Synthetic vaccines, consisting of subunits of pathogens, have the potential to address several challenges of conventional vaccines (live-attenuated or killed pathogens) such as instability, risk of infection, autoimmunity and manufacturing difficulties. However, synthetic vaccines have significant limitations including poor immunogenicity and fast degradation, necessitating co-administration with effective adjuvants and use of appropriate delivery systems. Current choices of effective and safe adjuvants and delivery systems are very limited, and formulations/conjugations of synthetic vaccines often do not lead to clinical success. Rational design and novel synthetic methods are in high demand to overcome obstacles in synthetic vaccine development. In this PhD research project, new antigen delivery platforms (lipopeptide-carbohydrate-antigen platforms) were developed by integrating a lipopeptide and a topology-controlling template to present and deliver synthetic antigens, which may serve as a generic strategy to develop safe and effective synthetic vaccines. The focus of this research was on the rational design and optimized chemical synthesis of the platforms. The design of the lipopeptide-carbohydrate-antigen platforms was inspired by the principle of self-adjuvanting lipopeptide vaccines that feature the covalent attachment of bacterial or synthetic lipids to peptide antigens. Lipopeptide vaccines have generally shown a good safety profile in human clinical trials and seen as a potential approach for human vaccination. The lipopeptide [C12-G-C12-C12-G; G: glycine; C12: 2-(R/S)-aminododecanoic acid] used in this study was derived from the lipid core peptide (LCP) system which has demonstrated high delivery capacity and synthetic flexibility in numerous applications. Aiming to introduce the topology-controlling feature to self-adjuvanting lipopeptide vaccines, this study introduced topological templates (carbohydrates) to the platforms, based on the concept of template assembled synthetic peptide (TASP) that has emerged as a de novo strategy to assemble peptides in a desired conformation. Five carbohydrate templates that varied in conformation and spacer were used in the platform design, providing an opportunity to optimize the antigen’s spatial arrangement. The ultimate aim of this research is to develop antigen delivery platforms which can be used for human vaccines. Therefore, optimized methods for the synthesis of the platforms were developed to achieve high purity and good yield of the final products. Two series of carbohydrate templates were first synthesized, with functional groups suitable for covalent conjugation to the lipopeptide moiety and multiple peptide antigens. The spatial conjugation positions and the length of the spacers of these templates were varied through modifications to the hydroxyl groups of glucose, galactose and mannose. To achieve an efficient conjugation of the sugar molecules and the peptides (lipopeptide and peptide antigens), techniques that were examined included solid phase peptide synthesis (SPPS) and native chemical ligation (NCL). The attempts using SPPS did not yield the target product when incorporating a model antigen (20-mer peptide epitope 8830; DNGKAIYERARERALQELGP; derived from group A streptococcal M protein) to a glucose-based template because of significant difficulties including low coupling efficiency and unsuccessful purification. In contrast, a series of lipopeptide-carbohydrate-8830 products were synthesized in high purity and good yield using NCL, after the solubility problem had been solved. The poor water solubility of the lipopeptide-carbohydrate constructs was significantly improved through co-lyophilization of the compounds with 1% (w/v) sodium dodecyl sulphate. Conformational studies of the synthesized vaccine candidates containing 8830 were performed using circular dichroism (CD). The data revealed that the attached antigenic peptides formed α-helix bundles in all cases. The result is in line with the common α-helix-inducing feature of carbohydrate templates. This approach was extended to incorporate synthetic J14i (14-mer peptide epitope; ASREAKKQVEKALE; derived from group A streptococcal M protein) in an effort to induce its α-helicity. Embedding this minimal epitope in helix-promoting peptide sequences was previously developed in literature to mimic J14i’s helical structure at the corresponding site of the native protein. However, the introduction of “foreign” peptide sequences is believed to have an unfavourable impact on the antigen specificity. This work employed a non-peptide approach, using the developed topological carbohydrate templates, to induce helical conformation of the peptide antigen. CD studies confirmed that the template-assembled peptide J14i formed α-helix bundles. This strategy also reduces the complexity and cost of vaccine production by simply reducing the peptide size. Preliminary immunological evaluation demonstrated antigen-specific immune responses elicited by the synthesized vaccine candidates containing 8830, with vaccines based on different carbohydrate templates exhibiting varied efficacy. This data suggested that the different spatial structure of the vaccine candidates had key influence on the vaccine efficacy, pointing out the future direction of customizing carbohydrate templates for specific antigens.

