Reniform nematode, Rotylenchulus reniformis is a semi-endoparasitic nematode infecting over 300 plant species including important fiber crops like cotton. Introgression of reniform nematode resistance from a distantly-related resistant species Gossypium longicalyx into cultivated upland species Gossypium hirsutum has been a challenge. An approach towards achieving nematode resistance in crop plants has been to identify candidate parasitism genes expressed in the nematode facilitating infection of host plant species, and silencing the same through reverse genetic approaches like RNAi. A cDNA library constructed from the sedentary female stage of reniform nematode revealed an EST coding for C-type lectins and occurring in high frequency. Identification and characterization of C-type lectins in reniform nematode is important in understanding the immune system of nematode and in planning strategies for the development of reniform nematode resistant cotton varieties. A total of 11 C-type lectin gene family members were identified across six life stages of reniform nematode, with each member expected to play a significant role in the development and parasitic establishment with the host plant. Conserved sites characteristic of C-type lectins found in other organisms have been identified in the C-type lectin genes in reniform nematode for binding of Ca+2 and mannose. The highest level of expression of C-type lectins was observed in the sedentary female stage indicating it to be possibly the most sensitive stage to microbial infection and so a likely stage to target for its management. The site of secretion of C-type lectins in the sedentary female stage could be identified by in situ hybridization as the hypodermal region of the exposed posterior body region which is not inserted into the host root tissue. Phylogenetic analyses of C-type lectin domains of various nematode groups placed the plant-parasitic nematodes in one group indicating the possibility of co-evolution and probably carrying out a similar function aiding in the establishment of parasitism with host plants. Our findings now extend the spectrum of known nematode C-type lectin genes and suggest that lectin activity might be a more general feature of parasitism which could be explored in better understanding the interactions occurring at the host-nematode and nematode-pathogen interfaces.

Members of the C-type lectin domain (CTLD) superfamily are metazoan proteins functionally important in glycoprotein metabolism, mechanisms of multicellular integration and immunity. This thesis presents the results of several computational and experimental studies of the CTLD structure, function and evolution.¶ Core structural properties of the CTLD fold were explored in a comparative analysis of the 37 distinct CTLD structures available publicly, which demonstrate significant structural conservation despite low or undetectable sequence similarity. Pairwise structural alignments of all CTLD structures were created with three different methods (DALI, CE and LOCK) and analysed manually and using a computational algorithm developed for this purpose. The analysis revealed a set of conserved positions and interactions, which were classified based on their role in CTLD structure maintenance.¶ The CTLD family is large and diverse. To organize and annotate the several thousand of known CTLD-containing protein sequences and integrate the information on their evolution, structure and function a local database and a web-based interface to it were developed. The software is written in Perl, is based on bioperl, bioperl-db and Apache::ASP modules, and can be used for collaborative annotation of any collection of phylogenetically related sequences.¶ Several studies of CTLD genomics were performed. In one such study, carried out in collaboration with the RIKEN structural genomics centre, CTLD sequences from the Caenorhabditis elegans genome were identified and clustered into groups based on similarity. The most representative members of the groups were then selected, which if characterized structurally would tell most about the C. elegans CTLDs and provide templates for homology modelling of all C. elegans CTLD structures.¶ In the other whole-genome study, the CTLD family in the puffer fish Fugu rubripes was analysed using the draft genome sequence. This work extended and complemented three genome-level surveys on human, C. elegans and D. melanogaster reported previously. The study showed that the CTLD repertoire of Fugu rubripes is very similar to that of mammals, although several interesting differences exist, and that Fugu CTLD-encoding genes are selectively duplicated in a manner suggesting an ancient large-scale duplication event. Another important finding was the identification of several new CTLDcps, which had mammalian orthologues not recognized previously.¶ CBCP, a novel CTLD-containing protein highly conserved between fish and mammals with previously unknown domain architecture, was predicted in the Fugu study based solely on ab initio gene models from the Fugu locus and cross-species genomic DNA alignments. To test if the prediction was correct, a full-length cDNA of the mouse CBCP was cloned, its tissue distribution characterized and untranslated regions determined by RACE. The full-length mCBCP transcript is 10 kb long, encodes a protein of 2172 amino acids and confirms the original prediction. The presence of a large N-terminal NG2 domain makes CBCP a member of a small but very interesting family of Metazoan proteins.

Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal β-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF,MIP-1α andMIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kB (IkB), enhancing NFkB nuclear translocation. These findings establish the first example of Syk-and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.

Pneumocystis jirovecii causes fungal pneumonia in immunocompromised patients and can be fatal if left untreated. The global mortality rate is estimated to be over 200 000 in AIDS patients. In non-AIDS patients there is an estimated mortality rate of 50 000 cases. This rate is increasing in developed countries, attributed to an increase in disorders which require immunotherapy. These include hematologic malignancies, organ transplant, inflammatory disorders and pre-existing lung disease. Immediate immunity is initiated by receptors that recognize pathogen associated molecular patterns on the surface of pathogenic fungi. Specifically, C-type lectin receptors (CLRs) have been shown to be the principal initiators of innate immune response during fungal infection. Limited studies have focused on the role of CLRs in Pneumocystis infection. Dectin1and Mincle have been shown to recognise Pneumocystis surface antigens with Dectin-1 recognizing β-glucans on the Pneumocystis cell wall leading to an effective immune response. However, the role of a newly described CLR, the Dendritic Cell Immunoactivating Receptor (DCAR) remains undefined. For this reason, we investigated the potential role of this receptor in a mouse model of Pneumocystis murina infection. Wild type and DCAR-deficient C57BL/6 mice were infected with P. murina organisms via intratracheal instillation. Fungal burden was measured in the lung using quantitative Polymerase Chain Reaction. DCAR-deficient mice had a significantly reduced burden compared to wild type mice at Day 7 and 14 post-infection. To identify the immune components involved in pathogen clearance in these mice we measured cellular recruitment and cytokine production at both early (48 hours) and late (7, 14 and 21 days) time points. Flow cytometry analysis showed an increase in alveolar macrophage, dendritic cells, inflammatory monocytes, eosinophils and T cell recruitment to the lung. While ELISA showed increased levels of IL-1β and IFN-γ at 48 hours, and later on in infection IL-1β and IL-12p40 levels were also elevated. Histology analysis determined the localization of the recruited cells, and v interestingly showed an increase in mucus production at day 21 in DCARdeficient mice. In conclusion, we have identified DCAR deficiency as a potential driver of protective immunity in mice during P. murina infection. This may be associated with increased levels of IL-1β in DCAR-deficient mice. Furthermore, DCAR may also be important in adaptive inflammatory response regulation, as DCAR-deficient mice have increased cellular recruitment and mucus production later in infection. The mechanism by which the deletion of this receptor affords these mice the ability to efficiently clear P. murina remains to be determined.

Natural killer cells play a significant role in the immune response against tumor and infected cells. NK cells express a wide variety of surface receptors, including NKRP1, a C-type lectin-like family of both activating and inhibitory receptors. Recently, ligands have been found for some of these previously orphan molecules, some of them lying within the same family. This is also the case of rat Clr-b as a cognitive ligand for rat NKRP1B. It has been shown that in rat, this inhibitory NKRP1B-Clr-b mutual receptor system is subverted by rat cytomegalovirus protein RCTL, a viral version of Clr-b, which serves as a decoy ligand for NK cells. The aim of my diploma thesis was cloning and production of the above mentioned C-type lectin-like proteins based on transient transfection of HEK293T cell line in a suspension culture. This expression system allows us not only to obtain proteins of our interest with a satisfactory yield but also in their native conformation, removing the need for time consuming and often fruitless refolding procedures required in case of using the E. coli expression system. Success was achieved in case of Clr-b and NKRP1B receptors from both WAG and SD strains. Proteins were purified using IMAC followed by gel filtration, identified by mass spectrometry and characterized by disulfide...

