Haemophilus influenzae type b (Hib) é uma importante bactéria Gram-negativa causadora de pneumonia, meningite e septicemia em crianças abaixo dos 5 anos de idade. A prevenção contra a doença pode ser alcançada pela imunização da população com vacina conjugada polissacarídeo-proteína, uma vez que a vacina de polissacarídeo não é eficiente. As vacinas conjugadas disponíveis comercialmente custam para o governo brasileiro cerca de US2,7 a dose, sendo necessárias no mínimo 3 doses para imunização completa. O presente estudo desenvolveu um novo método de conjugação de polissacarídeo capsular de Hib (PRP) com toxóide tetânico (TT). O método hidrazona baseia-se em 3 etapas simples: oxidação e derivatização de PRP com espaçador molecular e conjugação com TT na presença de uma carbodiimida e de um éster amino reativo. Após um estudo detalhado de cada etapa do método hidrazona, o novo processo mostrou excelentes resultados de rendimento mesmo após escalonamento. A imunogenicidade e o índice de avidez do conjugado hidrazona foram avaliados e os resultados encontrados foram comparáveis a vacina comercial Hiberix®. A técnica de HPSEC mostrou-se eficaz e o perfil cromatográfico do conjugado hidrazona foi muito similar ao da vacina Hiberix. Finalmente, o novo processo de conjugação de vacina permitiu o desenvolvimento de uma poderosa tecnologia constituindo uma excelente opção para o governo brasileiro. / Haemophilus influenzae type b (Hib) is an important encapsulated bacteria, which causes pneumonia, meningitis and septicaemia in infants. Prevention against infection is achieved by the currently available polysaccharide protein conjugate vaccine. However, due to its high production costs (around U$ 2,7 per dose) this formulation cannot be used in mass immunization programs in Brazil. In the present study, we developed a new method for the conjugation of Hib polysaccharide (PRP) and tetanus toxoid (TT). The hydrazone method is based on 3 singles steps: PRP oxidation, PRP derivatization with linker spacer and conjugation with TT in the presence of carbodiimide and an amino reactive ester. After detailed study of each step of method, the new process showed very good yield of conjugation even when it was scaled-up. The immunogenicity and the avidity index of hydrazone conjugate were evaluated and the results were comparable with those obtained with the commercial vaccine Hiberix®. The HPLC hydrazone profile was very similar to HPLC Hiberix profile. Finally, the new conjugation process allows the development of a powerful vaccine technology, constituting an excellent choice for the brazilian government.

Le biofilm est défini comme une communauté microbienne adhérant à une surface biotique ou abiotique, et engluée dans une matrice extracellulaire auto-produite. Les biofilms naturels sont composés de plusieurs espèces microbiennes et leurs interactions, qu'elles soient synergiques ou antagonistes, jouent un rôle important dans le développement, la composition et le fonctionnement des consortia impliqués. De plus, ces relations impliquent souvent la production de molécules antagonistes limitant la croissance ou l'adhésion bactérienne. Enfin la composition de la matrice extracellulaire joue un rôle important dans la robustesse du biofilm. Au cours de ce travail, l'étude des interactions au sein de biofilms mixtes constitués par K. pneumoniae et S. epidermidis a permis d'isoler un polysaccharide produit par K. pneumoniae, capable d'inhiber l'adhésion aux surfaces abiotiques de plusieurs autres espèces bactériennes à Gram-négatif et à Gram-positif. La caractérisation physico-chimique de cette molécules de haut poids moléculaire a permis de mettre en évidence qu'elle était composée de galactose, glucose, rhamnose et d'acide glucuronique. Par ailleurs, l'analyse d'extraits de mutants déficients pour la production de capsule a montré que ce polysaccharide correspondait à la capsule de K. pneumoniae. Son mode d'action consisterait à inhiber les interactions initiales entre bactéries et surface. Le suivi dans le temps de la formation de biofilm monospecies par K. pneumoniae avec la technique Biofilm Ring Test® a également permis de mettre en évidence un phénotype original. En effet, la détection initiale d'agrégats bactériens est suivie par une modification apparente de leur structure, qui serait liée à des changements de leur robustesse face aux forces d'aimantation magnétique. La présence d'ADN extracellulaire au sein de la matrice du biofilm pourrait jouer un rôle dans la survenue de ce phénotype comme l'indique les mesures effectuées en présence de l'enzyme DNAse I. En parallèle, l'observation de biofilm formés par K. pneumoniae dans des modèles expérimentaux cinétiques a mis en évidence des décrochements massifs de biomasse au cours de la maturation du biofilm, qui pourraient être corrélées aux modifications internes de robustesse de la matrice. L'ensemble de ces données permet de mieux caractériser les interactions intimes survenant à l'intérieur de biofilms constitués par K. pneumoniae et à terme de mieux caractériser et donc prévenir leur formation et dissémination. / Biofilms are defined as microbial communities adhering to biotic or abiotic surfaces and embedded in a self-produced extracellular matrix. Natural biofilms are composed of several microbial species and their interactions, synergistic or antagonistic, play important roles in development, composition and functioning of the consortia. Furthermore, the relationships often involve the production of antagonist molecules that impair competitors' growth or adhesion. The composition and evolution of the extracellular matrix plays also an important role in the biofilms' robustness. In this work, study of the interactions within biofilms formed by K. pneumoniae and S. epidermidis led to the isolation of a polysaccharide produced by K. pneumoniae able to inhibit the adherence to abiotic surfaces of several Gram-negative and Gram-positive bacterial species. The physico-chemical characterization of this high molecular weight molecule showed it was composed of galactose, glucose, rhamnose and glucuronic acid. This data, together with the analysis of extracts from capsule-deficient mutants, indicated that the capsule of K. pneumoniae was responsible for the biofilm inhibition phenotype, probably by inhibiting the initial interactions between bacteria and surface. By monitoring the formation of monospecies biofilm by K. pneumoniae with the Biofilm Ring Test® technique, we were able to detect an original phenotype. Indeed, detection of bacterial aggregates still occurred after a few hours of incubation but in a different way, probably related to changes of the biofilm robustness towards magnetic forces. The presence of extracellular DNA in the matrix of the biofilm is likely to play a role in the occurrence of this phenotype, as indicated by the assays performed in presence of the enzyme DNase I. At the same time, observations of biofilm formed by K. pneumoniae in kinetic experimental models showed massive detachment events during biofilm maturation, which could be correlated to changes in internal strength of the matrix. All these dtat contribute to a better characterization of the intimate interactions occuring within biofilms formed by K. pneumoniae and will ultimately lead to the development of efficient anti-biofilm strategies.

