aktiwiteit bestaan. Die Vv-AMP1-peptied is gegroepeer met subgroep B van die plantdefensin-superfamilie, 'n subgroep wat vir beide antifungiese en antibakteriese aktiwiteit gedokumenteer is. Die Vv-AMP1-seinpeptied het die groen fluoressensie-indikatorproteïen (GFP) na die apoplastiese areas van die plantselle gelei, met hoë vlakke van lokalisering in die vaatbundelweefsel en sluitselle van die huidmondjies. Die rekombinante Vv-AMP1-peptied is suksesvol geproduseer en uit 'n bakteriese produksieras gesuiwer, en het 'n molekulêre massa van 5.495 kDa gehad. Die gesuiwerde peptide het by lae konsentrasies 'n sterk aktiwiteit getoon teen 'n breë spektrum van fungiese patogene, wat Verticllium dahliae (IC50 van 1.8 μg mL-1) en die nekrotrofiese patogeen, B. cinerea (IC50 van 12-13 μg mL-1), ingesluit het. Vv-AMP1-aktiwiteit het geen ooglopende morfologiese veranderinge in die fungi-hifes veroorsaak nie, maar hulle aktiwiteit is verbind met 'n verhoogde membraandeurdringbaarheid in behandelde fungi-hifes. Suksesvolle intergrasie van Vv-AMP1 in die Nicotiana tabacum-genoom is deur Southern-kladontledings bevestig en 20 individuele transgeniese lyne is ontwikkel. Genetiese karakterisering van die transgeniese lyne het gewys dat Vv-AMP1 suksesvol geïntegreer is en ook in die transgeniese tabakomgewing uitgedruk word. Die peptied is uitgedruk onder beheer van sy eie seinpeptied, wat die aktiewe produk na die apoplastiese areas van die plantselle teiken. Die transgeniese tabaklyne is ook ontleed om te bepaal of die wingerdpeptied suksesvol geproduseer word en sy aktiwiteit in die transgeniese omgewing behou. Western-kladanalise van semi-gesuiwerde plantproteïenekstrakte het 'n positiewe sein gelewer in beide die kontroleplante en die transgeniese plantlyne. Bestudering van tabakgeenuitdrukkings-databasisse het drie nukleotiedvolgordes opgelewer wat homologie met Vv-AMP1 toon en moontlik verantwoordelik kan wees vir die positiewe sein in die ongetransformeerde kontroleplante. Kru proteïenekstrakte van die transgeniese tabaklyne het in vitro-aktiwiteit teen V. dahliae getoon. Geen oortuigende ooreenkoms kon egter gevind word tussen V. dahliae-inhibisie en die teenwoordigheid van die transgeniese Vv-AMP1-peptied nie, aangesien kontroleplante wat Southern-klad-positief is, maar nie geenuitdrukking toon nie, ook inhibisie van V. dahliae veroorsaak het. Soortgelyke resultate is met infeksiestudies verkry. Alle resultate dui daarop dat, al is daar suksesvolle integrasie en uitdrukking van die geen in tabak verkry, dat die Vv-AMP1 peptied óf onaktief óf onstabiel in die transgeniese tabakomgewing is. Die peptied behoort aan 'n subgroep peptiede met aktiwiteite wat, sodra sekere vlakke van peptied oorskry word, die moontlik streng kontrole op proteïenvlak in die gasheerplant kan uitlok. Sekere aspekte van die studie sal verder bestudeer moet word, aangesien die data teenstrydig is met data wat verkry is met soortgelyke plantdefensinpeptiede wat aan 'n ander subgroep behoort. Die studie het baie hulpbronne gegenereer wat vir die biotegnologiesektor belangrik kan wees, veral op ekonomiese gebied. Verder is die geenkonstrukte en plantlyne wat ontwikkel is waardevol om fundamentele vrae rondom die regulering en meganisme van aksie van defensinpeptiede, spesifiek plantpatogeeninteraksie, te beantwoord. Die nuwe plantdefensingene is na verskeie gasheerplante, insluitende wingerd, getransformeer waar die transgeniese lyne die volgende rondte van navorsing oor die bestudering oor die belangrike groep van antifungiese peptiede, sal aanvul.