Petroleum hydrocarbons are organic pollutants of major concern due to their wide distribution, persistence, complex composition, and toxicity. They can bioaccumulate in food chains where they disrupt biochemical or physiological activities and can affect genetic integrity of many organisms, resulting in carcinogenesis, mutagenesis and impairment of reproductive capacity. Polycyclic aromatic hydrocarbons (PAHs) have been recognized as priority pollutants due to their carcinogenic, mutagenic and teratogenic properties. Bioremediation, which utilizes the metabolic versatility of microorganisms such as bacteria and fungi to degrade or detoxify hazardous wastes into harmless substances has been recognized as a sustainable, economic, environmentally friendly and versatile alternative for the remediation of many contaminated environments; however its effectiveness is limited by low bioavailability of nonaquous phase and soil-bound PAHs and petroleum hydrocarbons due to their low aqueous solubility, high hydrophobicity and strong sorption to soil. The purpose of this study was to investigate the PAHs and petroleum hydrocarbons bioavailability and subsequent biodegradation enhancement potential of biosurfactants. Biosurfactants have steadily gained increased significance in environmental applications such as bioremediation dueto several advantages over surfactants of chemical origin, such as biodegradability, environmental compatibility, low toxicity, high selectivity and specific activity at extreme temperature, pH and salinity. A series of experiments was designed to investigate the bioavailability and subsequent biodegradation enhancement potential of the biosurfactants produced by the bacterial strains Bacillus subtilis CN2, Ochrobactrum intermedium CN3, Paenibacillus dendritiformis CN5 and Bacillus cereus SPL_4 in liquid culture and soil microcosms with PAH-enriched microbial consortium from chronically contaminated sites. The biosurfactants exhibited a high level of thermal stability, tolerance to extreme levels of salinity and a positive effect for increasing pH. They were identified after Fourier Transform Infrared (FT-IR) spectrometry, Thin Layer Chromatography (TLC) and Liquid Chromatography/Tandem Mass Spectrometry (LC MS/MS) analyses. The biosurfactants physicochemical characterization displayed vast structural diversity and potent surface active properties of surface tension reduction and emulsion formation with a range of hydrocarbons. The lipopeptide biosurfactants produced by CN3 and CN2 enhanced degradations of used motor oil and petroleum sludge in liquid culture. In a shake flask pyrene degradation study, lipopeptide supplementations at 600 and 300 mg L-1 enhanced pyrene degradation to 83.5% and 67% respectively in 24 days compared to 16% degradation in its absence. However, degradation of pyrene was reduced to 57% as the lipopeptide supplementation was raised to 900 mg L 1. This demonstrates that the biodegradation of pyrene was found to increase with an increase in the lipopeptide concentration up to a threshold level. In a soil bioremedial study, microcosms supplemented with 0.2 and 0.6% (w/w) lipopeptide, 51.2% of 4-ring and 55% of 5- and 6-ring PAHs, 64.1% of 4-ring and 79% of 5- and 6-ring PAHs were removed respectively, compared to, 29% of 4-ring and 25.5% of 5- and 6-ring PAHs removal in the surfactant free control after 64 days of incubation. However, there was no statistically significant change in the degradation rates of low molecular weight PAHs in surfactant amended and surfactant free controls. The degradation of 5 and 6 ring PAHs was significantly enhanced (p < 0.05) in the higher surfactant dosage compared to the lower dosage. The results of this work demonstrated that the use of biosurfactants is a viable option to reduce clean-up time and for effective remediation of soil and aqueous media contaminated with polycyclic aromatic and petroleum hydrocarbons. The study demonstrated potential applications of microbial surfactants and provided an insight for further investigation of their large scale production for commercial applications. / Thesis (PhD)--University of Pretoria, 2016. / Chemical Engineering / PhD / Unrestricted

