L'utilisation de biofongicides et de molécules stimulatrices des défenses des plantes (SDP) constituent des stratégies de lutte alternative permettant la réduction de l'utilisation des fongicides conventionnels. Toutefois, il n'existe que peu de produits homologués et appliqués sur blé et encore moins contre la septoriose. Dans le cadre de cette étude, nous avons testés et mis en évidence l'efficacité de protection du blé contre Zymoseptoria tritici de plusieurs molécules. En effet, 29 molécules biosourcées dérivées de la fonctionnalisation de l'acide pyroglutamique (PGA), contenu dans les mélasses de la betterave sucrière, ont été évaluées en plus de l'acide y-aminobutyrique GABA, pour la première fois contre Z. tritici. Le GABA et 16 molécules fonctionnalisées ont réduit significativement les pourcentages de la maladie. L'absence d'effets directs sur Z. tritici vérifiée in vitro confirme que ces protections sont dues exclusivement à un effet SDP. Pour ces molécules, l'étude de la relation structure activité a mis en évidence l'importance des différents groupements chimiques impliqués dans les protections observées. D'autre part, la conjugaison du PGA avec l'acide salicylique (SA) a permis l'obtention de 5 nouvelles molécules parmi lesquelles 4 étaient plus efficaces que les molécules mères et cette efficacité semble être également due à un effet SDP. Enfin, la saccharine ainsi que trois lipopeptides cycliques produits par Bacillus subtilis (mycosubtiline, surfactine et fengycine seuls ou en mélange) ont été évalués pour la première fois sur le pathosystème blé - Z. tritici et ont conféré des protections significatives dues à des propriétés SDP, à des activités antifongiques directes ou à des effets combinés. Cette étude a donc permis d'identifier de nouvelles molécules possédant différents modes d'action et qui pourraient être envisageables en lutte alternative contre la septoriose du blé, toutefois, des analyses complémentaires sont requises. / The use of biofungicides and elicitors of plant defenses are alternative control stategies that can reduce the use of conventional fungicides. However, there are few products registered and applied on wheat and even less against Septoria tritici blotch. As part of this study, we tested and demonstrated the protective efficacity of several molecules on wheat against Zymoseptoria tritici. In fact, 29 biosourced molecules derived from the functionalization of pyroglutamic acid (PGA) contained in the molasses of sugar beet were evaluated with y-aminobutyric acid GABA, for the first time against Z. tritici. GABA and 16 functionalized molecules significantly reduced the percentages of the disease. The absence of direct effects on the fungus tested in vitro confirms that these protections are due exclusively to an elicitation of plant defense mechanisms. For these molecules, the study of the structure activity relationship has highlighted the importance of different chemical groups involved in the observed protections. On the other hand, the conjugation of PGA with salicylic acid (SA) allowed the obtention of 5 new molecules among which 4 were more effective than the parent molecules and this efficiency seems to be also due to an eliciting effect. Finally, saccharin and three cyclic lipopeptides produced by Bacillus subtilis (mycosubtilin, surfactin and fengycin alone or as mixtures) were evaluated for the first time on the pathosystem wheat - Z. tritici and conferred significant protection due to eliciting properties, direct antifungal activities or combined effects. This study has thus made it possible to identify new molecules with different modes of action and which could be considered in alternative control against wheat leaf blotch, however, additional analyses are required.

Résumé Certaines bactéries de la rhizosphère (PGPR, rhizobactéries promotrices de la croissance des plantes) exercent un effet bénéfique sur la croissance des plantes en stimulant des mécanismes de défense inductibles chez lhôte, rendant celle-ci moins susceptible vis-à-vis dune infection ultérieure par un agent pathogène. Ce phénomène appelé résistance systémique induite (ISR) a été mis en évidence chez plusieurs plantes pour lutter contre une gamme relativement large de pathogènes fongiques, bactériens ou viraux. Cependant, les bases moléculaires des mécanismes de défense proprement dits stimulés lors de lISR restent assez méconnues malgré les nombreux travaux réalisés cette dernière décennie. Dans ce contexte, des souches de PGPR (Bacillus subtilis et Pseudomonas putida) capables de protéger certaines plantes via linduction de lISR sont étudiées depuis plusieurs années au laboratoire. Dans le cadre de cette thèse de doctorat, nous avons étudié l´effet protecteur de ces bactéries dans deux pathosystèmes différents, tomate/Botrytis cinerea et concombre/Colletotrichum lagenarium. Nos résultats ont montré la capacité de P. putida BTP1 à induire l´ISR chez la tomate et le concombre sur base de la réduction des symptômes de maladie observée et sur base de la séparation spatiale des deux agents, bénéfique au niveau racinaire et pathogène au niveau foliaire. Cette résistance a été clairement associée avec la stimulation de la voie des oxylipines chez la tomate. Linduction de cette voie métabolique a dabord été mise en évidence biochimiquement par une augmentation des activités lipoxygénase et lipide hydroperoxydase dans les feuilles des plantes traitées avec Pseudomonas. De plus, au niveau moléculaire, les analyses par northern blot nous ont permis d´identifier un nouvel isoforme de gène Lox chez la tomate qui est exprimé différentiellement chez les plants traités par la bactérie. Ce gène dénommé LoxF