In the post smallpox era, vaccinia virus (VACV) has emerged as an important candidate vaccine vector. As yet, the binding receptors and entry mechanisms utilised by the two infectious forms, IMV and EEV, in dendritic cells (DCs) are unknown. We have investigated the interactions between VACV and C-type lectin receptors (CLRs) that are known to be utilised by many other viruses for binding and entry in DCs. Using a variety of CLR ligands and inhibitors we were unable to inhibit IMV or EEV binding to MDDCs and we conclude that they do not bind to CLRs. We have also investigated VACV entry in MDDCs and show that both IMV and EEV enter MDDCs via an endocytic pathway. Using a variety of drugs that inhibit cellular processes we found IMV and EEV entry to be actin- and calcium-dependent. EEV entry was also cholesterol- and energy-dependent, whereas IMV entry was only partially dependent on these factors. Both IMV and EEV colocalised with endolysosomal markers. This data suggests that EEV may enter DCs via caveolin-mediated endocytosis whereas IMV entry can occur via multiple complementary mechanisms, including endocytosis and fusion. Macropinocytosis may also constitute a minor route of entry for IMV as entry was partially inhibited by dimethyl amiloride and the virus colocalised with dextran. Finally we have provided a comprehensive flow cytometric analysis of Toll-like receptor (TLR) expression at the protein level in MDDCs and monocyte-derived Langerhans cells (MDLCs) as models for different myeloid DC subsets. We found TLR expression to be cell type-specific and MDDCs expressed the full repertoire of TLRs 1-9, including small amounts of TLR8 and TLR9 on the cell surface. The expression of these TLRs that recognise nucleic acids on the surface of cells may constitute an early warning system for signalling the presence of viral invaders that would normally subvert the function of DCs. We also found TLR expression in mature cells to be dependent on the nature of the maturation stimulus (lipopolysaccharide versus cytokine/prostaglandin cocktail) and VACV infection induced profound down-regulation of all TLRs. These findings will have important implications for the rational design of VACV-vectored vaccines.

In the post smallpox era, vaccinia virus (VACV) has emerged as an important candidate vaccine vector. As yet, the binding receptors and entry mechanisms utilised by the two infectious forms, IMV and EEV, in dendritic cells (DCs) are unknown. We have investigated the interactions between VACV and C-type lectin receptors (CLRs) that are known to be utilised by many other viruses for binding and entry in DCs. Using a variety of CLR ligands and inhibitors we were unable to inhibit IMV or EEV binding to MDDCs and we conclude that they do not bind to CLRs. We have also investigated VACV entry in MDDCs and show that both IMV and EEV enter MDDCs via an endocytic pathway. Using a variety of drugs that inhibit cellular processes we found IMV and EEV entry to be actin- and calcium-dependent. EEV entry was also cholesterol- and energy-dependent, whereas IMV entry was only partially dependent on these factors. Both IMV and EEV colocalised with endolysosomal markers. This data suggests that EEV may enter DCs via caveolin-mediated endocytosis whereas IMV entry can occur via multiple complementary mechanisms, including endocytosis and fusion. Macropinocytosis may also constitute a minor route of entry for IMV as entry was partially inhibited by dimethyl amiloride and the virus colocalised with dextran. Finally we have provided a comprehensive flow cytometric analysis of Toll-like receptor (TLR) expression at the protein level in MDDCs and monocyte-derived Langerhans cells (MDLCs) as models for different myeloid DC subsets. We found TLR expression to be cell type-specific and MDDCs expressed the full repertoire of TLRs 1-9, including small amounts of TLR8 and TLR9 on the cell surface. The expression of these TLRs that recognise nucleic acids on the surface of cells may constitute an early warning system for signalling the presence of viral invaders that would normally subvert the function of DCs. We also found TLR expression in mature cells to be dependent on the nature of the maturation stimulus (lipopolysaccharide versus cytokine/prostaglandin cocktail) and VACV infection induced profound down-regulation of all TLRs. These findings will have important implications for the rational design of VACV-vectored vaccines.

β-Glucans are structural components of fungal cell walls which are involved in the immune recognition of fungal pathogens and possess beneficial immunomodulatory activities in isolated form. Here we have developed a soluble chimeric form of the major mammalian β-glucan receptor, Dectin-1, and demonstrate its application for the detection and characterisation of soluble and insoluble β-glucans, including fungal particles, using ELISA, flow cytometric and fluorescence-based microscopy assays.