Ongoing antibiotic drug discovery is vital as antimicrobial resistance continues to be a significant issue faced in the clinic. Natural products have long been a highly productive source to mine for new antimicrobials. While it has been challenging to discover new and unique antimicrobial natural products, numerous drugs have been derived from studying how natural products function as secondary metabolites. Previous studies suggested that screening natural product extract fraction libraries for antimicrobials can be more productive than screening crude extracts alone. These studies from large industrial enterprises are generally not directly portable to an academic setting due to significant infrastructure costs. We developed a screening platform consisting of low pressure reversed-phase chromatographic separation of methanolic extracts of bacteria and fungi to generate a prefractionated natural product library. This platform is suitable for academic labs to screen for antimicrobial compounds. A large growth inhibitor screen against multiple pathogens and lab strains of microbes was conducted to assess the validity of the advantages of screening fraction libraries versus crude extract libraries and to search for potential new drug-like compounds. Hits were investigated for reproducibility, isolated, and purified. One compound was discovered in an antifungal screen which may be a novel lipopeptide. / Thesis / Master of Science (MSc) / To combat the growing antibiotic resistance crisis, new strategies for drug discovery must be investigated and implemented. Natural products from bacteria and fungi have long been a source of critically important drugs. Prior research suggests that looking for vital natural products in fraction libraries can be more productive than screening crude extracts. Here the development and assessment of a fractionation library suitable for an academic lab's ingrained limitations are described. Assessing the library indicates an increased hit rate on screening fractions compared to crude extract. Furthermore, pursuing these hits may have revealed a novel antifungal lipopeptide.

Le milieu marin représente une source d'inspiration pour les chimistes, grâce à l'incroyable diversité structurale des composés isolés des organismes et micro-organismes marins. Parmi eux, la laxaphycine B, un lipopeptide cyclique isolée de la cyanobactérie Anabaena torulosa est comme la plupart des peptides marins produit selon une voie de biosynthèse non ribosomale « NRPS /PKS » et présente une forte activité cytotoxique sur différentes lignées cellulaires cancéreuses. Une des problématiques de ce lipopeptide est l'absence d'information sur son mécanisme d'action. Afin d'identifier des cibles potentielles, mais surtout d'entreprendre des études des relations structure-activités, la confirmation de sa structure est nécessaire. C'est dans cette optique que nous avons entrepris la synthèse de la laxaphycine B en synthèse peptidique en phase solide (SPPS). Dans un premier temps, nous avons entrepris la synthèse des quatre aminoacides non ribosomaux. Dans un second temps, nos efforts se sont concentrés sur le développement d'une stratégie de synthèse pour l'obtention de ce lipopeptide. En dernier lieu, nous avons étudié la structure secondaire possible de la laxaphycine B, afin d'apporter une explication sur son mécanisme d'action. / Marine environment is a source of inspiration for chemists, thanks to the incredible structural diversity isolated from marine organisms and microorganism's compounds. Among them, laxaphycine B, a cyclic lipopeptide isolated from Anabena torulosa cyanobacteria, as like most marine peptides produced by a non-ribosomal biosynthesis pathway "NRPS/PKS”. Furthemore, this peptide has shown a strong cytotoxic activity on various cancer cell lines. One of the problems of this lipopeptide, is the lack of information on its mechanism of action. To identify potential targets and also to study in structure activity relationships, confirmation of its structure is necessary. It is in this context that we undertook laxaphycine B's synthesis using SPPS. In a first step, the four non-ribosomal aminoacids have been synthesized. In a second step, our efforts have focused on the development of a synthesis strategy to obtain laxaphycine B. Lastly, we studied the laxaphycin B's secondary structure to understand its mechanism of action.

Tübingen, Univ., Diss., 2005.