montre une homologie de 82% avec un des cinq isoformes connus chez cette plante, LoxC. LoxF est essentiellement surexprimé dans les feuilles de tomates pré-inoculées avec BTP1 suite à l´infection par le pathogène. Il en est de même pour lactivité enzymatique correspondante de la lipoxygénase qui naugmente significativement chez les plants traités par rapport aux témoins quaprès infection par Botrytis. Ces résultats sous-entendent un phénomène de « priming » ou « mise en alerte » étroitement associé avec linduction de résistance systémique chez les plantes. La reconnaissance de la bactérie au niveau racinaire met en alerte la plante sans que des bouleversements majeurs au niveau métabolique ou génétique ne soient observés. Lhôte réagit alors plus fortement et plus rapidement pour mettre en uvre ses mécanismes de défense une fois le pathogène perçu. Par biotest sur TLC et analyses HPLC, CPG et LC-MS, nous avons mis en évidence laccumulation dune molécule dans les feuilles de tomate prétraitées avec BTP1. Cette molécule semble être de nature apolaire mais non phénolique et ne correspond pas aux phytoalexines connues de la tomate. Des études complémentaires doivent être réalisées pour lidentifier chimiquement mais sa cinétique daccumulation dans les tissus de la plante est étroitement associée à celle de la stimulation de la lipoxygénase. Elle pourrait donc dériver de cette voie métabolique. Des réactions de défense similaires ont été observées suite au traitement avec Bacillus subtilis chez la tomate et ces essais ont permis de mettre en évidence le rôle des lipopeptides produits en tant quéliciteurs impliqués dans linduction du phénomène de lISR par cette souche. Par contre, bien que les deux souches soient capables dinduire la résistance chez le concombre, aucune accumulation claire de phytoalexines ni de stimulation significative de la voie de la lipoxygénase nont pu y être associées chez cette plante. Globalement, nos résultats suggèrent que les voies métaboliques activées dans le cadre de lISR varient en fonction de lespèce végétale même si le microorganisme inducteur est identique. Abstract Some plant growth promoting rhizobacteria (PGPR) are able to stimulate inducible defence mechanisms that render the host plant less susceptible to a subsequent pathogen attack. This phenomenon called induced systemic resistance (ISR) can occur in several plant species against a wide range of bacterial, viral and fungal pathogens. Despite extensive work carried out this last decade, many aspects of the molecular basis underlying this rhizobacteria-mediated ISR remain unclear. In this context, Bacillus subtilis and Pseudomonas putida strains able to protect plants via induction of ISR have been studied for several years in our laboratory. In this thesis, we first aimed at evaluating the protective effect of these strains in different pathosystems and second at identifying the associated defence mechanisms. Our results showed the capacity of P. putida BTP1 to induce ISR in the tomato/Botrytis cinerea and cucumber/Colletotrichum lagenarium pathosystems on the basis of reduction of disease symptoms and on the basis of the spatial separation of the two agents, beneficial at the root level and pathogenic at the leaf level. This resistance was clearly associated the stimulation of the oxylipin pathway in tomato. The induction of this metabolic pathway was evidenced biochemically by an increase in the lipoxygenase and lipid hydroperoxydase activities in the leaves of plants treated with Pseudomonas compared to controls. Moreover, at the molecular level, northern blot analyses enabled us to identify a new isoforme of Lox gene in tomato which is expressed differentially in plants treated with the bacterium. This gene called LoxF shows a homology of 82% with one of the five isoformes known in this plant, LoxC. LoxF is mainly expressed in tomato leaves pre-inoculated with BTP1 after infection by pathogen. The same applies for the lipoxygenase enzyme activity which increases significantly in treated plants only after infection by Botrytis. These results imply a phenomenon of "priming" closely associated with the systemic resistance induction in plants. The recognition of the bacterium at the root level primes the plant but in most cases major changes at the metabolic or genetic level are not observed. The host then reacts more strongly and more quickly to express defence mechanisms once the pathogen is perceived. In addition, by combining the use of TLC-based biotests and analytical methods such as HPLC, CPG and LC-MS analyses, we highlighted the accumulation of a molecule in BTP1-pretreated tomato leaves. This molecule seems to be of a non-polar nature but not phenolic and does not correspond to any phytoalexin known in tomato. Complementary studies must be carried out to identify its structure but its kinetic of accumulation in the plant tissues is closely associated with the stimulation of the lipoxygenase enzyme. It could thus derive from this metabolic pathway. Similar defence reactions were observed in tomato following treatment with Bacillus subtilis and through these tests, we also highlighted the role of some lipopeptides produced by this strain as elicitors responsible for the induction of the ISR phenomenon. On the other hand, although the two strains are able to induce resistance in cucumber, no clear accumulation of phytoalexins nor of significant stimulation of the lipoxygenase pathway could be associated with disease reduction in this plant. All together, our results suggest that the metabolic pathways activated during ISR vary in function of the plant and pathogen species even if the inducing micro-organism is identical.