Les lectines de type C (CLRs) sont des récepteurs impliqués dans la reconnaissance d’oligosaccharides et principalement exprimés à la surface des cellules présentatrices d’antigène (APCs) et notamment des cellules dendritiques (DCs), véritable sentinelle de notre système immunitaire. Elles sont impliquées dans la reconnaissance de motifs spécifiques exprimés à la surface d’agents pathogènes et sont capables de stimuler le système immunitaire afin de déclencher une réponse adaptée. Ce rôle crucial joué par les CLRs dans l’équilibre de la réponse immunitaire confère aux interactions CLR/glycane des perspectives d’applications pharmaceutiques. L’objectif à long-terme du projet de recherche dans lequel cette thèse s’intègre consiste à utiliser ces CLRs pour modeler les réponses du système immunitaire. A cette fin, des néoglycoconjugués spécifiques de chaque CLR doivent être développés. Au cours de cette thèse, 9 CLRs ont été produits BDCA2, DC-SIGN, DC-SIGNR, dectin-1, dectin-2, langerin, LSECtin, MCL and Mincle. Différentes stratégies de production ont été testées en parallèle, incluant des techniques d’adressage au périplasme en vue d’obtenir des protéines solubles et fonctionnelles et de l’expression cytoplasmique, sous forme de corps d’inclusion suivie d’étapes de renaturation qui s’est révélé la plus efficace au final. Une stratégie permettant de construire des tétramères artificiels de CLRs, appelés TETRALEC, a été mise au point. Cet outil permettant le criblage et la caractérisation des lectines a été obtenu avec DC-SIGNR par un marquage spécifique de la lectine. Le complexe TETRALEC a été caractérisé au niveau structural et des tests fonctionnels ont été menés sur des puces à glycanes et des cellules pathogènes. La série de CLRs que nous avons produites a été utilisée pour cribler des puces à glycanes et à glycomimétiques. Ces études nous ont permis de mettre en évidence des interactions dépendantes de l’environnement du glycane et d’identifier de nouveaux glycanes ou glycomimétiques spécifiques de certains CLRs. En effet, de manière étonnante, plusieurs des CLRs testés sont capables, pour un glycane donné, de discriminer des isomères de position ouvrant ainsi de nouveaux questionnements sur la signification biologique de cette sélectivité. De plus des glycomimétiques reconnaissant préférentiellement dectin-2 par rapport à DC-SIGN, DCSIGNR et langerin ont été identifiés. Le choix des glycomimétiques et l’évaluation des étapes de leur optimisation ont été permis par diverses études biophysiques qui ont quantifié la force et la spécificité des interactions. Ceci a permis le développement d’un ligand optimisé sélectif de DC-SIGN. La co-cristallisation de la protéine avec ce ligand a révélé un intéressant mode de liaison qui amène également de nouvelles questions. Simultanément à l’optimisation de ligands monovalents, un premier pas a été réalisé vers la conception d’une molécule pour permettre une vaccination contre le cancer médiée par les CLRs. Les résultats de SPR ont identifié des candidats potentiellement intéressants et des tests biologiques préliminaires ont été réalisés. / C-type Lectin Receptors (CLRs) are carbohydrate-binding proteins mainly expressed on Antigen Presenting Cells (APCs), including dendritic Cells (DCs), the sentinel of the innate immune system. They recognize pathogens or damaged cells by interacting with glycan features and the encounter between the CLR and its ligand constitutes a necessary step for the activation of the adaptive immune system. This crucial role played by CLRs in the balance of immune responses offers to CLR-glycan interactions pharmaceutical applications. The long-term objective of the research project in which this PhD is included is to use these CLRs as modulators in order to tailor the immune system responses. To do so, neoglyco-conjugates selective to each individual CLR have to be developed.Nine different CLRs were produced in this work: BDCA2, DC-SIGN, DC-SIGNR, dectin-1, dectin-2, langerin, LSECtin, MCL and Mincle.Several approaches have been explored in parallel for CLR production, ranking from bacterial periplasmic targeting, aiming to express soluble and functional protein, to inclusion bodies production into the bacterial cytoplasm, with subsequent protein refolding. Our collection of CLRs were used to screen glycan and glycomimetic arrays, highlighting context-dependent binding and identifying natural ligands or glycomimetics selective to each CLRs. Thus, several CLRs were surprisingly able to differentiate between positional isomers of a given N-Glycan, which opens new questions regarding the biological significance. Moreover, glycomimetics with a selectivity towards dectin-2 over DC-SIGN, DC-SIGNR and langerin CLRs have been identified.To guide the choice of the glycomimetics and estimate their optimisation, diverse biophysical studies were performed to evaluate the strength and specificity of the interaction. This enabled the development of an ultimate ligand selective towards DC-SIGN. A co-crystallised structure of the protein with this ligand revealed an interesting binding mode that also opens new questions.Simultaneously to monovalent ligand optimization, a first step towards the design of a highly defined molecule for cancer vaccination by CLR targeting was made. SPR results revealed potential candidates to exploit and preliminary biological assays were performed. Finally, a strategy for tetrameric lectin engineering as been explored, termed TETRALEC. This tool for screening and lectin characterization, has been obtained with one the lectin of the study, DC-SIGNR, by a site-specific labelling of the lectin. The TETRALEC complex was structurally characterised and functional assays were performed on glycan array and pathogen cells.

