CORREIA, G. R. B. Efeito inibitório in vitro do composto farnesol frente ao biofilme de Burkholderia pseudomallei. 2015. 88 f. Dissertação (Mestrado em Microbiologia Médica) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-05-02T15:58:57Z No. of bitstreams: 1 2015_dis_grbcorreia.pdf: 1260823 bytes, checksum: 8ebde2fdd525176303b72981cc66cfa0 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-05-02T15:59:07Z (GMT) No. of bitstreams: 1 2015_dis_grbcorreia.pdf: 1260823 bytes, checksum: 8ebde2fdd525176303b72981cc66cfa0 (MD5) / Made available in DSpace on 2016-05-02T15:59:07Z (GMT). No. of bitstreams: 1 2015_dis_grbcorreia.pdf: 1260823 bytes, checksum: 8ebde2fdd525176303b72981cc66cfa0 (MD5) Previous issue date: 2015-07-14 / The intrinsic antimicrobial resistance of Burkholderia pseudomallei is a serious challenge to the treatment of melioidosis. Many studies have searched for adjuvants that increase susceptibility bacteria to antimicrobials. In this context, the antimicrobial activity of farnesol against B. pseudomallei in planktonic growth has been reported. Thus, the aim of this study was to analyze the in vitro activity of farnesol alone against Burkholderia pseudomallei biofilms, as well as its combination with the antibacterials amoxicillin, doxycycline, ceftazidime and sulfamethoxazole-trimethoprim. Susceptibility was assessed by the broth microdilution test and cell viability was read with the oxidation-reduction indicator dye resazurin. The interaction between farnesol and antibacterial drugs against B. pseudomallei biofilms was evaluated through the calculation of the fractional inhibitory concentration index. The minimum biofilm erradication concentration (MBEC) for farnesol was 75 to 2400 mM. In addition, farnesol significantly reduced the MBEC values for ceftazidime,amoxicillin, doxycycline and sulfamethoxazole-trimethoprim by 256, 16, 4 and 4 times respectively (P<0.05). Optical, confocal and electronic microscopic analyses of farnesol treated B. pseudomallei biofilms demonstrated that this compound damages biofilm matrix,facilitating antimicrobial penetration in the biofilm structure. This study demonstrated the effectiveness of farnesol against B. pseudomallei biofilms and its potentiating effect on the activity of antibacterial drugs, in particular ceftazidime, amoxicillin, doxycycline and sulfamethoxazole-trimethoprim. / A intrínseca resistência apresentada pela bactéria Burkholderia pseudomallei é um grave empecilho para o tratamento da melioidose. Muitas pesquisas focam na busca de adjuvantes que aumentem a sensibilidade desta bactéria aos antimicrobianos. Nesse contexto, a ação inibitória do farnesol frente às cepas de B. pseudomallei, na forma planctônica, já foi relatada em estudo prévio. Diante disso, esse estudo objetivou analisar a atividade in vitro do farnesol contra cepas de B. pseudomallei na forma de biofilme. Aliado à análise da ação do farnesol isoladamente, foi investigada a combinação desse composto com os antimicrobianos amoxicilina, ceftazidima, doxiciclina, imipenem e sulfametoxazol/trimetoprim frente ao biofilme. A sensibilidade foi avaliada por meio do teste de microdiluição em caldo e a leitura da viabilidade celular feita com a resazurina. A concentração inibitória mínima em biofilme (CEMB) para o farnesol foi de 75 a 2400 mM. Ademais, o farnesol reduziu em até 256, 16, 4 e 4 vezes os valores de CEMB para ceftazidima, amoxicilina, doxiciclina e sulfametoxazol/trimetoprim, respectivamente (P<0.05). Por meio de técnicas de microscopia, tais como óptica, confocal e eletrônica, observou-se que o farnesol foi capaz de causar danos na matriz do biofilme, facilitando assim, a penetração dos antibióticos. Deste modo, o presente estudo mostrou a eficácia do farnesol contra biofilmes de B. pseudomallei e seu efeito potenciador, em especial com ceftazidima, amoxicilina, doxiciclina e sulfametoxazol/trimetoprim.