Dans le lagon de Moorea, en Polynésie Française, nous avons identifié un écosystème constitué de deux producteurs primaires (les cyanobactéries filamenteuses Lyngbya majuscula et Anabaena cf. torulosa), trois mollusques herbivores (Stylocheilus striatus, S. longicauda, et Bulla orientalis), un nudibranche carnivore (Gymnodoris ceylonica) et un crabe carnivore (Thalamita coerulipes). L. majuscula et A. cf torulosa prolifèrent sur de vastes zones jusqu’à épiphyter les coraux ; elles sont des producteurs importants de métabolites secondaires, principalement des lipopeptides cycliques, qui peuvent être toxiques ou répulsifs. Cependant, ces composés n’empêchent pas le lièvre de mer S. striatus de consommer les cyanobactéries. S. striatus, décrit comme un prédateur spécialiste de L. majuscula, est connu pour séquestrer et/ou biotransformer les métabolites secondaires de L. majuscula. Cependant nous avons également observé S. striatus, sur A. cf torulosa où il semble moins exposé à la prédation du nudibranch G. ceylonica que quand il est sur L. majuscula. Dans cet écosystème modèle, nous avons combiné le profilage des métabolomes des deux cyanobactéries et des expériences en écologie dans le but d’étudier le rôle des médiateurs chimiques dans la structuration de cet écosystème ; nous avons complété la caractérisation des profils métaboliques des deux cyanobactéries, étudié les transmissions verticale et horizontale des métabolites secondaires produits par les cyanobactéries le long de la chaine trophique, et étudié le rôle de ces composés dans les relations prédateurs-proies. De A. cf torulosa, nous avons isolé cinq analogues acyliques et deux analogues cyliques des laxaphycines que nous avons caractérisés par RMN (1D et 2D RMN : COSY, TOCSY, HSQC, HMBC, NOESY), spectrométrie de masse (spectrométrie de masse à haute résolution et fragmentation en MSn), ainsi que par dégradation chimique avec la méthode de Marfey. La présence de laxaphycines acycliques n’a jamais été décrite auparavant. Nous avons montré que les peptides de L. majuscula sont séquestrés sans biotransformation par les herbivores, alors que les herbivores présents sur A. cf torulosa biotransforment deux laxaphycines en quatre composés nouveaux que nous avons caractérisés. Il ne semble pas que la séquestration et la biotransformation soient opérées dans le but d’améliorer les défenses chimiques des herbivores mais plutôt comme un mécanisme de tolérance. Nous avons également montré que les mollusques herbivores utilisent les composés produits par les cyanobactéries comme signaux chimiques pour détecter à distance les cyanobactéries et pour le choix de leur nourriture. Ces expériences de choix semblent indiquer que S. striatus et B. orientalis sont des herbivores généralistes bien que l’influence des molécules des cyanobactéries suggère un comportement adaptatif permettant au mollusque de retrouver l’hôte sur lequel il a été prélevé. / In the lagoon of Moorea in French Polynesia, we have identified a relatively simple tropical marine ecosystem consisting of two primary producers (two filamentous cyanobacteria, Lyngbya majuscula and Anabaena cf. torulosa), three herbivorous molluscs (Stylocheilus striatus, S. longicauda and Bulla orientalis), a carnivorous nudibranch (Gymnodoris ceylonica) and a carnivorous crab (Thalamita coerulipes). L. majuscula and A. cf torulosa, that bloom ephemerally across wide sandy areas and even on corals, are prolific producers of secondary metabolites, mainly cyclic lipopeptides, which may either be toxic or act as feeding deterrents to potential consumers. However, these compounds do not prevent the sea hare S. striatus, feeding on cyanobacteria. S. striatus, considered as L. majuscula specialist, is known to sequester and transform some secondary metabolites produced by L. majuscula,. However we found also S. striatus feeding on A. cf torulosa and in this case it was less susceptible to predation by the nudibranch G. ceylonicasa than when it fed on L. majuscula. In the study of this model ecosystem, we combine cyanobacterial metabolome profiling and ecological bioassays in order to study the cascading effects of chemical mediators in multi-trophic relationships; we completed the metabolic profile characterization of the two cyanobacteria, we studied vertical and horizontal transmissions of the cyanobacterial secondary metabolites along the trophic web, and studied the role of these compounds in predator-prey relationships. Focusing our attention on A. cf torulosa we isolated seven new lipopeptides, derived from the known laxaphycins, and characterized them using extensive NMR experiments (1D and 2D NMR: COSY, TOCSY, HSQC, HMBC, NOESY), mass spectrometry (HR-MS and fragmentation by MSn) and Marfey’s advanced method. It is the first time that acyclic analogs of laxaphycins have been described. Although the peptides from L. majuscula are found intact in herbivores, some lipopeptides from A. cf torulosa are biotransformed by sea hares into four new compounds we characterized. The sequestration and biotransformation by the herbivores may be considered as a tolerance mechanism rather than a defense mechanism. We demonstrate also that the herbivores use cyanobacterial compounds as chemical cues for cyanobacteria tracking and feeding choice. Our experiments suggest that S. striatus and B. orientalis are generalist consumers, although the influence of cyanobacterial chemical cues on their foraging preferences may suggest an adaptive behavior enabling the mollusc to track their host of origin.