Bacterial biofilms represent a challenge to the healthcare system because of their resilience against antimicrobials and immune attack. Biofilms consist of bacterial aggregates embedded in an extracellular polymeric substance (EPS) composed of polysaccharides, nucleic acids and proteins. We hypothesised that carbohydrates could contribute to immune recognition of Pseudomonas aeruginosa biofilms by engaging C-type lectins. Here we show binding of Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), mannose receptor (MR, CD206) and Dectin-2 to P. aeruginosa biofilms. We also demonstrate that DC-SIGN, unlike MR and Dectin-2, recognises planktonic P. aeruginosa cultures and this interaction depends on the presence of the common polysaccharide antigen. Within biofilms DC-SIGN, Dectin-2 and MR ligands appear as discrete clusters with dispersed DC-SIGN ligands also found among bacterial aggregates. DC-SIGN, MR and Dectin-2 bind to carbohydrates purified from P. aeruginosa biofilms, particularly the high molecular weight fraction (HMW; >132,000 Da), with Ks in the nM range. These HMW carbohydrates contain 74.9-80.9% mannose, display α-mannan segments, interfere with the endocytic activity of cell-associated DC-SIGN and MR and inhibit Dectin-2-mediated cellular activation. In addition, biofilm carbohydrates reduce the association of the DC-SIGN ligand Lewis, but not fucose, to human monocyte-derived dendritic cells (moDCs), and alter moDC morphology without affecting early cytokine production in response to lipopolysaccharide or P. aeruginosa cultures. This work identifies the presence of ligands for three important C-type lectins within P. aeruginosa biofilm structures and purified biofilm carbohydrates and highlights the potential for these receptors to impact immunity to P. aeruginosa infection.

Les sucres ont un rôle dans de nombreux phénomènes biologiques dont, notamment, la reconnaissance cellulaire. Par exemple, les cellules cancéreuses expriment des antigènes composés de sucres qui leurs sont propres, modifiant ainsi leur identité glycosidique par rapport à celle d’une cellule saine. Ces sucres sont reconnus spécifiquement pas des protéines que l’on nomme des lectines. Ces interactions sont faibles et non-covalentes donc difficiles à capturer. L’objectif de ce projet est de concevoir un outil permettant de capturer ces interactions ce qui en permettra une meilleure compréhension. Les stratégies déjà utilisées consistent en l’utilisation de photoréticulants. Ces molécules permettent de former un lien covalent entrer la protéine d’étude et son substrat. Bien que de nombreuses améliorations ont été effectuées depuis la création de ces outils, il reste difficile de pouvoir étudier ces interactions. Pour pallier à cela, nous avons ajouté une dimension visuelle a notre outil en incorporant un motif coumarine à notre photoréticulant qui, une fois soumise à une irradiation UV, permet de créer une liaison covalente entre notre sonde et notre protéine d’intérêt et de laisser en même temps, une trace fluorescente sur ladite protéine. Nous avons ensuite utilisé notre coumarine en présence d’une fucolectine, la BambL, en conditions dénaturantes pour laisser une trace fluorescente sur celle-ci. Notre sonde a également été utilisé pour cibler des protéines d’intérêts dans des lysats cellulaires. / Carbohydrates are involved in many biological phenomena, such as cellular recognition. As an example, cancer cells expressed there on sugared antigens and it’s modifiying their glycosidic identity compare to an healthy cell. These sugars can be recognized by proteins which are also called lectins. Interactions between the biomolecules are weak and difficult to capture. The aim of the project is to design a tool which let us able to capture and have a better understanding of theses interactions. Previous studies have shown the usefulness of the photocrosslinkers. These molecules can create a covalent bond between the protein of interest and its substrat. Although all the improvements since the creation of these tools, it is still difficult to underline sugar-protein interactions. To overcome the problem, visual parameters were incorporated to our probe with a coumarin scaffold whom, after UV irradiations, can generate a nitrene and formed a covalent bond between our probe and our targeting probe letting a fluorescent tag on the protein. The probe has been tested in presence of a fucolectine; the BambL, in denaturing conditions to check the fluorescent tag. Our probe was also tested in cell lysats conditions.