Ce travail a contribué à la compréhension des bases moléculaires de l'immunité de l'huître creuse par la caractérisation la diversité de trois effecteurs antimicrobiens de C. gigas et par l'appréhension du rôle de cette diversité dans les mécanismes de défense. Des analyses phylogénétiques de deux peptides antimicrobiens (AMPs), Cg-Défensines (Cg-Defs) et Cg-Proline rich peptide (Cg-Prp), et d'une protéine de type Bactericidal Permeability Increasing protein, Cg-BPI, nous a permis montrer la grande diversité pour les 2 AMPs, qui est générée par plusieurs mécanismes génétiques et par des pressions de sélection directionnelles, suggèrant une diversité fonctionnelle des variants. L'importance biologique de cette diversité a été étudiée pour trois variants de Cg-Defs. Une forte activité antimicrobienne a été mise en évidence contre les bactéries à Gram positive, mais celle-ci diffère selon les variants. De plus, nous avons démontré que le mécanisme d'action des Cg-Defs contre S. aureus repose sur l'inhibition de la biosynthèse du peptidoglycane par le piegeage de son précurseur, le lipide II. Finalement, l'expression des transcrits et la localisation de ces effecteurs en réponse à une infection par un Vibrio pathogène ont montré un réseau complexe des profils d'expression des différents antimicrobiens, au niveau des populations hémocytaires et des tissus d'huître, suggérant une interaction entre les antimicrobiens du fait de leur colocalization. La combinaison entre les familles ou entre les variants d'une même famille produit de fortes activités synergiques qui élargissent les spectres d'activité. Ainsi, la diversité produit par la coévolution entre hôte et pathogènes pourrait améliorer l'activité des AMPs d'huître, lui conférant une plus grande protection contre les pathogènes de son environnement. / This work contributed to the knowledge of the molecular bases of oyster immunity by the characterization of the diversity of three antimicrobials of C. gigas and the understanding of the role played by their diversity in the oyster antimicrobial response. Phylogenetic analyses of two antimicrobial peptides (AMPs), Cg-Defensins (Cg-Defs) and Cg-Proline rich peptide (Cg-Prp), and one Bactericidal Permeability Increasing protein, Cg-BPI, led us to the identification of a high diversity for both AMPs. Further analyses showed that this diversity is generated by gene duplication, allelic recombination and directional selection pressures, suggesting their functional diversification. The biological meaning of AMP diversity was investigated for the three major variants of Cg-Defs, which revealed a strong but variable potency against Gram-positive bacteria. We evidenced that oyster defensins kill S. aureus through binding to the cell wall precursor lipid II, resulting in the inhibition of peptidoglycan biosynthesis. Finally, transcript expression and localization of oyster antimicrobials after a pathogenic infection evidenced a complex network in their expression profiles in hemocyte populations and oyster tissues, suggesting a potential interplay between antimicrobials as a result of their colocalization. Indeed, the combination of oyster antimicrobials produced strong synergistic activities that enlarged their antimicrobial spectra. Thus, the diversity of oyster antimicrobials may provide significant means in acquiring functional divergence, probably concerned in the evolutionary arms race between hosts and their pathogens.From our data, it would provide oysters with a higher protection against the potential pathogens from their environment.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

La prévention du risque infectieux représente un enjeu de sécurité majeur pour l'Etablissement Français du Sang. A l'heure actuelle, le risque bactérien constitue le risque infectieux majoritaire dans les pays développés. Le risque bactérien ne se limite pas au domaine de la transfusion. La résistance aux antibiotiques constitue un problème majeur de santé publique. Les peptides antimicrobiens sont des armes importantes de l'immunité innée qui représentent une alternative intéressante aux antibiotiques classiques. Chez l'Homme, les α-défensines 1, 2 et 3 (HNPs 1-3) sont présentes dans les granules azurophiles des polynucléaires neutrophiles. Nous avons développé un procédé innovant de purification des HNPs 1-3 à partir des filtres de déleucocytation utilisés lors de la préparation des produits sanguins labiles. Ce procédé permet la production d'un cocktail pur d'HNPs 1-3 dont l'activité antibactérienne a été démontrée dans ce travail. Les défensines HNPs 1-3 ont par ailleurs été utilisées comme élément biorécepteur dans une approche innovante de détection bactérienne. Dans un premier temps, un immunocapteur électrochimique a été développé exploitant des microparticules magnétiques fonctionnalisées par des anticorps commerciaux. Dans un second temps, nous avons fonctionnalisé des microparticules magnétiques par les défensines HNPs 1-3 purifiées par notre protocole. Nous avons obtenu une première preuve de concept attestant de la capture