Os compostos pertencentes ao grupo caracterizado como BTEX (sigla para designar Benzeno Tolueno Etilbenzeno e Xileno) sÃo largamente utilizados na indÃstria como matÃria prima para diversos produtos como herbicidas tintas gasolina corantes borracha dentre outras substÃncias que podem ser praticamente inertes ou potencialmente poluidoras A utilizaÃÃo da atividade microbiana como alternativa aos tratamentos convencionais de despoluiÃÃo destas substÃncias tem sido bastante estudada como forma de remoÃÃo destes compostos visto que à um processo viÃvel e que muitas vezes deixa pouco ou nenhum resÃduo secundÃrio Este trabalho teve como objetivo analisar a capacidade de biodegradaÃÃo por meio de curvas de crescimento do perfil proteÃmico e do potencial de formaÃÃo de biofilme de Burkholderia SMF090 submetida ao crescimento em meio mÃnimo BH suplementado com gasolina comercial Curvas de crescimento foram feitas em meio BH suplementados com 2000 ppm do poluente A anÃlise proteÃmica foi feita a partir de crescimentos em meio BH suplementados com 2000 ppm de gasolina comercial As proteÃnas bacterianas do referido crescimento foram extraÃdas a fim de realizar a eletroforese bidimensional Foram analisadas as proteÃnas diferencialmente expressas entre tratamento e controle (crescimento apenas em meio BH) anÃlise da tendÃncia de pI e massa molecular das proteÃnas obtidas e identificaÃÃo das mesmas a partir do Expert Protein Analysis System (Expasy) As curvas de crescimento mostraram que à medida em que a bactÃria à submetida ao poluente seu crescimento à mais rÃpido sugerindo uma adaptaÃÃo ao poluente pela cÃlula bacteriana As proteÃnas encontradas na anÃlise proteÃmica sÃo principalmente proteÃnas relacionadas à sÃntese protÃica e ao metabolismo energÃtico bacteriano Nos gÃis bidimensionais foram encontrados tambÃm proteÃnas pertencentes à via de degradaÃÃo de compostos BTEX presentes na gasolina As anÃlises prÃvias do potencial de formaÃÃo de biofilme demonstraram alta capacidade da cÃlula de formar esta estrutura inclusive nÃo havendo diferenÃas na formaÃÃo de biofilme em presenÃa e ausÃncia de gasolina comercial Os resultados sugerem que Burkholderia SMF 090 tem alto potencial de bidegradaÃÃo dos compostos BTEX presentes na gasolina podendo futuramente ser estudada como possÃvel alternativa biotecnolÃgica para fins de biorremediaÃÃo

The bacterium Burkholderia strain 2.2 N is a soil isolate and a member of a group of non-obligative predator bacteria that can prey on other microorganisms or grow saprophytically. The bacterium has anti-bacterial, anti-fungal, anti-yeast and anti-protozoan activities. Burkholderia strain 2.2 N culture shows hydrolysis on Milk Casein Agar, indicating the bacterium also produces a protease. Azocoll hydrolysis was used to detect and measure protease activity. Protease activity was two-fold higher at pH of 7.5 than pH 9.0 and 25-fold at pH 4.0. Cultures grown in media containing 1.0 % yeast extract (YE), tryptic soy, tryptone or beef extract had protease activity, whereas activity was absent in cultures grown in media containing peptone, soytone, casitone, or tryptone as sole protein source. Addition of 1.0 % sucrose or glucose to 1.0 % YE medium increased protease activity 1.8-fold and 1.4-fold, respectively. Protease activity was 2-fold higher in cultures grown in media containing 1.0 % YE and 10 mM MgCl₂ or FeCl₂, than in 1.0 % YE medium lacking metals or containing 10 mM MnCl2 or CaCl2. The 1.0 % YE medium containing either ZnCl₂ or CuCl₂ lacked protease activity (< 5.0 %). In cultures grown in 1.0 % YE at 30°C with rotation at 120 rpm, protease activity was higher in stationary phase (0.38 units /mg protein) than in exponential phase (0.04 units/mg protein). The Burkholderia strain 2.2 N protease is evidently exported from cells because 86 % of the total proteolytic activity of cells was found in the cell-free culture medium. The cell free filtered culture supernatant medium assayed at 4°C had protease activity, however at a three-fold lower specific activity compared to the same supernatant assayed at 3°C. Protease activity was lower in filtered culture supernatants stored at 4°C, room temperature, and 30°C. Protease activity in samples stored at 4°C was only 40 % (24 hours) and 15 % (48 hours) of activity at time zero. Protease activity in samples stored at room temperature was only 45 % (24 hours) and 35 % (48 hours) of activity at time zero. Protease activity in samples stored at 30°C was only 78 % (24 hours) and 9 % (48 hours) of activity at time zero. Purification of the protease from filtered culture supernatant medium by ammonium sulfate precipitation, increased the protease activity 20-fold. An eluted protein fraction from DEAE-Sepharose column chromatography had 50-fold higher protease activity. Protease activity was inhibited by 10 mM 1-10-phenathroline, EDTA and EGTA, all