Dynamic systems for screening, control and identification of different protein-ligand interactions are presented. Dynamic chemistry is used to produce new compounds/constituents in situ that can interact with a target molecule. Several entities can be introduced at the same time and interact with one another. These molecules make a dynamic combinatorial library (DCL) which is used in dynamic combinatorial chemistry (DCC). DCC is a recently introduced approach to generate dynamically interchanging libraries of compounds. These libraries are made of different building blocks that reversibly interact with one another and spontaneously assemble to encompass all possible combinations. If a target molecule, for instance a receptor is added to the system and one or more molecules show affinity to the target species, these compounds will, according to Le Châtelier´s principle, be amplified on the expense of the other non-bonding constituents. To further advance the technique, especially when biological systems are targeted, new reaction types and new screening methods are necessary. This thesis describes the development of different reversible reactions, thiol/disulfide interchange, transthiolesterification and the nitroaldol (Henry) reaction as means of generating reversible covalent bond reactions. Two different types of target proteins are used, enzymes belonging to the hydrolase family and the plant lectin Concanavalin A. Dynamic combinatorial resolution (DCR) is presented. This new concept relies on the consecutive kinetic resolution of dynamic combinatorial libraries, leading to complete amplification and control of dynamically interchangeable processes. By applying a kinetically controlled step to a thermodynamically controlled system, complete transformation and amplification can be obtained. The concept has been demonstrated by developing transthiolesterification and nitroaldol exchange reactions to generate diversity, forming libraries under thermodynamic control, and used in one-pot processes with kinetically controlled enzyme-mediated resolution. The results demonstrate that the reaction types are useful for the generation of dynamic libraries, and that the dynamic combinatorial resolution concept is highly valuable for efficient substrate identification, asymmetric synthesis, and library screening. The thesis also describes three other dynamic chemistry protocols. The first one describes dynamic kinetic resolution (DKR) of nitroaldol adducts by combined lipase catalysis. The second one describes finding lectin inhibitors from a glycodisulfide library and the third one describes finding an inhibitor of acetylcholinesterase using a tandem driven dynamic self-inhibition approach. / <p>QC 20100818</p>

A Nattectina é uma toxina isolada do veneno de Thalassophryne nattereri que possui homologia com lectinas tipo C. Este estudo consistiu em avaliar a ação da Nattectina sobre as funções de macrófagos e a influência de citocinas Th1/Th2 nessa ativação. Nossos resultados mostram que a Nattectina induziu o aumento da expressão de moléculas coestimuladoras dependente da sinalização MAPK p38 e PI3K em macrófagos derivados da medula óssea, e também um aumento na capacidade endocítica e expressão de MHC de classe II dependentes da sinalização ERK1/2 e da ligação da Nattectina ao carboidrato. Verificamos ainda que alterações induzida pela Nattectina nos macrófagos são consistentes com a ativação clássica M1 dependente de IL-12 e IFN-<font face=\"Symbol\">g, e regulada negativamente por IL-4, IL-10 e IL-13. Por fim, verificamos uma ação coadjuvante da IL-4 com IFN-<font face=\"Symbol\">g na mobilização de SPMs (Small Peritoneal Macrophages) para cavidade peritoneal e na ativação de macrófagos. Dessa forma, a Nattectina pode ser considerada um importante agente imunomodulador capaz de modular as funções de macrófagos. / Nattectin is a toxin isolated from Thalassophryne nattereri fish venom that has homology with C-type lectin. This study was carried out to evaluate the action of Nattectin on macrophage functions and the influence of Th1/Th2 cytokines in this activation. Our results show that Nattectin induced the increased expression of costimulatory molecules dependent on MAPK p38 and PI3K signaling in bone marrow-derived macrophages, and also an increase in endocytic capacity and MHC class II expression dependent on ERK1/2 signaling and binding of Nattectin to carbohydrate. We also found that modifications induced by Nattectin in macrophages are consistent with classical (M1) activation of macrophages dependent on IL-12 and IFN-<font face=\"Symbol\">g cytokines, and negatively regulated by IL-4, IL-10 and IL-13. Finally, it was demonstrated an adjuvant role of IL-4 with IFN-<font face=\"Symbol\">g in mobilization of SPMs (Small Peritoneal Macrophages) to peritoneal cavity and also in macrophage activation. Thus, Nattectin can be considered an important immunomodulatory agent able to modulate macrophage functions.

Espécies do gênero Paracoccidioides spp são fungos patogênicos, termodimórficos, agentes etiológicos de doença endêmica em diversas regiões da América Latina. O indivíduo infectado desenvolve uma resposta específica que, quando associada à alta produção de TNF-? e IFN-?, favorece a resistência ao fungo. Componentes de alguns fungos patogênicos foram caracterizados, por técnicas de knockdown gênico, como importantes para a virulência fúngica. Nosso grupo identificou paracoccina (PCN) como um componente de leveduras de P. brasiliensis; trata-se de uma proteína com um domínio enzimático, dotado de atividade quitinase e um domínio lectínico, ligante de GlcNAc. PCN é dotada das seguintes propriedades: (a) contribui para o crescimento do fungo; (b) promove a adesão da levedura à matriz extracelular, por ligar-se à laminina; (c) interage com N-glicanas de TLR2 e TLR4 e promove ativação celular; (d) estimula macrófagos a produzirem mediadores pró-inflamatórios como IL- 12, TNF-? e NO; (e) promove a polarização M1 de macrófagos; (f) induz atividade fungicida em neutrófilos, bem como formação de NETs e supressão da apoptose, eventos que se mostraram dependentes da síntese de novo de proteínas pelos neutrófilos estimulados. Dada a relevância das atividades biológicas de PCN, promovemos recentemente o silenciamento do gene que codifica essa proteína, através de metodologia que