des bactéries par cette approche innovante. La stabilité des peptides, associée aux performances des biocapteurs électrochimiques permettrait d'élaborer un test générique de détection de bactéries dans les produits sanguins labiles. / The prevention of the infectious risk is a major issue for the Etablissement Français du Sang. Currently, bacterial contamination is the most infectious risk in developed countries. The bacterial risk is not limited to blood transfusion safety. The antimicrobial resistance is a major public health problem. Antimicrobial peptides are important arm of the innate immune system which represents an interesting alternative to antibiotics. Human neutrophil peptides 1, 2 and 3 (HNPs 1-3) are found in the azurophilic granules of neutrophils. We have developed an original approach of HNPs 1-3 purification from leucodepletion filters used in blood processing. This process allows the production of a pure cocktail of HNPs 1-3 displaying high antibacterial activity as demonstrated by this work. HNPs 1-3 have also been used as bioreceptor in an innovative approach for bacterial detection. Initially, an electrochemical immunosensor was designed, exploiting magnetic microparticles coated with commercially available antibodies. In a second step, magnetic microparticles have been coated efficently with the HNPs 1-3 purified according our protocol. We have obtained a first proof of concept showing the bacterial capture by this innovative approach. The peptides stability combined with the electrochemical biosensors performances would allow the development of a generic bacteria detection assay in labile blood products.

Le peptide antimicrobien Cateslytine (bCGA RSMRLSFRARGYGFR ) inhibe la libération des catécholamines des cellules chromaffines. Des études biologiques ont montré que ce peptide est capable d’inhiber aussi la croissance de nombreux microorganismes notamment des bactéries, des levures ainsi que le parasite Plasmodium falciparum responsable de la malaria. Cependant, le mode d’action moléculaire demeurait inconnu. Afin de mieux comprendre le ciblage et la sélectivité de ce peptide sur les membranes de mammifères ou de microorganismes, nous avons donc envisagé la reconstitution du système biologique composé initialement de peptides en contact avec des cellules, en le substituant par des modèles de membrane, de composition mimant celle des différents microorganismes. Des études structurales ont été menées en utilisant la technique d’ATR-FTIR polarisé, le dichroïsme circulaire et la RMN à haute résolution. La dynamique membranaire a été étudiée en utilisant la RMN des solides du phosphore et du deutérium. Des expériences de patch-clamp ont été effectuées afin de mesurer des flux d’ions au travers de la membrane. Enfin, de la simulation par ordinateur a permis de comprendre cette interaction au niveau moléculaire. Trois résultats principaux sont ressortis de cette approche pluridisciplinaire : i) Des flux ioniques au travers de la membrane attestent de la présence de cannaux. ii) La formation de domaines membranaires rigides constitués de lipides chargés négativement est démontrée. iii) Une structuration des peptides en feuillets ß antiparallèles est observée sur des membranes chargées négativement mimant les microorganismes. L’ensemble de ces résultats conduit à la proposition d’un mode d’action dans lequel la déstabilisation membranaire est induite par les domaines rigides stabilisés par les agrégats de peptides structurés en feuillets ß. / The antimicrobial peptide Cateslytin (bCGA RSMRLSFRARGYGFR ) is a five positively charged arginin rich peptide known to inhibit the release of catecholamine in chromaffin granules. Although biological data showed that it is able to inhibit the growth of several microorganisms such as bacteria, yeast and Plasmodium falciparum parasite involved in malaria, the mechanism of action has not been yet studied. In order to better understand both targeting and selectivity of this peptide towards microorganisms, model membranes of variable compositions have been chosen to respectively mimic microorganisms or mammalian membranes. Structural studies have been performed using polarised ATR-FTIR, circular dichroïsm and high resolution NMR Membrane dynamics has been followed using deuterium labelled lipids and solid state NMR Patch clamp experiments were also performed on lipid vesicles to measure channe conductivity. All-atom molecular dynamics on hydrated peptide-lipid membrane systems was also used to assess the interaction from the atomic level. Main results from this interdisciplinary approach are three-fold. i) Electric current passages through membranes demonstrate permeation akin to pore formation. ii) Peptide-induced formation of rigid domains mainly made of negatively charged lipids is found. iii) Peptide antiparallel ß-sheets are observed preferentially with negatively charged lipids mimicking microorganism membranes. The general picture leads to the proposal that membrane destabilization/permeation is promoted by rigid domains stabilised by peptide ß-sheets.