metalloprotease inhibitors. Purified protease activity inactivated with 10 mM 1-10-phenanthroline or 10 mM EGTA was regained through the addition of Ca2+ or Mg2+. Protease activity was reduced by exposure to dithiothreitol (29 % with 1 mM and 84 % with 10 mM), a disulfide bond inhibitor. Protease activity was not inhibited by leupeptin or phenylmethylsulphonyl flouride. Casein polyacrylamide zymography revealed a band of hydrolysis at approximately 60,000 Da. SDS-PAGE resolved a doublet band present at 60,000 Da present in both the filtered culture supernatant sample and the ammonium sulfate / DEAE-Shepharose column chromatography purified protease sample. Burkholderia strain 2.2 N protease is a metalloprotease with a broad temperature range of activity. It has a molecular weight of approximately 60,000 Da. / Master of Science

The aim of this study was to identify and characterise peptidyl-prolyl cis-trans isomerases (PPIases) from the bacterium Burkholderia pseudomallei, the causative agent of the disease melioidosis. The longer term goal was to assess their potential as vaccine candidates or antimicrobial targets. Using bioinformatic approaches, six putative FK506-binding proteins (FKBPs) proteins and three putative parvulin proteins were identified in B. pseudomallei. Of these, six were expressed and purified as recombinant proteins. The purified proteins were used to immunise BALB/c mice, with some providing protection against a subsequent B. pseudomallei infection. These proteins could therefore be proposed as potential vaccine candidates. Homologues of Mip or SurA, which are associated with virulence in other bacterial species, were identified in B. pseudomallei and closely related B. thailandensis. Recombinant Mip or SurA homologues from B. pseudomallei were shown to have characteristic PPIase enzyme activity. To evaluate the role of the Mip homologue from B. pseudomallei in virulence, an unmarked deletion mutant was constructed. The mutant had reduced intracellular survival; defects in putative virulence mechanisms and attenuated virulence in mice. To assess the role of a SurA homologue, closely related B. thailandensis was used as a model organism, with deletion of the gene resulting in defects in intracellular infection, outer membrane integrity and virulence. This indicates that PPIases from B. pseudomallei and B. thailandensis represent novel virulence determinants and potential antimicrobial targets for therapeutics against melioidosis.

Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for host cell invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. The aims of this study are twofold. The first is to expand the repertoire of known effector proteins using modern proteomics techniques. The second is to explore the function of a subset of effector proteins to better understand their interaction with host cells. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hyper-secreting mutants of B. pseudomallei with the isogenic parent strain as well as a mutant incapable of effector protein secretion. This study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei. To determine the possible function of two effector proteins, BipC and BapA, a yeast two-hybrid system was used to identify host cell proteins the effectors interact with. The proteins were screened against a library of human proteins for interactions. BapA interacted with 2 proteins while BipC interacted with 14. Both BapA and BipC were shown to interact with human C1QBP, a mitochondrial protein involved in inflammation, immunity and autophagy. Finally, the Bsa T3SS protein BipC was characterised in its ability to interact with actin. This study is the first evidence that BipC has the ability to bind to filamentous actin, but not monomeric actin. This binding is direct and no intermediate proteins are required for the interaction. Ectopic expression of BipC in eukaryotic cells caused cytoskeletal rearrangements consistent with an actin-binding protein. The core secretome represents a substantial resource of targets that will be mined for improved diagnostic assays and vaccines. Diagnostics that will detect early stages of disease to allow for more effective antimicrobial intervention are currently lacking. Furthermore, there is scope to design diagnostic assays with dual use such as to detect both melioidosis and infection of cystic fibrosis patients with the closely related opportunistic pathogen B. cepacia. The description of novel T3SS effector proteins is also of considerable value since T3SS proteins are often potent B- and T- cell antigens representing promising components of sub-unit vaccines. Such effector proteins commonly modulate cellular processes such as phagocytosis, inflammasome activation and cell cycle progression, hence the function of the predicted T3SS effectors will provide a series of future research opportunities.