usa RNA anti-sense e transformação mediada por Agrobacterium tumefaciens (ATMT). Uma vez PCN silenciada, a levedura perdeu a capacidade de fazer a transição para micélio e diminuiu a resistência à atividade fungicida de macrófagos. A infecção de camundongos com as cepas silenciadas, em comparação com as WT, causou doença de menor gravidade, com carga fúngica reduzida e baixa taxa de mortalidade. Essas observações sugerem de que PCN funcione como um fator de virulência em P. brasiliensis, que afeta a patogênese da infecção. Neste trabalho, ampliamos as ferramentas moleculares de manipulação do fungo e viabilizamos a superexpressão de PCN em leveduras de P. brasiliensis, tendo como objetivos estudar seu papel na virulência e na patogênese da infecção, bem como determinar os mecanismos responsáveis por tais atividades. A inoculação de leveduras que superexpressam PCN (ov-PCN) em camundongos causou doença pulmonar muito grave, em comparação à doença leve e moderada causada por leveduras silenciadas em PCN e leveduras WT, respectivamente. Nesse sentido, nossos esforços se dedicaram à busca dos mecanismos dos mecanismos através dos quais PCN influencia o curso da infecção experimental. Na tentativa de identificar o papel exercido pelo domínio quitinase da PCN, coletamos o sobrenadante de culturas de leveduras ov-PCN e WT. Partículas de quitina presentes nesses sobrenadantes foram purificadas por afinidade à lectina WGA (wheat germ agglutinin). Através de medida da área das partículas capturadas, através de microscopia eletrônica e aplicação do programa ImageJ, verificamos que a superexpressão de PCN resultou em clivagem mais eficiente da quitina da parede de leveduras, uma vez que apenas partículas muito pequenas (mediana das medidas = 2 nm2) foram detectadas, enquanto as áreas das partículas de quitina obtidas de leveduras selvagens (WT) forneceram mediana 3 vezes maior (6 nm2). As partículas de quitina foram então utilizadas para estimular macrófagos a produzirem citocinas. As obtidas de ov-PCN estimularam preponderantemente a secreção da citocina antiinflamatória IL-10, enquanto os macrófagos estimulados com partículas de leveduras WT produziram mais TNF-? e IL-1?, ambas de efeito pró-inflamatório. Esses resultados permitiram a identificação de um mecanismo importante para que a superexpressão de PCN se associe à ocorrência de doença pulmonar muito grave: o microambiente anti-inflamatório criado pelo estímulo de macrófagos por PCN leva ao desenvolvimento de resposta imune não protetora do tipo Th2 e lesões mais graves. Um segundo mecanismo foi identificado ao compararmos a resistência de leveduras ov-PCN e WT às respostas efetoras de macrófagos. A superexpressão de PCN associou-se à maior internalização das leveduras e maior resistência à atividade fungicida exercida por macrófagos. O estudo demonstra que diferentes níveis da expressão de uma quitinase (como PCN) levam à resistência a atividades antifúngicas de macrófagos e a diferentes graus de clivagem de quitina. A clivagem, por sua vez, pode alterar a estrutura da parede celular fúngica e a geração de fragmentos de quitina, cujos tamanhos e concentrações influenciam a produção de citocinas pelos macrófagos. Sob a ação de citocinas pró- ou antiinflamatórias liberadas pelos macrófagos e, consequentemente, a montagem de respostas adaptativas pode ser decisiva para haver suscetibilidade ou resistência à infecção por P. brasiliensis. Este trabalho proporciona um importante avanço no conhecimento do papel de quitinases na resposta anti-fúngica do hospedeiro. / Species of the genus Paracoccidioides spp are thermodymorphic fungi that cause a systemic disease, which is endemic in several regions of the Latin America. The infected individual develops a specific response that, when associated with the high production of TNF-? and IFN- ?, favors resistance to the fungus. Components of some pathogenic fungi were characterized by gene knockdown techniques as important the for fungal virulence. Our group has identified a component of P. brasiliensis, named Paracoccin (PCN); it is a bifunctional protein with an enzymatic domain, endowed with chitinase activity and a lectin domain, which binds GlcNAc and chitin, a GlcNAc polymers. PCN has the following properties: (a) contributes to the fungus growth; (b) promotes the yeast adhesion to the extracellular matrix, by binding to laminina glycans; (c) interacts with TLR2 and TLR4 N-glycans, which triggers cell activation; (d) stimulates macrophages to produce proinflammatory mediators, such as IL-12, TNF-? and NO; (e) promotes the M1 polarization of macrophages; (f) induces the neutrophils fungicidal activity, NETs formation, and suppression of neutrophils apoptosis, which are depending events on the de novo protein synthesis by neutrophils. Given the relevant biological activities exerted by PCN, we have performed recently the silencing of the gene that codes for this protein through a system that uses RNA anti-sense and Agrobacterium tumefaciens mediated transformation (ATMT). Once having the PCN gene silenced, yeast lost the ability of doing the transition to mycelium and decreased its resistance to macrophages fungicidal activities. Mice infection with PCN-silenced yeasts, compared to the infected with WT yeasts, exhibited a milder pulmonary disease with reduced fungal burden and low mortality rate. These observations suggest that PCN acts as a P. brasiliensis virulence factor that affects the pathogenesis of the fungal infection. In the present study, we expanded the molecular tools for the fungus manipulation and enabled the overexpression of PCN in P. brasiliensis yeasts, aiming to elucidate the PCN role in the fungus virulence and the infection pathogenesis, as well as determining the responsible mechanisms for the PCN activities. Inoculation of the PCN overexpressing yeasts (ov-PCN) into mice caused a very severe lung disease, compared to the mild and moderate diseases caused by PCN-silenced and WT yeasts, respectively. Then our efforts became dedicated to the search of mechanisms through which PCN influences the course of the experimental fungal disease. In an attempt to identify the role of the PCN chitinase domain, we harvested the supernatant of the ov-PCN and WT yeasts cultures. Chitin particles contained in the supernatants have been captured by affinity to the immobilized