Many biosurfactants show antimicrobial activity and some are found to be superior for isolating membrane proteins. This study was aimed towards a general understanding of the interactions of biosurfactants with lipid membranes on a molecular level. To this end, a new, fluorescence lifetime-based membrane leakage assay has been established that does not only quantify membrane permeabilization more precisely but reveals also the leakage mechanism. This mechanism, referred to as graded or all-or-none leakage, is crucial for interpreting potential biological activities and modes of action. Lipopeptides of the surfactin, fengycin, and iturin families as produced by Bacillus subtilis were studied along with synthetic surfactants. Their membrane permeabilizing activity and selectivity mirrored, to some extent, the active concentrations and fungicidal selectivity of the compounds in vivo. Furthermore, the effects of co-surfactants and co-solvents (glycerol, urea, DMSO) have been investigated to better understand and predict means of improving the performance of fungicidal products as well as conditions for membrane protein solubilization.

Many biosurfactants show antimicrobial activity and some are found to be superior for isolating membrane proteins. This study was aimed towards a general understanding of the interactions of biosurfactants with lipid membranes on a molecular level. To this end, a new, fluorescence lifetime-based membrane leakage assay has been established that does not only quantify membrane permeabilization more precisely but reveals also the leakage mechanism. This mechanism, referred to as graded or all-or-none leakage, is crucial for interpreting potential biological activities and modes of action. Lipopeptides of the surfactin, fengycin, and iturin families as produced by Bacillus subtilis were studied along with synthetic surfactants. Their membrane permeabilizing activity and selectivity mirrored, to some extent, the active concentrations and fungicidal selectivity of the compounds in vivo. Furthermore, the effects of co-surfactants and co-solvents (glycerol, urea, DMSO) have been investigated to better understand and predict means of improving the performance of fungicidal products as well as conditions for membrane protein solubilization.

Submitted by Geyciane Santos (geyciane_thamires@hotmail.com) on 2015-12-07T20:08:17Z No. of bitstreams: 1 Dissertação - Adriana Spirotto Stein Mesquita.pdf: 3914675 bytes, checksum: a98f72f70f2e72b3c17fddd0248824e7 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-01-19T17:58:08Z (GMT) No. of bitstreams: 1 Dissertação - Adriana Spirotto Stein Mesquita.pdf: 3914675 bytes, checksum: a98f72f70f2e72b3c17fddd0248824e7 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-01-19T17:59:14Z (GMT) No. of bitstreams: 1 Dissertação - Adriana Spirotto Stein Mesquita.pdf: 3914675 bytes, checksum: a98f72f70f2e72b3c17fddd0248824e7 (MD5) / Made available in DSpace on 2016-01-19T17:59:14Z (GMT). No. of bitstreams: 1 Dissertação - Adriana Spirotto Stein Mesquita.pdf: 3914675 bytes, checksum: a98f72f70f2e72b3c17fddd0248824e7 (MD5) Previous issue date: 2015-09-15 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The endophytic microrganisms, particularly bacteria and fungi live in the plants, including thein roots apparently cause no harm. Bacteria and fungi are potentially useful in agriculture and industry, particularly in the food and pharmaceutical industries, as they produce different chemical classes and can participate in various industrial biotechnological processes. Among the compounds produced by bacteria, lipopeptides belong to a class that has attracted great interest for its antifungal, antibiotic and surfactant among others. In this stud 50 strains were selected of Amazonian endophytic bacteria for antimicrobial evoluction and the production of lipopeptides by ESI-ITMS2. Of these, 25 strains produced lipopeptides, six plant isolated from Gustavia genus, nine Macrophyte, five Duguetia, one Pothomorpha, two Rollina and one Bryophyllum. The fragmentation analysis by ESI-MS2 in negative mode identified eight extracts the lipopeptide surfactin homologs of C11 - C18, including C11, C17 and C18 not been reported in the literature. In biological assays paired cultures, 48 strains showed mycoparasitism antimicrobial activity against Gram-negative bacteria P. aeruginosa and E. coli, 27 strains against Gram-positive bacteria S. aureus, E. faecalis, 29 strains against and 28 against C. albicans, also presenting competition activity against E. faecalis 21 strains and C. albicans strains against 23 S. aureus strains and 2 against P.aeruginosas and E. coli. No strain with antibiotic activity observed. In assessing the bioactivity of lipopeptide extracts, 13 of the extracts strains showed antibiosis against S. aureus, P. aeruginosa and C.albicans. / Os microrganismos endofíticos, especialmente bactérias e fungos, são assim denominados por viverem no interior das plantas, habitando inclusive suas raízes, sem lhes causar aparentemente nenhum dano. As bactérias e os fungos são potencialmente úteis na agricultura e na indústria, particularmente na alimentícia e farmacêutica, pois produzem inúmeras classes de substâncias e podem participar de processos biotecnológicos industriais diversos. Entre as substâncias produzidas por bactérias, os lipopeptídeos são uma classe que tem atraído grande interesse por suas atividades antifúngica, antibiótica e surfactante entre outras. No presente trabalho foram selecionadas 50 linhagens de bactérias endofíticas amazônicas para serem avaliadas por ensaios antimicrobianos e quanto à produção de lipopeptídeos por ESI-ITMS2. Destas, 25 linhagens produziram lipopeptídeos, sendo seis isoladas de planta do gênero Gustavia, nove de Macrofita, cinco de Duguetia, uma de Pothomorpha, duas de Rollina e uma de Bryophyllum. As análises de fragmentação por ESI-MS2 no modo negativo dos extratos lipopeptídicos identificaram oito homólogos da surfactina de C11 - C18, entre os quais o C11, C17 e C18 ainda não foram relatados na literatura. Em ensaios biológicos de culturas pareadas, 48 linhagens apresentaram atividade antimicrobiana de microparasitismo contra as bactérias Gram-negativas P. aeruginosa e E. coli, 27 linhagens contra as bactérias Gram-positiva S. aureus, 29 linhagens contra E. faecalis e 28 contra C. albicans, apresentando também atividade de competição frente a 21 linhagens contra E. faecalis e C. albicans, 23 linhagens contra S. aureus e 2 linhagens contra P.aeruginosa e E. coli. Não foi observada nenhuma linhagem com atividade antibiótica. Na avaliação da bioatividade dos extratos lipopeptídicos, 13 extratos destas linhagens apresentaram antibiose frente a S. aureus, P. aeruginosa e C. albicans.