Spreading drug resistances among Gram-negative pathogens and the paucity of new agents on the antibacterial drug market against these tenacious bacteria create a pressing need for the development of new antibiotics. The bacterial fatty acid biosynthesis pathway FAS-II, especially the enoyl-ACP reductase catalyzing the last step of the elongation cycle, is an established drug target against tuberculosis but has not been extensively exploited for drug design against other bacterial pathogens. In this thesis the enoyl-ACP reductases of the Gram-negative biothreat organisms Burkholderia pseudomallei and Yersinia pestis were targeted in a structure-based drug design approach. The structure of the most recently identified enoyl-ACP isoenzyme FabV was characterized by X-ray crystallography and could be determined in three different states. FabV from B. pseudomallei was obtained in the apo-form of the enzyme, whereas FabV from Y. pestis was characterized in a binary complex with the cofactor NADH as well as in a ternary complex with NADH and the triclosan-based 2-pyridone inhibitors PT172 and PT173. Analysis of the FabV structure revealed the typical fold of the short chain dehydrogenase/reductase superfamily with the NADH-binding Rossmann fold and a substrate-binding pocket with a conserved active site geometry compared to the related isoenzyme FabI. Additional structural elements of FabV are located around the active site. The monomeric form of the enzyme is thereby stabilized and the substrate-binding loop is kept in a closed, helical conformation. The ternary complexes of FabV exhibited a similar inhibitor-binding mode as observed for triclosan inhibition in FabI and point to a potential substrate-binding mechanism. B. pseudomallei possesses FabI as an additional enoyl-ACP reductase isoenzyme, which was structurally characterized in the apo form and in ternary complexes with NAD+ and the diphenyl ether inhibitors triclosan, PT02, PT12 or PT404 as well as the 4-pyridone inhibitor PT155. The structural data of the ternary enoyl-ACP reductases complexes of B. pseudomallei and Y. pestis hold the promise for the possibility to develop antibacterials targeting FabV or even both isoenzymes, FabI and FabV, based on the triclosan scaffold. / Die Ausbreitung von Antibiotikaresistenzen in Gram-negativen Pathogenen sowie der Mangel neuer Medikamente auf dem Arzneimittelmarkt gegen diese hartnäckigen Bakterien weist einen dringenden Bedarf an neuen Antibiotika auf. Die bakterielle Fettsäurebiosynthese (FAS-II), speziell die Enoyl-ACP-Reduktase, welche den finalen Schritt des Elongationszyklus katalysiert, ist ein etablierter Angriffspunkt in der Tuberkulosetherapie. Sie wurde jedoch bisher noch nicht für die gezielte Wirkstoffentwicklung gegen andere bakterielle Krankheitserreger genutzt. In dieser Dissertation waren die Enoyl-ACP Reduktasen aus Burkholderia pseudomallei und Yersinia pestis Gegenstand des strukturbasierten Wirkstoffdesigns. Die Struktur des zuletzt gefundenen Isoenzyms der Enoyl-ACP-Reduktase, FabV, wurde röntgenstrukturanalytisch charakterisiert und konnte in drei verschiedenen Zuständen bestimmt werden. Die Struktur des FabV Proteins aus B. pseudomallei wurde in der Apo-Form gelöst, während FabV aus Y. pestis in binären und ternären Komplexen mit NADH bzw. NADH und einem Triclosan-basierten 2-Pyridon-Inhibitor, PT172 bzw. PT173 charakterisiert wurde. FabV weist die typische Struktur eines Mitglieds der Short-Chain-Dehydrogenase/Reduktase Superfamilie mit einer NADH-bindenden Rossmann-Faltung und einer Substratbindetasche auf mit einer, im Vergleich zu dem verwandten Isoenzym FabI, konservierte Geometrie des aktiven Zentrums. Zusätzliche strukturelle Elemente sind um das aktive Zentrum gefaltet und stabilisieren damit das Enzym in seiner monomeren Form. Darüber hinaus halten sie den Substratbindeloop in einer geschlossenen helikalen Gestalt. Die ternären FabV Komplexe zeigen Übereinstimmungen mit dem bekannten Bindungsmechanismus des Inhibitors Triclosan in FabI und deuten auf einen möglichen Substratbindungsmechanismus hin. B. pseudomallei besitzt FabI als zusätzliches Isoenzym der Enoyl-ACP-Reduktasen. Dieses Isoenzym wurde in der Apo-Form und in ternären Komplexen mit NAD+ und den Diphenylether-Inhibitoren Triclosan, PT02, PT12 und PT404 sowie dem 4-Pyridon-Inhibitor PT155 strukturell charakterisiert. Die strukturellen Daten der ternären Enoyl-ACP-Reduktase Komplexe von B. pseudomallei und Y. pestis stellen die Möglichkeit in Aussicht Antibiotika zu entwickeln, welche FabV oder auch beide Isoenzyme, FabI und FabV, inhibieren.