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20314号 / 薬科博第83号 / 新制||薬科||9(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 掛谷 秀昭, 教授 高須 清誠, 教授 大野 浩章 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Fusarium head blight is a devastating fungal disease of cereals caused by the pathogen Fusarium graminearum that leads to important economic losses due to diminished yields and grain downgrading. F. graminearum deploys several secondary metabolites known as virulence factors to facilitate its invasion of host tissues. These include the gramillins, a group of bicyclic lipopeptide ionophores that cause cell death and increased virulence in Arabidopsis, maize, and barley, but not wheat. Ionophores are involved in many plant-microbe interactions, but current knowledge of the molecular mechanisms governing host response to these molecules is limited. Susceptibility to gramillin varies among cultivars of affected species, but the basis for insensitivity has not yet been described, nor has the function of gramillin during infection. Here, we establish ion leakage as a method to survey Canadian barley for sensitivity, demonstrate that insensitivity to gramillin is likely mediated by a plant protease rather than inducible immune responses, and suggest a possible function of gramillin in positively regulating the expression of other fungal virulence factors during infection. This contributes to deepening our understanding of cyclic lipopeptide ionophores and their role during plant-microbe interactions.

Les substances synthétisées par les bactéries du genre Bacillus présentent un grand intérêt en raison de leurs nombreuses activités antimicrobiennes. L’objectif de ce travail a consisté à mettre au point un protocole analytique fiable pour extraire ces substances, et les caractériser par spectrométrie de masse et par leurs activités biologiques. Une première approche par spectrométrie de masse MALDI-ToF ne nous a pas donné de résultats satisfaisants en raison d’un phénomène de suppression spectrale. Une seconde stratégie d’étude combinant deux techniques analytiques a alors été envisagée. Les composés et les familles lipopeptidiques ont d’abord été séparés par chromatographie liquide analytique et analysés en ligne en mode de couplage ESI-IT. Ensuite les fractions collectées ont été caractérisées par spectrométrie de masse MALDI-Q-ToF (et ESI-Q-ToF) pour déterminer les masses exactes et obtenir des spectres de fragmentation complémentaires des premiers. Ce procédé nous a permis d’établir des critères d’identification des composés et des familles de lipopeptides de structures connues. Cette caractérisation est basée sur des temps de rétention en chromatographie, des mesures de masses exactes et sur des schémas de fragmentations. Nous avons fait de même avec les composés non identifiés et ensuite évalué leur activité biologique en réalisant des tests de diffusion sur agar. Cette stratégie d’étude permet de faire un criblage de l’ensemble des biomolécules produites par des souches de Bacillus et de relier une structure à une activité biologique. Ce protocole, mis au point pour des cultures réalisées en milieu liquide, a ensuite été adapté aux cultures sur boîtes de Pétri pour pouvoir analyser les composés produits à l’échelle de la colonie bactérienne. / Compounds produced by Bacillus bacteria present a major interest because of their biological activities. The aim of this work was to develop a reliable analytical methodology to extract these compounds, and to characterize them by mass spectrometry and by their biological activities. A first approach by MALDI-ToF mass spectrometry doesn’t give satisfactory results because of strong spectral suppression effects. Therefore, we have designed a second strategy which combined two analytical technologies. First, compounds and lipopeptides families were separated by analytical liquid chromatography and analysed by ESI-IT. Then, collected fractions were characterized by MALDI-Q-ToF (and ESI-Q-ToF) in order to determine accurate mass measurements and obtain complementary product ion spectra. This process led us to establish identification criteria of compounds and lipopeptides families which have known structures. This characterization is based of retention times in chromatography, accrurate mass measurements and fragmentation schemes. We have achieved the same experiments with non identified compounds and then assessed their biological activity by agar well diffusion test.This strategy allows to obtain a screening of whole of the biomolecules produced by bacillus strains and establishes a link between structure and biological activity. This methodology, designed for cultures in liquid medium, was then adapted to cultures in Petri disches in order to analyse compounds producted at the bacterial colony scale.