Bacillus amyloliquefaciens FZB42 ist ein im Boden weit verbreitetes Bakterium. Es kolonisiert Pflanzenwurzeln und werde als Biodünger verwendet, da sie in der Lage sind, Pflanzenwachstum zu fördern. Der domestizierte Stamm B. subtilis 168 ist eng verwand mit B. amyloliquefaciens FZB42, fördert jedoch kein Pflanzenwachstum. Als ein erster Ansatz zur Ermittlung von Gendifferenzen zwischen FZB42 und B. subtilis 168 - wobei zum damaligen Zeitpunkt nur die Genomsequenz letzteren Organismus bekannt war - wurde die Supression Subtractive Hybridisation (SSH) angewandt. Hierdurch wurden mehrere einzigartige Gene in B. amyloliquefaziens identifiziert. Unterdessen beteiligte sich unser Labor in Kollaboration mit dem GenoMik Network in Göttingen an einem Projekt, dessen Ziel die komplette Sequenzierung des Genoms von B. amyloliquefaciens war. Der Hauptanteil der Arbeit, sowie die Koordination des gesamten Projekts wurden von Xiao-Hua Chen und mir selbst durchgeführt. B. amyloliquefaciens FZB42 besitzt die srf, fen, pks1 (bae), bac und dhb Operons, welche für die Synthese von Surfactin, Fengycin, Bacillaene, Bacilysin und Bacillibactin verantwortlich sind und die ebenfalls im Genom von B. subtilis 168 enthalten sind. Das Genom von B. amyloliquefaciens FZB42, beinhaltet die bmy Gencluster, die die Synthese von Bacillomycin D kontrolliert. Ein weiteres in dieser Arbeit verfolgtes Ziel war die Identifizierung der regulatorischen Wege, die die Expression von Bacillomycin D steuern. Es wurde gezeigt, dass globale Regulatoren, wie beispielsweise DegU, DegQ und ComA, die alternativen Sigmafaktoren sigB und sigH und ein neuartiges Rap-Protein die transkriptionale Aktivität des in dieser Arbeit identifizierten Hauptpromotors des bmy-Operons beeinflussen. Es wurde gezeigt, dass DegU seine Effekte nach direkter Bindung an zwei unterschiedliche Regionen im bmy-Promotor ausübt. Es wurde außerdem gezeigt, dass DegU abgesehen von der Aktivierung des Hauptpromoters des bmy-Operons eine zusätzliche, vermutlich eine post-transkriptionale Rolle spielt. Auf ähnliche Weise erwies sich YczE, ein Membranprotein unbekannter Funktion, das neben sfp kodiert liegt, als essentiell für die Bacillomycin D-Produktion. Der Effekt wurde auf einem post-transkriptionalen Level ausgeübt und war unabhängig von DegU. / Bacillus amyloliquefaciens FZB42 is widely distributed in the soil. It colonizes the plant roots and is used as bio-fertilizer, since they can promote plant growth.The domesticated strain of B. subtilis 168 is closely related to B. amyloliquefaciens FZB42, but does not promote plant growth. As a first approach to detect gene differentiation between FZB42 and B. subtilis 168, and since only the genome sequence of the latter was known at that point, Suppression Subtractive Hybridization (SSH) was employed. Thereby, several unique genes of B. amyloliquefaciens FZB42 could be identified. Meanwhile, our laboratory became engaged in a project aiming to define the complete genome sequence of B. amyloliquefaciens FZB42, in collaboration with the GenoMik Network in Göttingen. The major part of the work and the co-ordination of the whole process were performed by Xiao-Hua Chen and myself. B. amyloliquefaciens FZB42 possesses the srf, fen, pks1 (bae), bac and dhb operons, which are also shared by B. subtilis 168. In addition, the genome of B. amyloliquefaciens FZB42 contains the bmy gene clusters, which controls the synthesis of bacillomycin D. A further issue pursued in this work was to identify the regulatory pathways that govern the expression of bacillomycin D. Global regulators, such as DegU, DegQ and ComA, the alternative sigma factors, sigB and sigH, and a novel Rap protein were found to affect the transcriptional activation of the main promoter of the bmy operon identified in this work. In particular, DegU was shown to mediate its effects, after binding directly to two sites at the bmy promoter region. DegU was shown to play an additional role on bacillomycin D production, presumably a post-transcriptional one. Similarly, YczE, a membrane protein of unknown function, encoded adjacently to sfp proved to be essential for bacillomycin D production, but dispensable for the production of the rest peptide antibiotics produced by B. amyloliquefaciens FZB42. The effect was mediated at a post-transcriptional level and was independent of DegU.