O desenvolvimento de resistência antimicrobiana e a consequente seleção de microrganismos multirresistentes consolidam-se como grandes ameaças à saúde global. Neste contexto, a busca por novas drogas antimicrobianas/microbicidas é fundamental e compostos como os peptídeos antimicrobianos (AMPs) tornaram-se alvos atraentes. Os AMPs são compostos químicos de massa molar média e grande diversidade estrutural, produzidos por todos os seres vivos e com capacidade de inibir o crescimento de e/ou matar microrganismos. O AMP Cheferina I (Chef I) foi isolado das raízes de Capsella bursa-pastoris e é resultado da proteólise de uma proteína da família das proteínas ricas em glicina, que em plantas estão relacionadas às funções de defesa e cicatrização. O nosso grupo de pesquisa foi pioneiro no desenvolvimento e estudo de análogos truncados amidados deste AMP atípico rico em glicina (67,9%) e histidina (28,6%), que se mostraram ativos frente às diferentes cepas de Candida e a S. cerevisiae pela internalização/ação celular acompanhada de manutenção da integridade da membrana plasmática; o análogo amidado (Chef Ia) e o análogo marcado com 5(6)-carboxifluoresceína/FAM (FAM-Chef Ia) tiveram as suas atividades antifúngicas potencializadas por íons Zn2+. Este trabalho deu continuidade ao estudo do efeito dos íons metálicos divalentes Zn2+, Cu2+, Ca2+ e Mg2+ nas atividades anticandida/fungistática e candidacida/fungicida a diferentes pHs e forças iônicas, estruturas e localizações intracelulares destes análogos. Os resultados na ausência de íons em pH 5,1 revelaram maior atividade do análogo fluorescente em relação à do não fluorescente. Neste mesmo pH, as atividades anticandida e candidacida de Chef Ia foram influenciadas negativamente pelos íons Ca2+ e Mg2+ (2-4 vezes) enquanto que, na presença de íons Zn2+ as atividades anticandida de ambos os análogos foram aumentadas (Chef Ia: 8-64 vezes; FAM-Chef Ia: 4-32 vezes). Os íons Cu2+ aumentaram a atividade anticandida de Chef Ia (2-4 vezes), mas não a do análogo fluorescente, mas as atividades candidacidas de ambos foram melhoradas (Chef Ia: 2-8 vezes; FAM-Chef Ia: 2 vezes). Em pH 5,1, os íons Zn2+ mantiveram a atividade anticandida de Chef Ia em alta força iônica, mas só FAM-Chef Ia exibiu atividade candidacida. Em pH 7,4 ambos análogos foram inativos em baixa e alta forças iônicas na ausência e presença de Zn2+ ou Cu2+. As maiores porcentagens de folhas-β-antiparalelas e dobras foram observadas no espectro de DC de Chef Ia em pH 7,4, sendo que aqueles registrados em pH 5,1 e 7,4 em presença de íons Zn2 e Cu2+ indicaram a formação de quelatos estruturalmente distintos. Ambos os peptídeos são bioquelantes em potencial, sendo as proporções peptídeo: íon obtidas as seguintes: FAM-Chef Ia = 1:2 para Cu2+, 1:10 para Zn2+; Chef Ia = 1:1 para Cu2+. A análise da internalização celular de FAM-Chef Ia permitiu a suposição de dois mecanismos de internalização (translocação direta e endocitose), sendo que nas células vivas a presença de Zn2+ afetou negativamente a translocação direta (p 0,0343) e potencializou a endocitose (p 0,0002). / The development of antimicrobial resistance and the consequent selection of multiresistant microorganisms have become major threats to global health. In this context, the search for new antimicrobial/microbicidal drugs is crucial and the antimicrobial peptides (AMPs) have been seen as attractive targets. AMPs are chemical compounds of medium molecular mass and high structural diversity produced by all living beings, capable of inhibiting the growth of microorganisms and killing them. The AMP Shepherin I (Shep I) was isolated from the roots of Capsella bursa-pastoris, being a bioactive peptide encrypted in a glycine-rich protein from a family that in plants are strictly related to defense and healing functions. Our research group has pioneered the development and study of amidated truncated analogues of this atypical glycine- (67.9%) and histidine-rich (28.6%) AMP, which has shown activity against different strains of Candida and S. cerevisiae through cellular internalization with maintenance of the plasma membrane integrity. The amide analogue (Chef Ia) and its fluorescent analog labeled with 5 (6) - carboxyfluorescein / FAM (FAM-Chef Ia) had their antifungal activities potentiated by Zn2+ ions, so the present work continued examining the effect of the divalent metallic ions Zn2+, Cu2+, Ca2+ and Mg2+ on the anticandidal/fungistatic and candidacidal/fungicide activities at different pHs and ionic forces, structures and intracellular locations of these analogues. The results in the absence of those ions at pH 5.1 revealed that the fluorescently labelled analog was more potent than the nonfluorescent. At the same pH, Shep Ia anticandidal and candidacidal activities were negatively influenced by Ca2+ and Mg2+ ions (2-4 fold), whereas in the presence of Zn2+ ions the anticandidal activities of both analogues were increased (Shep Ia: 8-64 fold, FAM- Shep Ia: 4-32 fold). Cu2+ ions increased Shep Ia anticandidal activity (2-4 fold) but not that of FAM-Shep Ia, nevertheless, the candidacidal activities of both analogues were increased (Shep Ia: 2-8 fold, FAM-Shep Ia: 2 fold). Also at pH 5.1, the Zn2+ ions helped retaining the anticandidal activity of Shep Ia at high ionic strength, although only FAM-Shep Ia exhibited candidacidal activity. At pH 7.4 both analogues were inactive at low and high ionic strengths in the absence or presence of Zn2+ or Cu2+. The highest percentages of antiparallel β-sheet and turns were observed in Shep Ia CD spectrum at pH 7.4, while those recorded at pH 5.1 and 7.4 in the presence of Zn2+ or Cu2+ ions indicated the formation of structurally different chelates. Both peptides are potential biochelates, with the following peptide:ion ratios: FAM-Shep Ia = 1: 2 for Cu2+, 1:10 for Zn2+; Shep Ia = 1: 1 for Cu2+. The analysis of the cellular internalization of FAM-Chef Ia allowed the assumption of two mechanisms of internalization (direct translocation and endocytosis) and in the living cells the presence of Zn2+ negatively affected the direct translocation (p 0.0343) and potentiated endocytosis (p 0.0002).