Biofertilisers are rhizosphere microorganisms inoculated to reduce the need for N or P fertiliser application and maximise plant growth and nutrition, resulting in greater grain yield and N or P content. This study aimed to evaluate the effectiveness of diazotrophic bacteria isolated from the rhizosphere of wheat in Victoria, Australia. This thesis shows that N2-fixing Burkholderia species have great potential as biofertilisers on wheat productivity. In Chapter 2, strains of bacteria were isolated from wheat-growing soils in main Victoria wheat belt at Horsham and Birchip in North West Victoria. Strains were identified as Burkholderia spp. by their closest matches in the 16S DNA and by morphology and physiology. In Chapter 3, one selected strain from each of Birchip and Horsham were used to inoculate wheat in a pot trial in a glasshouse during winter-spring. Soil was collected on site from wheat fields. Pots were inoculated with these strains to evaluate the effects of Burkholderia inoculum as biofertiliser on the plant growth and yield. Different nitrogen sources (urea 46% N and ammonium sulphate 21% N) were used as fertiliser at one of four levels (0, 50, 100 and 150 kg N/ ha). There was a greater effect in Birchip than in Horsham soil and with ammonium sulphate than with urea due to waterlogying in Horsham soil. In Chapter 4, field-grown wheat was inoculated with the same strains of Burkholderia. Three experiments were carried out in plots at two sites, dryland and irrigated fields at Horsham and a dryland field at Birchip, during the winter wheat season of 2006, to evaluate the effect of Burkholderia species inoculum and different types of nitrogen source at one of four levels of added N (0, 50, 100 and 150 kg N/ha) on wheat growth and yield. The effects of both bacterial inoculation and N fertiliser on growth promotion and grain yield. Since 2006 was a year of drought, dry land crops were unsuccessful. Grain %N as well as total N content in grain per area in the Horsham irrigated field increased with increasing N fertiliser levels up to 100 kg N/ha. In Chapter 5, acetylene reduction (ARA) activity was measured in the pots for both inoculated and uninoculated plants at various growth stages and populations of nitrogen-fixing bacteria associated with the wheat roots and bulk soil were measured in addition to biomass and N content of plants and grain. Molecular tracing using specific primers showed that the inoculum was present only in inoculated treatments. Up to 60% of the increased N content of the grain in inoculated plants was potentially derived from nitrogen fixed by the inoculum in the rhizosphere. It was concluded that the most significant result due to inoculation was the consistent maximal increase of N content in grain in inoculated treatments with ammonium sulphate fertiliser at 100 kg N/ha. Inoculation with Burkholderia consistently increased %N in wheat grain, with the potential benefit of decreasing the production cost and reducing use of chemical fertilisers.