Vaccination comes second in importance after introduction of clean water as a public health intervention which has largely contributed in the reduction of deaths from infectious diseases. Success in the development of a group A streptococcal (GAS) vaccine is expected to save 517 000 deaths per annum according to a recent independent review commissioned by the world health organization (WHO) and would offer an ideal means to prevent rheumatic heart disease (responsible for the greatest health burden) and other GAS-associated diseases which affect the health of 600 million. Traditional vaccine approaches (killed or live attenuated) have demonstrated great success against many bacterial and viral infectious diseases, crowned by the global eradication of smallpox announce by the WHO in 1980 and near-to-be announced eradication of polio viral disease. However, application of traditional techniques in many cases such as HIV/AIDS, malaria, GAS and Mycobacteria tuberculosis, has not shown the same success. Risk associated with the use of live–attenuated pathogens, such as recurrence of virulence (e.g. HIV), development of autoimmune diseases (e.g. GAS), and difficulties of manufacture hindered the use of such approaches. Other vaccine approaches such as subunit vaccines (recombinant proteins) and carrier conjugated vaccine are also hindered by the lack of suitable adjuvants, carriers and delivery systems. The current thesis focused on the design, synthesis and evaluation of novel adjuvants and vaccine delivery systems against GAS. The first chapter reviews recent approaches in the field of GAS vaccine design and new findings in immunology which represent the basis of our novel strategies. The second chapter describes the design, synthesis and evaluation of a novel library of lipopeptides as self-adjuvanting GAS vaccine candidates, composed of: (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2d) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment, or incorporation of P25 and J14 into a lipid-core peptide system (LCP) on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side-chain. Mucosal surface of the nasal-oral route is a primary site of GAS infections. An ideal GAS vaccine would have to elicit both mucosal as well as systemic immune responses and hence would not only prevent the development of GAS-associated diseases but also would prevent primary GAS infections. Therefore, the nasal route is considered a highly promising route of vaccine administration to provide local as well as systemic immune responses against pathogens that utilize mucosal surface as site of infection. The third chapter includes immunological assessment of the lipopeptide vaccine library described in the second chapter following intranasal immunization of B10BR (H-2k) mice. The whole library was first investigated in a small scale experiment (5 mice per group) to select promising candidates which demonstrate the best local and systemic J14-specific antibodies. Four selected lipopeptides were further investigated in a larger scale experiment (15 mice per group) followed by intranasal challenge of vaccinated mice with a virulent GAS M1 strain. The best local and systemic immune responses were demonstrated by a lipopeptide featuring a lipid moiety consisting of two 16 carbon chains incorporated at the C-terminus of the lipopeptide. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide candidate featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side-chain. A possible explanation for these results was investigated where antibodies elicited by the former candidate was found to better recognize the minimal B-cell epitope in the native p145 sequence of the M protein. Circular dichroism study of lipopeptides used in the previous experiment demonstrated that the former candidate features α-helical conformation which is required to produce protective J14-specific antibodies. Further studies are needed to explain structural features required to achieve both α-helicity and strong mucosal immune responses shown by the previously mentioned two lipopeptides. Signaling through toll-like receptors expressed by immune cells was recently shown to result in a robust immune response and was investigated as a possible mode of action for our novel lipopeptides. The fourth chapter introduces our lipopeptide vaccine approach as novel synthetic ligands targeting TLR2. A lipid moiety consisting of two alkyl chains of 16 carbons was found to achieve optimal TLR2 signaling regardless of the position of lipid attachment. Carbohydrates as polyhydroxy compounds provide an easily accessible class of compounds to design scaffolds (carriers) to attach lipids and peptide epitopes in different number and stereochemical positions which makes glycolipopeptides an attractive target for adjuvant research and structure-adjuvanticity relationships studies. The Fifth chapter reports immunological assessment of two series of glycolipopeptides as GAS vaccine candidates and novel vaccine delivery systems. The first series: lipid carbohydrate core peptide system (LCCP); represents a modification of the classical LCP system where polylysine dendrimer is replaced by different monosccharides as carriers for peptide antigens. LCCP analogues induced proper humoral immune responses against incorporated epitopes comparable to the LCP delivery system and as strong as the immune response elicited by CFA mixtures. Moreover, LCCP delivery system has been proved to be tolerant to the use of different epitopes as well as changing carbohydrate cores. Design of novel carbohydrate cores with different orthogonal protecting groups is needed to explore the potential advantage of various stereochemical arrangements provided by monosaccharides. The second series of glycolipopeptides incorporates various glycolipid moieties (self-adjuvanting activity) covalently coupled to the N-terminus of J8 (a model epitope). The new glycolipopeptide vaccine candidates (containing only one copy of J8) bear comparison with an LCP analogue (containing four copies of J8) which would improve the ease of synthesis, purification and cost of vaccine production. The slight difference in immunogenicity among these glycolipopeptides was difficult to be explained due to intervening effects of both the number and orientation of lipids on immunological activity. Further investigation is needed to determine the contribution of each factor.