As society draws closer to the post-antibiotic era and the pipeline for alternatives dries, there is an urgent need for the development of novel antimicrobial therapies that do not promote bacterial resistance, particularly for immunocompromised chronic wound patients. Antimicrobial peptides (AMPs), including human-derived LL37, show considerable promise as broad spectrum alternatives that also have wound healing properties; however, few have been clinically implemented as novel antimicrobials due to their cytotoxicity stemming from a poor understanding of their mechanisms and low stability in vivo. It has been suggested that tethering, or attaching AMPs onto surfaces, is a viable strategy of delivering bioactive AMPs to surfaces while reducing cytotoxicity and improving stability. Thus, we designed new chimeric versions of LL37 with collagen-binding domains (CBD), derived from collagenase (cCBD-LL37) and fibronectin (fCBD-LL37) for non-covalent tethering onto collagen, a prevalent biopolymer in commercially available wound dressings and scaffolds. Our overall hypothesis was that CBDs would mediate stable tethering of broadly active, non-cytotoxic CBD-LL37 onto collagen-based scaffolds. We first studied the loading, release and bioactivities (e.g. antimicrobial activity and cytotoxicity) of each CBD-LL37 on commercially available 100% collagen type I PURACOL® wound scaffolds. We found that both cCBD-LL37 and fCBD-LL37 bound highly to collagen, were active against relevant wound pathogens, demonstrated stable activity after 14 days of release, and were not cytotoxic to human fibroblasts. The addition of different CBDs onto LL37 also markedly altered their soluble bioactivities. Using similar methods, we then studied the loading, release and bioactivity of each CBD-LL37 on a commercially available FIBRACOL® wound scaffolds, comprised of 90% collagen type I and 10% calcium alginate biopolymers. We found that both cCBD-LL37 and fCBD-LL37 also bound highly to and retained on collagen for 14 days, but were only active against Gram-negative P. aeruginosa. This suggested that the presence other biopolymers in addition to collagen, which is common among commercial wound dressings, could cause significant differences in binding, retention and bioactivities of CBD-LL37. To better understand how CBD modification affected CBD-LL37 structure leading to different bioactivities, we studied the CBD sequence-, peptide structure-, concentration-, time-, and bilayer composition-dependent interactions of soluble CBD-LL37 and compared these findings with the properties of unmodified LL37. Using Molecular Dynamics (MD) simulations, circular dichroism (CD) spectroscopy, quartz crystal microbalance with dissipation (QCM-D), and fluorescent bilayer imaging we determined the structural basis behind CBD alterations in bioactivities. MD and CD, in addition to other intrinsic CBD properties (helicity, amphiphilicity, charge) we hypothesized that cCBD-LL37 utilized similar mechanisms as unmodified LL37 while fCBD-LL37 demonstrated based primarily on surface adsorption. We used QCM-D and Voigt-Kelvin viscoelastic modeling to determine the time- and concentration-dependent interactions of unmodified LL37 with model mammalian lipid bilayers, the mechanisms of which are still controversial in literature despite being widely studied. These results were used to propose a model for the interaction mechanism of LL37 with zwitterionic bilayers that aligned with its bioactive concentrations. LL37 adsorbed at concentrations where it is immunomodulatory until reaching a threshold which corresponded with its antimicrobial concentrations. The threshold was correlated to lipid bilayer saturation, after which LL37 formed transmembrane pores. We observed collapse of the bilayer into a rigid proteolipid film at concentrations higher than the reported cytotoxic threshold of LL37. The mechanistic and structural information for each CBD-LL37 and unmodified LL37 provided a baseline for QCM-D and Voigt-Kelvin viscoelastic modeling to further elucidate the time-, concentration-, lipid composition- and CBD sequence-dependent basis behind the observed bioactivities of cCBD-LL37 and fCBD-LL37. We found that similar to LL37, cCBD-LL37 demonstrated pore formation mechanisms likely due to their similar charges, structural content and amphiphilicity. fCBD-LL37 demonstrated time-dependent, adsorption-based mechanism likely due to its anchoring aromatic residues, low charge, and low amphiphilicity. Knowledge gained from this study allowed mechanistic predictions of two newly designed hypothetical CBD-LL37 peptides. Results from this study contribute to a better understanding of a new class of antimicrobial, non-cytotoxic therapies based on collagen-tethered CBD-LL37, bringing it closer to clinical implementation in chronic wound applications and demonstrate the viability of biopolymer tethering as a platform toward using AMPs to quench the resistance crisis.