WGA (wheat germ agglutinin) lectin. By measuring through electron microscopy and application of the ImageJ program the area of the isolated chitin particles, we verified that the overexpression of PCN resulted in a more efficient cleavage of whole chitin molecules contained in the yeast cell wall, since only very small particles (median of the measurements = 2 nm2) were detected, while the the chitin particles areas obtained from WT-yeasts provided a median 3 fold higher (6 nm2). Then, the preparations of chitin particles were taken to stimulate macrophages to produce cytokines. The particles obtained from ov-PCN have stimulated preponderantly the secretion of the anti-inflammatory cytokine IL-10, whereas the macrophages stimulated with WT yeast particles have produced higher concentrations of TNF-? and IL-1?, which are known proinflammatory cytokines. These results allowed the identification of an important mechanism for the association of PCN overexpression to the occurrence of very severe pulmonary disease: the anti-inflammatory microenvironment created by the macrophages stimulation with PCN leads to the development of a non-protective Th2-type immune response and the more severe pulmonary injury. A second mechanism was identified as implicated in the severity of the lung disease associated to PCN overexpression. We compared the sensitivity of ov-PCN and WT yeasts to macrophages effector functions. PCN overexpressing yeasts were better internalized by macrophages and more resistant to the fungicidal activity of these cells, events that contributes for the high pulmonary fungal load verified in mice infected with ov-PCN yeasts. The study demonstrates that different levels of a chitinase (PCN) expression and enzymatic activity lead yeasts to change their sensitivity to macrophages antifungal activities as well as to different grades of chitin cleavage. The cleavage, in its turn, leads to changes in the structure of the fungal cell wall and generation of chitin fragments, whose sizes and concentrations influence the cytokines production by macrophages. Under the influence of pro-inflammatory or anti-inflammatory cytokines released by macrophages, the mounted adaptative responses can be decisive in conferring susceptibility or resistance to the P. brasiliensis infection. This study provides an important advance in the knowledge on the role of a chitinase in the host antifungal response.

Speichelglykane können einerseits spezifisch an bakterielle Lektine binden und damit deren Adhäsion an orale Oberflächen vermitteln, andererseits eine Antiadhäsion bedingen. Sie stellen ein Schutzsystem für orale Oberflächen dar. Bei vorhandener Karies bzw. Parodontitis ist die Konzentration bestimmter Glykokonjugate verändert. Ziel dieser Studie war es, die Reaktivität der Glandulae majores bzgl. ihrer Sekretion von Glykanen in Abhängigkeit einer experimentellen Gingivitis zu ermitteln. 14 gesunde Probanden enthielten sich 9 Tage der Mundhygiene. Neben der Erhebung des PBI und QH wurde drüsenspezifisch Speichel gewonnen. Die Konzentrationen an die Lektine PNA, GS1, VVA, SNA und AAA bindender Komponenten und deren drüsenspezifische Sekretionsraten wurden bestimmt. Bei allen Probanden stiegen PBI und QH im Versuchsverlauf signifikant an. Gleiches galt für die Speichelmenge nach Stimulation sowie zum Ende der Kontrollreihe. Die Konzentrationen der verschiedenen Glykane verhielten sich unabhängig von der Speichelmenge und unabhängig voneinander. Meist ergab sich eine erhöhte Glykansekretion spezifisch für das untersuchte Lektin. Neben dem Konzentrationsgefälle der einzelnen Drüsen war auch eine Verschiebung nach erfolgter Stimulation zu beobachten. Da genetische und externe Einflüsse für diese Studie weitgehend ausgeschlossen werden konnten bzw. als konstant einzuordnen waren, darf die Veränderung als Reaktion auf die orale Bakterienbelastung angesehen werden. Der Rückgang bestimmter terminaler Strukturen könnte als Folge der vermehrten Synthese anderer, in Bezug auf die veränderte Bakterienflora effektiverer Speichelbestandteile eingeordnet werden. Basierend auf dem Modell, dass freie Glykane die Adhäsion von Mikroorganismen inhibieren können, ließe sich die gemessene Reaktion der Speicheldrüsensekretion als ein gesteigerter Schutzmechanismus im Sinne einer ´first line of defence´ interpretieren. Dieser könnte z.B. in Bezug auf Prophylaxe und Therapie genutzt werden. / Salivary glycans can bind specificly to bacterial lectins. Consequently, bacterial adhesion to oral surfaces is mediated or inhibited by glycans. It is known that the concentration of certain glycans changes in the presence of caries or periodontitis. Therefore this study examines the reactivity of the major salivary glands with respect to the secretion of glycans as conditioned by an experimentally induced gingivitis. 14 healthy subjects refrained from all oral hygiene measures for 9 days. On 5 days a plaque and bleeding index as well as pure glandula saliva with and without stimulation were obtained. The collected salivary samples were examined for their concentration of certain structures that bind to the lectins ´PNA´, ´GS1´, ´VVA´, ´SNA´ and ´AAA´. All subjects developed a gingivitis as measured by the plaque and bleeding index. Salivary flow increased after stimulation and compared to baseline at the end of the trial. The concentration of glycans was neither related to one of the glands nor to the salivary flow. Besides to the differentials of concentration after stimulation there was no symmetrical development between the concentrations of salivary lectin-specific components compared one lectin to another. Genetic and external influences could be largely excluded or considered to be stable during the trial. Therefore the observed results can be regarded as a reaction to the increased bacterial load. The decrease of certain terminal structures in saliva might be explained by a raised synthesis of other components, which are more effective in defending the body against bacterial adhesion. The observed changes in salivary secretion might be interpreted as a mechanism in order to protect the human organism within the meaning of a ´first line of defence´. This mechanism would be able to respond more quickly than the immune system and might be used in future, for example, for preventive and therapeutical strategies.