<p>This thesis describes the synthesis of structures related to the capsular polysaccharide of <i>Neisseria meningitidis</i> serogroup A and the synthesis of analogues of mycothiol, a compound produced by <i>Mycobacterium</i> <i>tuberculosis</i>. The first part of the thesis describes the synthesis of structural elements present in the native capsular polysaccharide of <i>Neisseria</i> <i>meningitidis</i> serogroup A. In this part, an improved synthesis of 2-azido-2-deoxy-D-mannopyranose is included. The second part of the thesis describes the formation of stable C-phosphonate analogues related to the capsular polysaccharide. The last part outlines the formation of analogues of mycothiol, where the syntheses of a bicyclic analogue and a thioglycosidic analogue are described.</p>

This thesis describes the synthesis of structures related to the capsular polysaccharide of Neisseria meningitidis serogroup A and the synthesis of analogues of mycothiol, a compound produced by Mycobacterium tuberculosis. The first part of the thesis describes the synthesis of structural elements present in the native capsular polysaccharide of Neisseria meningitidis serogroup A. In this part, an improved synthesis of 2-azido-2-deoxy-D-mannopyranose is included. The second part of the thesis describes the formation of stable C-phosphonate analogues related to the capsular polysaccharide. The last part outlines the formation of analogues of mycothiol, where the syntheses of a bicyclic analogue and a thioglycosidic analogue are described.

UDP-galactopyranose mutase (EC 5.4.99.9; UGM), the product of the glf gene, is an enzyme that catalyzes the conversion of uridine diphosphate galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UGM activity is found in bacteria, parasites and fungi, however is absent in higher eukaryotes. This enzyme is essential for the viability of many pathogenic organisms, such as Mycobacterium tuberculosis and Escherichia coli, due to the broad distribution of Galf in crucial structures such as the cell wall or capsular polysaccharide. Not surprisingly, galactofuranose biosynthesis has become an attractive antimicrobial target due to the absence of these sugars in higher eukaryotes. The UGM homologue, UDP-Nacetylgalactopyranose mutase (UNGM), was identified in Campylobacter jejuni 11168, encoded for by the cj1439c gene. UNGM is known to function as a bifunctional mutase, which catalyzes the reversible ring contraction between the pyranose-furanose forms of UDPgalactose (UDP-Gal) and UDP-N-acetylgalactosamine (UDP-GalNAc). UNGM is essential for the virulence of C. jejuni, due to the incorporation of UDP-N-acetylgalactofuranose into the capsular polysaccharide. We report the first structure of UNGM determined by X-ray crystallography, to a resolution of 1.9 Å. Analysis of the dimeric, holoenzyme structure of UNGM has identified that the cofactor flavin adenine dinucleotide is bound within each monomer of the enzyme. Comparative analysis with UGM homologues has confirmed the conserved active site residues involved in the binding of various substrates. Docking studies suggest that UNGM binds its natural substrates in a productive binding mode for catalysis with the flavin cofactor, which is consistent with the proposed mechanism for UNGM. The mobile loops are essential for substrate binding, and we have identified that the conserved arginine residue, Arg169, and the neighboring Arg168, function to stabilize the diphosphate region of UDP, although not concurrently. The non-conserved arginine residue, Arg168, appeared to favor the stabilization of N-acetylated sugars, which is in agreement with the enzyme’s higher binding affinity for UDP-GalNAc over UDP-Gal by a factor of 0.9. We have also identified that the active site Arg59 exists in two conformations in the structure of UNGM, with one conformation directed toward the active site. Arg59 is 2.5 to 3.0 Å from the acetamido moiety of GalNAc, which is favorable for stabilization and is believed to confer specificity for this substrate.