Vaccination comes second in importance after introduction of clean water as a public health intervention which has largely contributed in the reduction of deaths from infectious diseases. Success in the development of a group A streptococcal (GAS) vaccine is expected to save 517 000 deaths per annum according to a recent independent review commissioned by the world health organization (WHO) and would offer an ideal means to prevent rheumatic heart disease (responsible for the greatest health burden) and other GAS-associated diseases which affect the health of 600 million. Traditional vaccine approaches (killed or live attenuated) have demonstrated great success against many bacterial and viral infectious diseases, crowned by the global eradication of smallpox announce by the WHO in 1980 and near-to-be announced eradication of polio viral disease. However, application of traditional techniques in many cases such as HIV/AIDS, malaria, GAS and Mycobacteria tuberculosis, has not shown the same success. Risk associated with the use of live–attenuated pathogens, such as recurrence of virulence (e.g. HIV), development of autoimmune diseases (e.g. GAS), and difficulties of manufacture hindered the use of such approaches. Other vaccine approaches such as subunit vaccines (recombinant proteins) and carrier conjugated vaccine are also hindered by the lack of suitable adjuvants, carriers and delivery systems. The current thesis focused on the design, synthesis and evaluation of novel adjuvants and vaccine delivery systems against GAS. The first chapter reviews recent approaches in the field of GAS vaccine design and new findings in immunology which represent the basis of our novel strategies. The second chapter describes the design, synthesis and evaluation of a novel library of lipopeptides as self-adjuvanting GAS vaccine candidates, composed of: (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2d) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment, or incorporation of P25 and J14 into a lipid-core peptide system (LCP) on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side-chain. Mucosal surface of the nasal-oral route is a primary site of GAS infections. An ideal GAS vaccine would have to elicit both mucosal as well as systemic immune responses and hence would not only prevent the development of GAS-associated diseases but also would prevent primary GAS infections. Therefore, the nasal route is considered a highly promising route of vaccine administration to provide local as well as systemic immune responses against pathogens that utilize mucosal surface as site of infection. The third chapter includes immunological assessment of the lipopeptide vaccine library described in the second chapter following intranasal immunization of B10BR (H-2k) mice. The whole library was first investigated in a small scale experiment (5 mice per group) to select promising candidates which demonstrate the best local and systemic J14-specific antibodies. Four selected lipopeptides were further investigated in a larger scale experiment (15 mice per group) followed by intranasal challenge of vaccinated mice with a virulent GAS M1 strain. The best local and systemic immune responses were demonstrated by a lipopeptide featuring a lipid moiety consisting of two 16 carbon chains incorporated at the C-terminus of the lipopeptide. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide candidate featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side-chain. A possible explanation for these results was investigated where antibodies elicited by the former candidate was found to better recognize the minimal B-cell epitope in the native p145 sequence of the M protein. Circular dichroism study of lipopeptides used in the previous experiment demonstrated that the former candidate features α-helical conformation which is required to produce protective J14-specific antibodies. Further studies are needed to explain structural features required to achieve both α-helicity and strong mucosal immune responses shown by the previously mentioned two lipopeptides. Signaling through toll-like receptors expressed by immune cells was recently shown to result in a robust immune response and was investigated as a possible mode of action for our novel lipopeptides. The fourth chapter introduces our lipopeptide vaccine approach as novel synthetic ligands targeting TLR2. A lipid moiety consisting of two alkyl chains of 16 carbons was found to achieve optimal TLR2 signaling regardless of the position of lipid attachment. Carbohydrates as polyhydroxy compounds provide an easily accessible class of compounds to design scaffolds (carriers) to attach lipids and peptide epitopes in different number and stereochemical positions which makes glycolipopeptides an attractive target for adjuvant research and structure-adjuvanticity relationships studies. The Fifth chapter reports immunological assessment of two series of glycolipopeptides as GAS vaccine candidates and novel vaccine delivery systems. The first series: lipid carbohydrate core peptide system (LCCP); represents a modification of the classical LCP system where polylysine dendrimer is replaced by different monosccharides as carriers for peptide antigens. LCCP analogues induced proper humoral immune responses against incorporated epitopes comparable to the LCP delivery system and as strong as the immune response elicited by CFA mixtures. Moreover, LCCP delivery system has been proved to be tolerant to the use of different epitopes as well as changing carbohydrate cores. Design of novel carbohydrate cores with different orthogonal protecting groups is needed to explore the potential advantage of various stereochemical arrangements provided by monosaccharides. The second series of glycolipopeptides incorporates various glycolipid moieties (self-adjuvanting activity) covalently coupled to the N-terminus of J8 (a model epitope). The new glycolipopeptide vaccine candidates (containing only one copy of J8) bear comparison with an LCP analogue (containing four copies of J8) which would improve the ease of synthesis, purification and cost of vaccine production. The slight difference in immunogenicity among these glycolipopeptides was difficult to be explained due to intervening effects of both the number and orientation of lipids on immunological activity. Further investigation is needed to determine the contribution of each factor.