Glykane aus exokrinen Drüsen stellen ein Schutzsystem der Schleimhautoberflächen dar, indem sie an mikrobielle Adhäsine binden und dadurch Einfluß auf die mikrobielle Besiedelung und Invasion des Wirtes nehmen. 11 Patienten mit rasch fortschreitender Parodontitis (RPP) wurde über jeweils 20 Minuten in Ruhe und unter adrenerger Belastung Speichel aus den großen Speicheldrüsen entnommen und in einem kompetitiven Lektinbindungsinhibitionstest auf die Bindungsfähigkeit an 8 verschiedene Planzenlektine untersucht. Patienten mit RPP zeigen ein anderes Verteilungsmuster der antiadhäsiven Glycane als die Kontrollgruppe. Sie zernieren u.a. aus den Unterkieferdrüsen konstant signifikant mehr Glykane mit endständigen Mannosegruppen, aus der Parotis dagegen nur unter adrenerger Stimulation. Dies zeigt die unterschiedliche Funktion der Drüsen bei der Beeinflussung des Milieus in der Mundhöhle als auch einen unterschiedlichen Erregungszustand des Schleimhautschutzsystems bei Erkrankten und Kontrollen. Aus den Ergebnissen können sich diagnostische Aspekte für das Risiko und den Aktivitätszustand einer parodontalen Erkrankung ergeben. / Glycans from exocrine glands create a defense system of the mucosal surfaces by binding to microbial adhesins and interfering with colonisation and invasion of the host. Saliva from 11 patients with rapidly progressive periodontitis (RPP) was collected over a period of 20 minutes each in rest and during adrenergic stimulation. The samples were tested for binding properties to 8 different plant lectins by a competitive lectin binding inhibition test. A pattern of antiadhesive glycans different from the control group is secreted in patients with RPP. The latter constantly secrete significantly more glycans with terminal mannose from the mandibular glands. In the parotis this is only the case during adrenergic stimulation. This demonstrates the different purpose of the glands in maintaining the oral milieu as well as different states of activity of the mucosal defense system in RPP patients and controls. Diagnostic aspects for risk and activity of periodontal diseases can be drawn from these findings.

Lectinas são proteínas que possuem pelo menos um domínio não catalítico que se liga reversível e especificamente a um monossacarídeo ou oligossacarídeo. A capacidade de ligação a diferentes tipos de açúcares torna essas moléculas ferramentas úteis no estudo de diversos processos celulares específicos. Embora as lectinas de plantas sejam amplamente estudadas, aquelas referentes à família Apocynaceae ainda são pouco exploradas. Resultados prévios obtidos pelo nosso grupo de pesquisa mostraram que extratos brutos de súber do caule da apocinácea Tabernaemontana hystrix Steud apresenta atividade hemaglutinante. Além de aglutinar eritrócitos do sistema ABO, a putativa aglutinina foi capaz de estimular a síntese de RNAm de IL-6 e TGF- beta em células esplênicas de camundongos. À vista disso, no presente projeto tivemos como objetivo identificar, caracterizar bioquimicamente e avaliar o possível potencial imunoestimulador da aglutinina de T. hystrix. Os extratos de T. hystrix obtidos por meio da farinha de raspas do súber apresentaram atividade hemaglutinante, o que não foi observado no extrato do caule destituído de súber e no extraído das folhas. Para comprovar que se tratava da atividade observada anteriormente, obtivemos a inibição da hemaglutinação com a glicoproteína fetuína, mas não houve inibição por monossacarídeos. Foi determinado um protocolo de isolamento da hemaglutinina com precipitação do extrato do súber com sulfato de amônio, cuja atividade foi recuperada no material precipitado na faixa de 30 a 60% de saturação, seguido de cromatografias sequenciais por (1) interação hidrofóbica (HiTrap Octyl), (2) troca catiônica (HiTrap SP), (3) fase reversa (EC Nucleosil C18) e (4) afinidade (Blue Sepharose). Nessas colunas a atividade foi recuperada do (1) material não retido e dos eluatos (2 e 4) com 1M e 0,5M de NaCl, respectivamente, e (3) 83% de acetonitrila. Esse protocolo produziu uma preparação homogênea contendo um peptídeo cuja análise eletrofóretica revelou massa molecular (MM) aproximada de 3kDa e concentração hemaglutinante mínima de 50g/mL. A fim de determinar se esse peptídeo formava estrutura quaternária (dímeros, tetrâmetros, etc.), característica da maioria das lectinas de plantas, submeteu-se a preparação a uma eletroforese em gel nativo (PAGE), não sendo observadas mudanças na MM do peptídeo e nem a presença de outras moléculas com MM maiores que pudessem estar associadas a ele, o que sugere que a aglutinina de T. hystrix (denominado aqui de Tabhys) é um peptídeo de MM aproximada de 3kDa. O fato da heveína, um dos peptídeos lectínicos com atividade antifúngica mais estudado, ter especificidade por quitina nos motivou a tentar o isolamento do peptídeo em coluna desse polissacarídeo. Observou-se atividade hemaglutinante e presença de peptídeo com MM de 3kDa no material eluído com Ácido acético a 0,1M da coluna de quitina. Curiosamente, nenhuma de nossas preparações foram capazes de inibir o crescimento do fungo Trichophyton rubrum. O peptídeo purificado foi testado quanto a sua capacidade em induzir a proliferação celular e a produção de citocinas em células esplênicas murinas. Os resultados dos ensaios de RT-PCR em tempo real e citometria de fluxo demonstraram que o a aglutinina de T. hystrix não foi capaz de estimular a proliferação de linfócitos, entretanto, induziu o aumento de mensagem para a citocina TGF-beta, cujo pico de produção ocorreu em célula estimuladas com 37ng/mL. Neste estudo, relatamos a presença de um peptídeo no extrato de T. hystrix com atividade hemaglutinante, o que é relativamente raro e