The genome of B. phytofirmans OLGA172 has been sequenced by Next Generation sequencing methods. Over 42 kbp of its genome surrounding its 3CBA degradative genes, tfdCIDIEIFI, was assembled and annotated. The most important method used was the synteny method, which implies homology between the genes, and descent from a common ancestor (Guttman, 2008). The conserved gene order between B. phytofirmans PsJN, B. xenovorans LB400, and OLGA172 was used as a confirmation of annotation through BLASTn, enabled closing of the gaps in the sequencing data, and allowed prediction of genes further downstream. Though the whole genome is not yet assembled, a very significant region carrying a concentrated area of mobile genetic elements (MGE) has been found to surround the degradative genes in OLGA172. This thesis details the sequence evidence that, upon examination of closely related strains, OLGA172 and its related strain from pristine soils may be the ancestral chlorobenzoate degraders.

The genome of B. phytofirmans OLGA172 has been sequenced by Next Generation sequencing methods. Over 42 kbp of its genome surrounding its 3CBA degradative genes, tfdCIDIEIFI, was assembled and annotated. The most important method used was the synteny method, which implies homology between the genes, and descent from a common ancestor (Guttman, 2008). The conserved gene order between B. phytofirmans PsJN, B. xenovorans LB400, and OLGA172 was used as a confirmation of annotation through BLASTn, enabled closing of the gaps in the sequencing data, and allowed prediction of genes further downstream. Though the whole genome is not yet assembled, a very significant region carrying a concentrated area of mobile genetic elements (MGE) has been found to surround the degradative genes in OLGA172. This thesis details the sequence evidence that, upon examination of closely related strains, OLGA172 and its related strain from pristine soils may be the ancestral chlorobenzoate degraders.

La fibrose kystique (FK) est une maladie génétique causée par la mutation du gène codant pour la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Celle-ci présente un défaut sur le canal à ions chlorures affectant ainsi entre autres la viscosité des muqueuses au niveau du système respiratoire. Cet environnement est alors propice aux colonisations bactériennes opportunistes sous la forme de biofilm. Burkholderia cepacia, bacille Gram-négatif mobile, multi-résistant aux antibiotiques et hautement transmissible, s'avère d'une extrême virulence pour les patients atteints de FK. Cette bactérie pathogène désigne en fait un ensemble de neuf souches rassemblées sous le nom de « complexe B. cepacia » (CBC). Au moins huit de ces neuf souches produisent un exopolysaccharide nommé Cepacian. Celui-ci est constitué d'un motif de répétition heptasaccharidique composé notamment de l'enchaînement α-D-Rhap-( 1→4 )-α-D-GlcpA. Le D-rhamnose (ou 6-deoxy-D-mannose) est un composant de glycoconjugués des parois de bactéries pathogènes mais ce sucre rare est absent chez l'homme. Ce dernier présente donc un fort potentiel antigénique dans le cadre de la préparation d'un vaccin entièrement synthétique et spécifique contre B. cepacia. L'élaboration de celui-ci consiste en un activateur universel immunogénique peptidique (Tc-épitope), PADRE ou P2TT préalablement synthétisés, fonctionnalisé par une unité antigénique spécifique. Dans ce but, deux tri-O·saccharides constituants du LPS de B. cepacia et composés majoritairement de D-rhamnose, ainsi que des fragments du motif de répétition de l'exopolysaccharide (EPS) du CBC ont été synthétisés. Des méthodes de synthèses orthogonales ont été optimisées avec de très hauts rendements sur les cinq différents sucres constituant l'EPS ou les LPS du CBC, et plus spécifiquement sur le D-rhamnose. Une série de glycosylation suivie d'une conjugaison finale via une addition radicalaire ou une métathèse croisée a conduit à deux méthodes de conjugaison pour la synthèse de vaccins potentiels, ceci constituant le but final du projet. La synthèse linéaire en 21 étapes a conduit au trisaccharide, α-D-Rhap-(1→3)-α-D-Rhap-(1→2)-α-D-Rhap, avec 36% de rendement global (ou 19% en 18 étapes optimisables). Un rendement de 28% a été obtenu pour une synthèse séquentielle en 17 étapes pour le trisaccharide majeur du LPS, α-D-Rhap-(1→3)-α-D-Rhap-(1→4)-α-D-Galp. La conjugaison de ce dernier sur T-cell épitope constituera l'ultime étape afin d'obtenir un vaccin potentiel entièrement synthétique. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Vaccin synthétique, T-cell épitope, Burkholderia cepacia, Fibrose kystique, Oligosaccharide, D-rhamnose, Complexe B. cepacia.