5 Abstract Protein lipidation occupies an important place in the post-translational modification group. It plays an important role in the processes of cell differentiation or synaptic transmission. Changes in the presence of lipidated proteins may in some cases indicate the appearance of human diseases such as Huntington's disease, schizophrenia, or cancer. For this reason, their analysis has become desirable, but also problematic due to its specific characteristics. This work is focused on the development of conditions for measurement of lipopeptides produced by cleavage of proteins containing lipomodification. Two separation procedures were tested, in which the aim of work was to enrich the lipopeptides and remove as many other molecules as possible from the samples. The first test method - phase interface separation, using water and chloroform, was able to concentrate the lipopeptides on the phase interface and separate some of the added unmodified peptides from the mixture. The second method of separation was a solid phase using a C18 stationary phase. As a more suitable solid phase extraction variant, elution with solutions containing acetonitrile with trifluoroacetic acid was chosen as compared to solutions containing isopropyl alcohol. With the modified elution procedure, it was possible to separate...

Thesis (MEng) -- Stellenbosch University, 2014. / ENGLISH ABSTRACT: As overpopulation threatens the world’s ability to feed itself, food has become an invaluable resource. Unfortunately, almost a third of the food produced for human consumption is lost annually. Pests including insects, phytopathogens and weeds are responsible for more than a third of the annual major crop losses suffered around the world. The majority of current post-harvest control strategies employ synthetic agents. These compounds, however, have been found to be detrimental to the environment as well as human health, which has led researchers to investigate alternative strategies. Biocontrol agents are environmentally compatible, have a lower toxicity and are biodegradable, making them an attractive alternative to the synthetic control agents. The lipopeptides produced by Bacillus spp. in particular, have shown great potential as biocontrol agents against various post-harvest phytopathogens. Most biocontrol strategies apply the biocontrol organism, for example Bacillus, directly, whereas this study focused on the use of the lipopeptide itself as an antifungal agent. This is advantageous as the lipopeptides are less sensitive to their surroundings, such as temperature and pH, compared to living organisms, allowing for the production of a standardized product. This study investigated the production of the Bacillus lipopeptides surfactin, fengycin and iturin under controlled batch conditions. Parameters increasing lipopeptide production were quantified, focussing on antifungal lipopeptides (iturin and fengycin), and lipopeptide production was optimized. Experiments were performed in a fully instrumented 1.3 L bench-top bioreactor and lipopeptide analyses were performed via high pressure liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS). After screening four Bacillus spp., Bacillus amyloliquefaciens DSM 23117 was found to be the best antifungal candidate. This was based on it outperforming other candidates in terms of maximum antifungals produced, Yp/x,antifungals (yield per cells), and antifungal productivity. Nitrate, in the form of NH4NO3, was critical for lipopeptide production and an optimum concentration was observed above which the CDW (cell dry weight) no longer increased significantly and both μmax (maximum specific growth rate, h-1) and lipopeptide production decreased. For μmax, the optimum NH3NO4 concentration was 10 g/L and for lipopeptides it was 8 g/L. At these respective NH4NO3 concentrations μmax = 0.58 (h-1), the maximum antifungals (fengycin and iturin) were 285.7 mAU*min and the maximum surfactin concentration was 302 mg/L. The lipopeptides produced by B. amyloliquefaciens, the antifungals (fengycin and iturin) and surfactin, are secondary metabolites, regardless of the optimization treatment, i.e. increased NH4NO3 concentrations. Using 30% enriched air extended the nitrate utilization period, suggesting that when increasing supply concentration, more oxygen was available to act as electron acceptors, allowing nitrate to be used for lipopeptide production. The number of iturin and fengycin homologues generally increased with an increase in nitrate concentration. This suggested that process conditions, such as nitrate concentration, can be used to manipulate homologue ratios, allowing for the possibility to tailor-make biocontrol-agent upstream, during the production process, and possibly increase the efficacy of the biocontrol