novo. Devido a isso, este estudo pode proporcionar novas perspectivas e paradigmas nos estudos das lectinas a nível molecular e estrutural. / Lectins are proteins that have at least one non-catalytic domain that binds specifically and reversibly to a monosaccharide or oligosaccharide. This ability to bind to different types of sugars makes these molecules useful tools in the study of various specific cellular processes. Although the plant lectins are widely studied, those belong to Apocynaceae family are still little explored. Previous results obtained by our research group showed that bark crude extracts from Tabernaemontana hystrix Steud (Apocynaceae) had hemagglutination activity. Besides to agglutinate erythrocytes from ABO blood group system, the putative agglutinin induced the synthesis of IL-6 and TGF-beta mRNA in mouse spleen cells. Here we aim to identify, characterize biochemically and evaluate the possible immunostimulatory potential of T. hystrix agglutinin. The haemagglutination activity was obtained from crude extracts of bark flour, but not of flours of stems without bark and leaves. The activity of the bark extract was similar to that from the previous study, since the haemagglutination was inhibited by the glycoprotein fetuin, but not by monosaccharides. An isolation protocol was determined by using ammonium sulfate precipitation, with haemagglutination activity recovered in the range of 30-60% of saturation, and sequential chromatography procedures: (1) hydrophobic interaction (HiTrap Octyl), (2) cation-exchange (HiTrap SP), (3) reverse phase (EC Nucleosil) and (4) affinity (BlueSepharose) chromatography. From these columns the activity was recovered in the (1) unbound material, and eluates (2 and 4) with 1M and 0,5M of NaCl, respectively, and (3) 83% acetonitrile. On the basis of electrophoresis analysis, the protocol produced a preparation comprised of only band corresponding a peptide with molecular weight (MW) of about 3-kDa, with minimum haemagglutination concentration of 50g/ml. To determine if this molecule arrangement had a quaternary structure arrangement, a feature of most known lectins, we submitted the preparation to a native electrophoresis. Because there was neither change in migration pattern nor presence of molecules of higher molecular mass, we suggested that T. hystrix peptide (Tabhys) is a peptide with MW of about 3-kDa. Since hevein, which is a most studied lectin-like peptide with antifungal activity, binds specifically to chitin, we performed an affinity chromatography in the chitin column with bark extract. We observed haemagglutination activity and the presence of peptide with MW of 3-kDa in the material bound to column and eluted with 0,1M acetic acid. Curiously, this peptide was not able to inhibit the growth of the fungus Trichophyton rubrum. Thereafter, when the purified peptide was used to stimulate murine spleen cells, we detected the expression of TGF-beta message, with a peak production obtained in cell stimulated with 37 ng/mL of Tabhys. In the current study, we isolated a peptide from crude extract of T. hystrix bark with haemagglutination activity, providing new perspectives in molecular and structural researches of peptide lectins.

Espécies do gênero Paracoccidioides spp são fungos patogênicos, termodimórficos, agentes etiológicos de doença endêmica em diversas regiões da América Latina. O indivíduo infectado desenvolve uma resposta específica que, quando associada à alta produção de TNF-? e IFN-?, favorece a resistência ao fungo. Componentes de alguns fungos patogênicos foram caracterizados, por técnicas de knockdown gênico, como importantes para a virulência fúngica. Nosso grupo identificou paracoccina (PCN) como um componente de leveduras de P. brasiliensis; trata-se de uma proteína com um domínio enzimático, dotado de atividade quitinase e um domínio lectínico, ligante de GlcNAc. PCN é dotada das seguintes propriedades: (a) contribui para o crescimento do fungo; (b) promove a adesão da levedura à matriz extracelular, por ligar-se à laminina; (c) interage com N-glicanas de TLR2 e TLR4 e promove ativação celular; (d) estimula macrófagos a produzirem mediadores pró-inflamatórios como IL- 12, TNF-? e NO; (e) promove a polarização M1 de macrófagos; (f) induz atividade fungicida em neutrófilos, bem como formação de NETs e supressão da apoptose, eventos que se mostraram dependentes da síntese de novo de proteínas pelos neutrófilos estimulados. Dada a relevância das atividades biológicas de PCN, promovemos recentemente o silenciamento do gene que codifica essa proteína, através de metodologia que usa RNA anti-sense e transformação mediada por Agrobacterium tumefaciens (ATMT). Uma vez PCN silenciada, a levedura perdeu a capacidade de fazer a transição para micélio e diminuiu a resistência à atividade fungicida de macrófagos. A infecção de camundongos com as cepas silenciadas, em comparação com as WT, causou doença de menor gravidade, com carga fúngica reduzida e baixa taxa de mortalidade. Essas observações sugerem de que PCN funcione como um fator de virulência em P. brasiliensis, que afeta a patogênese da infecção. Neste trabalho, ampliamos as ferramentas moleculares de manipulação do fungo e viabilizamos a superexpressão de PCN em leveduras de P. brasiliensis, tendo como objetivos estudar seu papel na virulência e na patogênese da infecção, bem como determinar os mecanismos responsáveis por tais atividades. A inoculação de leveduras que superexpressam PCN (ov-PCN) em camundongos causou doença pulmonar muito grave, em comparação à doença leve e moderada causada por leveduras silenciadas em PCN e leveduras WT, respectivamente. Nesse sentido, nossos esforços se dedicaram à busca dos mecanismos