Le trypanosome sud américain, Trypanosoma cruzi, transmis par un insecte hématophage de type triatome est le protozoaire connus pour causer la maladie de Chagas chez l’Homme. Le cycle de vie de ce parasite alterne à la fois sur le type d’hôte, insecte ou hôte vertébré, et sur la forme :trypomastigote pour la forme quiescente et amastigote et épimastigote pour les formes prolifératives. Concernant la forme, seuls les parasites épimastigotes évoluent et prolifèrent dans le tube digestif des triatomes et possèdent un système endocytaire actif nécessaire à leur besoin énergétique. Toutefois, cette endocytose est restreinte à deux sites membranaires, la poche flagellaire et le cytostome, à partir desquels se créent des cargos endocytaires. Ces cargos endocytaires fusionnent ensuite avec un réseau vésiculaire endosomal qui délivre son contenu dans des réservosomes, compartiments similaires aux lysosomes.Chez le trypanosome africain (Trypanosoma brucei brucei), l’endocytose ne se réalise qu’au niveau de la poche flagellaire. Certaines protéines appartenant à cette voie endocytaire sont modifiées par de longues chaines de résidus poly-N-acétyllactosamine (pNAL) de manière post-traductionnelle. Initialement, il a été proposé que ces résidus puissent agir en tant que signal de tri dans le processus d’endocytose chez ces parasites.En nous basant sur les travaux qui ont été réalisés chez le trypanosome africain, nous nous sommes proposés d’approfondir les connaissances sur la voie endocytaire du trypanosome sud américain (Trypanosoma cruzi) qui est beaucoup moins étudié. Pour ce faire, à l’aide de deux lectines, la tomatolectine et la lectine de Griffonia simplicifolia qui présentent respectivement une affinité pour les résidus pNAL et les résidus N-acétylglucosamine (GlcNAc) en fin de chaine, nous avons pu enrichir et caractériser par LC-MS², 173 glycoprotéines putatives dont plus de 13% sont localisées dans la voie endocytaire. Parmi les protéines identifiées, en plus des nombreuses hydrolases lysosomiales, nous avons pu identifier une lectine de type C localisée dans la partie antérieure des parasites, au niveau des principaux sites endocytaires. Cette dernière possédant de nombreux résidus en commun avec les récepteurs de type scavengers, elle pourrait donc jouer un rôle important dans la fixation et l’endocytose de certains nutriments.Nos travaux ont ainsi permis d’établir que similairement aux trypanosomes africains, Trypanosoma cruzi possèdent des glycoprotéines modifiées par des N-glycanes contenant des pNAL. Nos travaux ont également permis d’établir que ces résidus s’associent préférentiellement aux glycoprotéines de la voie endocytaire (au niveau du cytostome et du réservosome) de la forme épimastigote. L’ensemble des résultats obtenus durant cette thèse tendent à montrer que les résidus pNAL des glycoprotéines présentes dans la voie endocytaire ont été conservées entre les deux parasites étudiés (Trypanosoma cruzi et Trypanosoma brucei brucei). / Doctorat en Sciences / info:eu-repo/semantics/nonPublished