strategy. The lipopeptides produced by B. amyloliquefaciens showed complete inhibition against Botryotinia fuckeliana and diminished the growth capabilities of Botrytis cinerea. No inhibition was observed against Penicillium digitatum. These results indicate potential of the biocontrol strategy, although scale-up and fed-batch studies are recommended, especially when considering commercial implementation. Studies regarding the lipopeptide application method, i.e. a single application or multiple applications, should also be investigated as this will influence the efficacy of the lipopeptides against the target organisms. / AFRIKKANSE OPSOMMING: Met oorbevolking wat die wêreld se vermoë om die groeiende bevolking te onderhou belemmer, het dit noodsaaklik geword om huidige voedselbronne te beskerm. Daar word beraam dat een derde van die voedsel wat wêreldwyd geproduseer word vir menslike verbruik verlore gaan elke jaar. Verder is insekte, plantpatogene en onkruide verantwoordelik vir meer as ‘n derde van die verliese rakend jaarlikse oeste. Meeste bestaande na-oes beheermetodes maak gebruik van sintetiese stowwe. Ongelukkig kan hierdie verbindings nadelig wees vir die omgewing sowel as menlike gesondheid. Navorsers het hulsef dus toespits daarop om alternatiewe beheermetodes te ondersoek. Bio-beheermetodes is omgewingsvriendelik sowel as bio-afbreekbaar, wat hulle ideale alternatiewe maak vir die sintetiese stowwe. Bacillus spp. lipopeptiede het veral hoë potensiaal getoon as bio-beheermiddels teen verskeie na-oes plantsiektes. Meeste bio-beheermetodes wend die biobeheer organisme, soos Bacillus, direk aan, waar hierdie studie op die gebruik van lipopeptiede as ‘n beheermiddel gefokus het. Die voordeel is dat lipopeptidiede minder sensitief is vir hul omgewings, soos temperatuur en pH, i.v.m. organismes en die moontlikheid bied van ‘n gestandardiseerde produk. Hierdie studie het die produksie van spesifieke Bacillus lipopeptide, naamlik surfactin, fengycin en iturin, onder beheerde lottoestande ondersoek. Parameters wat lipopeptied produksie verhoog is gekwantifiseer, spesifiek antifungiese middels (iturin en fengycin) en lipopeptied produksie is geoptimeer. Eksperimente is uitgevoer in ‘n 1.3 L bioreaktor en lipopeptiedanaliese is met behulp van hoë druk vloeistof chromatografie en vloeistofchromatografie-massa spektroskopie uitgevoer. Van die vier moontlike Bacillus spp., was Bacillus amyloliquefaciens DSM 23117 die mees belowende antifungus-produserende kandidaat. Dit het beter resultate gelewer in terme van maksimale antifungiese produksie, Yp/x,antifungies (opbrengs per sel) asook antifungiese produktiwiteit. Nitraat, in hierdie geval NH4NO3, was noodsaaklik vir lipopeptied produksie en ‘n optimale konsentrasie is waargeneem waarbo die seldigtheid nie meer beduidend toegeneem het nie en beide die μmax (maksimale spesifieke groei tempo, h-1) en lipopeptied produksie afgeneem het. Die optimale NH4NO3 konsentrasie vir μmax was 10 g/L en vir lipopeptiedproduksie was 8 g/L. By 10 g/L NH4NO3 was μmax = 0.58 (h-1) en by 8 g/L was die maksimale antifungiese produksie (fengycin en iturin) 285.7 mAU*min en die maksimale surfactin produksie 302 mg/L onderskeidelik. Die lipopeptide, die antifungiese middels (fengycin en iturin) en surfactin, geproduseer deur B. amyloliquefaciens is sekondêre metaboliete, ongeag van die optimerings-behandelinge wat toegepas word, soos ‘n verhoging in NH4NO3 konsentrasie. Die gebruik van 30% verrykte suurstof het die nitraat verbruikingsperiode verleng, wat voorgestel het dat met die verryking, meer suurstof beskikbaar was om te dien as finale elektron ontvanger en sodoende die nitraat beskikbaar te stel vir lipopeptied produksie. Iturin en fengycin homoloë, oor die algemeen, het toegeneem soos wat die nitraat konsentrasie verhoog is. Hierdie resultate dui daarop dat prosestoestande, soos nitraat konsentrasie, gebruik kan word om die verhouding waarin lipopeptied homoloë geproduseer word te manipuleer. Hierdie resultate dui op die potensiaal vir die stroomop produksie van ‘n unieke bio-beheermiddel, wat die effektiwiteit van die bio-beheermetode moontlik sal verhoog. Die geproduseerde lipopeptiede het totale inhibisie getoon teen Botryotinia fuckeliana en ook fungiese aktiwiteit belemmer met Botrytis cinerea. Geen inhibisie is getoon teen Penicillium digitatum nie. Hierdie resultate toon die potensiaal van die bio-beheermetode, maar ‘n opskalerings-studie asook ‘n voerlot studie word aanbeveel, veral met die oog op moontlike kommersiële implementering van die strategie. Verdere studies met betrekking tot die aanwendingsmetode van die lipopeptiede moet ook verder ondersoek word, m.a.w. enkel teenoor menigte aanwendigs, aangesien dit die effektiwiteit van die lipopeptiede teen die teikenorganismes sal beïnvloed.