Streptococcus pneumoniae (pneumococo) é um importante patógeno humano, responsável por graves infecções das vias respiratórias. O principal fator de virulência desse microrganismo é a cápsula polissacarídica (PS), antígeno das vacinas atuais, que são elaboradas com os PS purificados de cepas de pneumococo prevalentes na população. O objetivo desse trabalho foi desenvolver um processo de purificação do PS do sorotipo 14, responsável por 39% das doenças em crianças de 0-6 anos no Brasil. A metodologia de purificação envolveu separação celular por microfiltração tangencial e concentração do microfiltrado com membrana de ultrafiltração tangencial de 50 kDa. O produto dessa etapa foi diafiltrado com dodecil sulfato de sódio em membrana de ultrafiltração tangencial de 30 kDa, seguido de precipitação com ácido tricloroacético a 5%, precipitação por etanol (20% e 60%) e cromatografia de troca aniônica. A pureza do PS foi avaliada pelo conteúdo de proteínas e ácidos nucleicos remanescentes e o tamanho por cromatografia de exclusão molecular. O PS foi obtido com pureza e tamanho requeridos pelos órgãos regulatórios e o rendimento final do processo foi de 65%. / Streptococcus pneumoniae (pneumococcus) is an important human pathogen responsible for severe respiratory tract infections. The main virulence factor of this microorganism is the capsular polysaccharide (PS), which is the antigen of all current vaccines, which are prepared with purified PS of pneumococcal strains prevalent in the population. The objective of this work was to develop a new purification process for PS of serotype 14, responsible for 39% of diseases in children of 0-6 years old in Brazil. The purification method involved cell separation by tangential microfiltration and concentration of cell-free culture broth containing PS by tangential ultrafiltration (50 kDa). The product of this step was diafiltrated with sodium dodecyl sulfate by tangential ultrafiltration (30 kDa), following by 5% trichloroacetic acid precipitation, 20% and 60% ethanol precipitation and anion exchange chromatography. The PS purity was evaluated by the content of residual proteins and nucleic acids, and the molecular mass by size exclusion chromatography. The purity and molecular mass requirements were achieved and the process global yield was 65%.

Haemophilus influenzae type b (Hib) é uma importante bactéria Gram-negativa causadora de pneumonia, meningite e septicemia em crianças abaixo dos 5 anos de idade. A prevenção contra a doença pode ser alcançada pela imunização da população com vacina conjugada polissacarídeo-proteína, uma vez que a vacina de polissacarídeo não é eficiente. As vacinas conjugadas disponíveis comercialmente custam para o governo brasileiro cerca de US2,7 a dose, sendo necessárias no mínimo 3 doses para imunização completa. O presente estudo desenvolveu um novo método de conjugação de polissacarídeo capsular de Hib (PRP) com toxóide tetânico (TT). O método hidrazona baseia-se em 3 etapas simples: oxidação e derivatização de PRP com espaçador molecular e conjugação com TT na presença de uma carbodiimida e de um éster amino reativo. Após um estudo detalhado de cada etapa do método hidrazona, o novo processo mostrou excelentes resultados de rendimento mesmo após escalonamento. A imunogenicidade e o índice de avidez do conjugado hidrazona foram avaliados e os resultados encontrados foram comparáveis a vacina comercial Hiberix®. A técnica de HPSEC mostrou-se eficaz e o perfil cromatográfico do conjugado hidrazona foi muito similar ao da vacina Hiberix. Finalmente, o novo processo de conjugação de vacina permitiu o desenvolvimento de uma poderosa tecnologia constituindo uma excelente opção para o governo brasileiro. / Haemophilus influenzae type b (Hib) is an important encapsulated bacteria, which causes pneumonia, meningitis and septicaemia in infants. Prevention against infection is achieved by the currently available polysaccharide protein conjugate vaccine. However, due to its high production costs (around U$ 2,7 per dose) this formulation cannot be used in mass immunization programs in Brazil. In the present study, we developed a new method for the conjugation of Hib polysaccharide (PRP) and tetanus toxoid (TT). The hydrazone method is based on 3 singles steps: PRP oxidation, PRP derivatization with linker spacer and conjugation with TT in the presence of carbodiimide and an amino reactive ester. After detailed study of each step of method, the new process showed very good yield of conjugation even when it was scaled-up. The immunogenicity and the avidity index of hydrazone conjugate were evaluated and the results were comparable with those obtained with the commercial vaccine Hiberix®. The HPLC hydrazone profile was very similar to HPLC Hiberix profile. Finally, the new conjugation process allows the development of a powerful vaccine technology, constituting an excellent choice for the brazilian government.

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