Les protéines lipidées purifiées à partir d’extraits de pathogènes ou produites de façon recombinante ont démontré leur pouvoir immunogène. Malheureusement, ce type de protéines est généralement difficile à purifier et/ou à produire. Afin de résoudre ces difficultés, l’objectif de cette thèse a été de mettre au point un procédé de lipidation chimique d’antigènes protéiques d’intérêt vaccinal, présents naturellement sous forme lipidée mais produits de façon recombinante sous forme non lipidée. La lipidation chimique a ainsi été réalisée sur 2 protéines, à savoir rTbpB-dl provenant de N. meningitidis et rNWMN_A issue de S. aureus, par des lipopeptides synthétiques de structure minimale, Pam2CAG et Pam3CAG, ciblant respectivement les récepteurs TLR2/6 et TLR2/1. Ces lipopeptides ont été préalablement fonctionnalisés par des groupes réactifs aux fonctions thiols (maléimide ou bromoacétyle) afin de réaliser le couplage chimique. Des analyses physico-chimiques et biochimiques ont démontré que la modification des protéines antigéniques a bien été réalisée. La lipidation de rTbpB-dl et rNWMN_A par les lipopeptides a induit in vitro l’activation des cellules immunitaires murines et humaines via TLR2 et a également augmenté in vivo l’immunogénicité de ces protéines recombinantes, en présence ou non d’un adjuvant. De plus, ces protéines lipidées ont joué in vivo le rôle d’adjuvant en augmentant l’immunogénicité d'une protéine antigénique co-administrée. Notre procédé de lipidation chimique a été simple, rapide, de faible coût et répétable. Au final, ce procédé pourrait s’appliquer sur des antigènes protéiques d’intérêt vaccinal et de faible immunogénicité provenant de différents pathogènes et/ou sur des antigènes lipoprotéiques rencontrant des problèmes de production/purification. Il pourraitreprésenter un choix pertinent dans la mise au point et dans le développement de candidatsvaccins, en présence ou non d’adjuvant(s) et/ou d’autre(s) antigène(s) d’intérêt. / Lipidated proteins, purified from pathogens extracts or produced recombinantly, have demonstrated their immunogenic power. Unfortunately, this type of proteins is generally difficult to purify and/or to produce. To solve these difficulties, the objective of this thesis was to develop a process of chemical lipidation of protein antigens of vaccine interest, which are present naturally inlipidated form but produced recombinantly in non-lipidated form. The chemical lipidation was carried out on two proteins, namely rTbpB-dl from N. meningitidis and rNWMN_A from S. aureus, by minimum structure synthetic lipopeptides, Pam2CAG and Pam3CAG targeting receptors TLR2/6 and TLR2/1 respectively. These lipopeptides have been functionalized beforehand with thiolreactive groups (maleimide or bromoacetyl) for performing chemical coupling. Physicochemical and biochemical analysis have shown that the modification of the antigenic proteins has been achieved. The lipidation of rTbpB-dl and rNWMN_A by these lipopeptides induced in vitro activation of murine and human immune cells through TLR2. This lipidation also increased in vivo immunogenicity (mainly humoral) of the recombinant protein, in the presence or absence of an adjuvant. Furthermore, these lipidated proteins acted in vivo as an adjuvant by increasing immunogenicity of a co-administered antigen protein. Our process of chemical lipidation was simple, rapid, low-cost and repeatable. Taken together, this process could apply to antigens of protein nature, of vaccine interest and poorly immunogenic from different pathogens and/or to lipoprotein antigens encountering production/purification problems. It could be a relevant choice for the development of candidate vaccines, in the presence or absence of adjuvant(s) and/or other antigen(s) of interest.