dos mecanismos através dos quais PCN influencia o curso da infecção experimental. Na tentativa de identificar o papel exercido pelo domínio quitinase da PCN, coletamos o sobrenadante de culturas de leveduras ov-PCN e WT. Partículas de quitina presentes nesses sobrenadantes foram purificadas por afinidade à lectina WGA (wheat germ agglutinin). Através de medida da área das partículas capturadas, através de microscopia eletrônica e aplicação do programa ImageJ, verificamos que a superexpressão de PCN resultou em clivagem mais eficiente da quitina da parede de leveduras, uma vez que apenas partículas muito pequenas (mediana das medidas = 2 nm2) foram detectadas, enquanto as áreas das partículas de quitina obtidas de leveduras selvagens (WT) forneceram mediana 3 vezes maior (6 nm2). As partículas de quitina foram então utilizadas para estimular macrófagos a produzirem citocinas. As obtidas de ov-PCN estimularam preponderantemente a secreção da citocina antiinflamatória IL-10, enquanto os macrófagos estimulados com partículas de leveduras WT produziram mais TNF-? e IL-1?, ambas de efeito pró-inflamatório. Esses resultados permitiram a identificação de um mecanismo importante para que a superexpressão de PCN se associe à ocorrência de doença pulmonar muito grave: o microambiente anti-inflamatório criado pelo estímulo de macrófagos por PCN leva ao desenvolvimento de resposta imune não protetora do tipo Th2 e lesões mais graves. Um segundo mecanismo foi identificado ao compararmos a resistência de leveduras ov-PCN e WT às respostas efetoras de macrófagos. A superexpressão de PCN associou-se à maior internalização das leveduras e maior resistência à atividade fungicida exercida por macrófagos. O estudo demonstra que diferentes níveis da expressão de uma quitinase (como PCN) levam à resistência a atividades antifúngicas de macrófagos e a diferentes graus de clivagem de quitina. A clivagem, por sua vez, pode alterar a estrutura da parede celular fúngica e a geração de fragmentos de quitina, cujos tamanhos e concentrações influenciam a produção de citocinas pelos macrófagos. Sob a ação de citocinas pró- ou antiinflamatórias liberadas pelos macrófagos e, consequentemente, a montagem de respostas adaptativas pode ser decisiva para haver suscetibilidade ou resistência à infecção por P. brasiliensis. Este trabalho proporciona um importante avanço no conhecimento do papel de quitinases na resposta anti-fúngica do hospedeiro. / Species of the genus Paracoccidioides spp are thermodymorphic fungi that cause a systemic disease, which is endemic in several regions of the Latin America. The infected individual develops a specific response that, when associated with the high production of TNF-? and IFN- ?, favors resistance to the fungus. Components of some pathogenic fungi were characterized by gene knockdown techniques as important the for fungal virulence. Our group has identified a component of P. brasiliensis, named Paracoccin (PCN); it is a bifunctional protein with an enzymatic domain, endowed with chitinase activity and a lectin domain, which binds GlcNAc and chitin, a GlcNAc polymers. PCN has the following properties: (a) contributes to the fungus growth; (b) promotes the yeast adhesion to the extracellular matrix, by binding to laminina glycans; (c) interacts with TLR2 and TLR4 N-glycans, which triggers cell activation; (d) stimulates macrophages to produce proinflammatory mediators, such as IL-12, TNF-? and NO; (e) promotes the M1 polarization of macrophages; (f) induces the neutrophils fungicidal activity, NETs formation, and suppression of neutrophils apoptosis, which are depending events on the de novo protein synthesis by neutrophils. Given the relevant biological activities exerted by PCN, we have performed recently the silencing of the gene that codes for this protein through a system that uses RNA anti-sense and Agrobacterium tumefaciens mediated transformation (ATMT). Once having the PCN gene silenced, yeast lost the ability of doing the transition to mycelium and decreased its resistance to macrophages fungicidal activities. Mice infection with PCN-silenced yeasts, compared to the infected with WT yeasts, exhibited a milder pulmonary disease with reduced fungal burden and low mortality rate. These observations suggest that PCN acts as a P. brasiliensis virulence factor that affects the pathogenesis of the fungal infection. In the present study, we expanded the molecular tools for the fungus manipulation and enabled the overexpression of PCN in P. brasiliensis yeasts, aiming to elucidate the PCN role in the fungus virulence and the infection pathogenesis, as well as determining the responsible mechanisms for the PCN activities. Inoculation of the PCN overexpressing yeasts (ov-PCN) into mice caused a very severe lung disease, compared to the mild and moderate diseases caused by PCN-silenced and WT yeasts, respectively. Then our efforts became dedicated to the search of mechanisms through which PCN influences the course of the experimental fungal disease. In an attempt to identify the role of the PCN chitinase domain, we harvested the supernatant of the ov-PCN and WT yeasts cultures. Chitin particles contained in the supernatants have been captured by affinity to the immobilized WGA (wheat germ agglutinin) lectin. By measuring through electron microscopy and application of the ImageJ program the area of the isolated chitin particles, we verified that the overexpression of PCN resulted in a more efficient cleavage of whole chitin molecules contained in the yeast cell wall, since only very small particles (median of the measurements = 2 nm2) were detected, while the the chitin particles areas obtained from WT-yeasts provided a median 3 fold higher (6 nm2). Then, the preparations of chitin particles were taken to stimulate macrophages to produce cytokines. The particles obtained from ov-PCN have stimulated preponderantly the secretion of the anti-inflammatory cytokine IL-10, whereas the macrophages stimulated with WT yeast particles have produced higher concentrations of TNF-? and IL-1?, which are known proinflammatory cytokines. These results allowed the identification of an important mechanism for the association of PCN overexpression to the occurrence of very severe pulmonary disease: the anti-inflammatory microenvironment created by the macrophages stimulation with PCN leads to the development of a non-protective Th2-type immune response and the more severe pulmonary injury. A second mechanism was identified as implicated in the severity of the lung disease associated to PCN overexpression. We compared the sensitivity of ov-PCN and WT yeasts to macrophages effector functions. PCN overexpressing yeasts were better internalized by macrophages and more resistant to the fungicidal activity of these cells, events that contributes for the high pulmonary fungal load verified in mice infected with ov-PCN yeasts. The study demonstrates that different levels of a chitinase (PCN) expression and enzymatic activity lead yeasts to change their sensitivity to macrophages antifungal activities as well as to different grades of chitin cleavage. The cleavage, in its turn, leads to changes in the structure of the fungal cell wall and generation of chitin fragments, whose sizes and concentrations influence the cytokines production by macrophages. Under the influence of pro-inflammatory or anti-inflammatory cytokines released by macrophages, the mounted adaptative responses can be decisive in conferring susceptibility or resistance to the P. brasiliensis infection. This study provides an important advance in the knowledge on the role of a chitinase in the host antifungal response.

Les interactions protéines-carbohydrates sont à la base de nombreux processus biologiques physiologiques aussi bien que pathologiques. Ces interactions incluent la synthèse et la dégradation enzymatique des oligosaccharides, la cohésion des tissus, l'immunité, le cancer ou encore l'infection bactérienne et virale. L'inhibition de ce type d'interaction par des molécules multivalentes synthétiques telles les glycopolymères, glycodendrimères, glycoclusters, etc. fait l'objet d'études importantes depuis plusieurs décennies. L'obtention de telles molécules pourrait permettre de développer de nouvelles thérapies qui pourraient palier notamment la multi-résistance aux antibiotiques. De plus, la détection de telles interactions par des méthodes simples et faciles à mettre en oeuvre permettrait une amélioration de la compréhension de ces phénomènes, ainsi que le diagnostic rapide de la présence de microorganismes. C'est dans ce contexte, que nous avons développé une nouvelle classe de composés glycosylés multivalents à coeur triazine. Ces glycoclusters de basse valence, ont la particularité de présenter une double fonctionnalité : l'inhibition d'interactions lectine-sucre par des effets de multivalence ainsi que la détection de ces interactions. Nous présentons dans ce manuscrit, la synthèse d'une nouvelle famille de glycoclusters polysulfurés à coeur triazine portant des épitopes saccharidiques tels que D-glucose, D-galactose, D-mannose, L-fucose, ainsi que leurs évaluations biologiques réalisées sur des lectines de Pseudomonas aeuriginosa. Nous avons ainsi mis en évidence la possibilité de reconnaître et de détecter les interactions lectine-sucre dans un premier temps par association d'un cluster mixte portant un fluorophore, et de façon plus sophistiquée, grâce à un système à géométrie variable incorporant dans le scaffold même un switch photochimique variant l'arrangement des sucres dans l'espace / Protein-carbohydrate interactions mediate a wide range of biochemical processes. Amongst these is the process of bacterial infection, which often proceeds through carbohydrate-binding lectins involved in biofilm formation. Even if the individual associations result from weak interactions, the assembly of multiple carbohydrate-protein interactions, typically more than additive, confers to the system the required specificity and avidity for their biological functions. In order to study this « glycocluster effects », a number of scaffold systems presenting multivalent carbohydrate ligands have been prepared in the literature. Dendrimers, polymers, peptides, calixarenes, to name a few, have been used as core molecules for the synthesis of multivalent glycoconjugates. The purpose of this work is to design new glycoclusters which exhibit dual functionality: the inhibition of carbohydrate-protein interactions via a multivalency effect; and detection of the interactions via fluorescence spectroscopy. A first generation of polysulfurated glycoclusters, organized around a heteroaromatic core, was synthesized using click chemistry reactions, which provided a family of highly soluble and readily accessible clusters. The glycoclusters were evaluated for their ligand-lectin interactions, multivalency effects, thermodynamic parameters, and abilty to modulate biofilm formation by Pseudomonas aeruginosa, a major causative agent of lung infections in cystic fibrosis patients. We describe a new family of ‘switchable glycoclusters’ based on photochromic behavior. They are designed to generate a modulated fluorescence signal as well as a defined change in the three-dimensional arrangement of the sugar epitopes, and may eventually provide significantly improved probes for studying the distribution, dynamics, interactions, and activities of specific lectins