A crotoxina (CTX) é a principal toxina do veneno de serpentes Crotalus durissus terrificus, caracterizada como <font face=\"Symbol\">b-neurotoxina com ação principalmente pré-sináptica, inibindo a liberação de acetilcolina nas junções neuro-musculares. No entanto, vários dados de literatura têm mostrado que, adicionalmente à atividade neurotóxica, a CTX apresenta efeitos imunomodulatórios antiinflamatório, analgésico e anti-tumoral. Apesar destes dados, não foi ainda determinada a ação desta toxina sobre modelos experimentais de alergia, como por exemplo, a inflamação alérgica pulmonar. Este trabalho teve por objetivo investigar o possível efeito inibitório da CTX sobre a gênese e o desenvolvimento de resposta inflamatória pulmonar induzida em ratos pela sensibilização com Ovalbumina (OVA), bem como a sua possível interferência na alergia já estabelecida. Em conjunto, os resultados sugerem que a CTX interfere com a inflamação pulmonar acarretada pela sensibilização e desafio por OVA, modulando mecanismos indutores e efetores da resposta alérgica. / Crotoxin (CTX) is the main toxin of the venom of South American rattlesnake Crotalus durissus terrificus, corresponding to 65% of the protein content of the crude venom. CTX is a <font face=\"Symbol\">b-neurotoxin that displays mainly pre-synaptic action, inhibiting the release of acetylcholine in the neuromuscular junctions. However, some experimental and clinical data have shown that, in addition to the neurotoxic activity, CTX displays immunomodulatory, antiinflammatory, analgesic and anti-tumoral properties. Despite these observations, the possible inhibitory action of this toxin in experimental models of allergy, such as lung allergic inflammation, was not determined yet. Therefore, this study aims to investigate the inhibitory effect of CTX on the genesis and development of lung inflammation induced in rats by sensitization with Ovalbumin (OVA). Together, these results suggest that CTX inhibits lung inflammation induced by sensitization and challenge with OVA, interfering with the initiators and effectors mechanisms of allergic inflammation.

Neste trabalho avaliamos se as Natterinas (NTR), proteínas majoritárias presentes no veneno do peixe peçonhento T. nattereri, são as responsáveis pela a ação inibitória do recrutamento de leucócitos durante a inflamação e os mecanismos moleculares envolvidos neste processo. Utilizando análises de microscopia intravital tratamos os animais com as NTR e 6 h depois induzimos o rolamento de leucócitos com KC, LPS ou agonista de PAR-4 sobre a microcirculação. Os dados mostraram que o tratamento com as NTR inibem o rolamento e a migração de leucócitos induzido pela KC e pelo LPS. Além disso, o efeito inibitório das NTR depende parcialmente da sua atividade proteásica e não está relacionado com a clivagem proteolítica da quimiocina KC ou com a indução de reguladores endógenos como IL-10, corticóides, a enzima HO-1 ou IL-1Ra. O efeito inibitório parece ser dependente da ativação da sinalização TLR/MyD88 e PI3K e das proteínas serina/treonina fosfatases, contribuindo de forma significativa na sobrevida de animais induzidos a endotoxemia por LPS. / In this work we evaluated whether Natterins (NTR) the majority proteins present in the venom of Thalassophryne nattereri venomous fish are responsible for the inhibitory action of leukocyte recruitment during inflammation and the molecular mechanisms involved in this process. Using intravital microscopy analysis we treated the animals with NTR and 6 h after we induced the leukocyte rolling by topical of KC, LPS or agonist of PAR-4 on the microcirculation. The data showed that treatment with NTR inhibited the leukocyte rolling and migration induced by LPS and KC. Moreover, the inhibitory effect of NTR depends partly on its proteinase activity and is not related to the proteolytic cleavage of the chemokine or KC nor with the induction of endogenous regulators such as IL-10, corticoids, the HO-1 or IL-1Ra. The inhibitory effect is dependent on the activation of PI3K and TLR/MyD88 signaling and protein serine/threonine phosphatases, contributing significantly to the survival of animals endotoxemia induced by LPS.

Les maladies des plantes causées par les champignons sont un problème majeur en agriculture. Pour les contrôler, les gènes de résistance (R) qui permettent de développer des variétés de plantes résistantes sont des éléments clés. La majorité des gènes R codent pour des protéines NLRs caractérisées par la présence d'un domaine de liaison aux nucléotides (NB-ARC) et un domaine de répétitions riches en leucines (LRR). Ces protéines agissent comme des récepteurs immunitaires intracellulaires et reconnaissent des facteurs de virulence des agents pathogènes appelés effecteurs. Les champignons phytopathogènes possèdent de vastes répertoires d'effecteurs qui contiennent centaines de protéines sécrétés, de petites tailles et sans similarités de séquence entre elles.La première question abordée dans ma thèse concerne l’origine de l'immense diversité des effecteurs fongiques. Une analyse structurale a identifié une famille d’effecteurs de séquences différentes mais qui possèdent une structure conservée. Cette famille a été appelée MAX-effectors (Magnaporthe Avrs and ToxB like) et elle est particulièrement importante chez Magnaporthe oryzae, l'agent causal de la pyriculariose du riz. Par des analyses d'expression, j'ai confirmé que la majorité des effecteurs MAX de M. oryzae sont spécifiquement exprimés durant la phase précoce de l'infection, suggérant une fonction importante durant la colonisation de la plante. Les effecteurs MAX constituent la première famille d'effecteurs fongiques définis par leur structure. Cette étude apporte donc de nouvelles pistes pour l'identification d'effecteurs chez les champignons et contribue à une meilleure compréhension de l'évolution des effecteurs. En effet, le scénario observé chez les effecteurs MAX suggère que beaucoup d’effecteurs fongiques appartiennent à un nombre restreint de familles d'effecteurs définies par leur structure. La seconde question que j’ai abordée durant ma thèse est le mécanisme moléculaire de la reconnaissance des effecteurs par les NLRs. J'ai abordé cette question en étudiant la reconnaissance de l'effecteur AVR-Pia par le couple de NLRs RGA4/RGA5. Des travaux précédents ont montré que RGA5 agit comme récepteur et se lie directement à AVR-Pia tandis que RGA4 agit comme élément de signalisation constitutivement actif, qui, en absence de l’agent pathogène, est réprimé par RGA5. Un domaine de RGA5, normalement absent chez les protéines NLR et similaire à la chaperonne du cuivre ATX1 (domaine RATX1), interagit physiquement avec AVR-Pia. Il a été suggéré que ce domaine RATX1 puisse agir comme un leurre de la cible de virulence d’AVR-Pia. Ce leurre, intégré dans la structure de RGA5, permettrait de « piéger » l’effecteur par interaction directe et jouerait donc un rôle crucial dans sa reconnaissance spécifique. Grâce à une analyse structurale détaillée d’AVR-Pia j’ai pu confirmer le rôle central de l'interaction AVR-Pia-RATX1 dans la reconnaissance de cet effecteur ce qui conforte le modèle du « leurre intégré ». De plus, j’ai caractérisé la surface d'interaction avec laquelle AVR-Pia lie le domaine RATX1. De plus, j'ai détecté des interactions entre AVR-Pia et d'autres parties de RGA5, indépendantes du domaine RATX1, notamment les domaines NB-ARC et LRR. Ceci a permis de développer un modèle qui explique comment la liaison d’un effecteur à un récepteur NLR comportant un leurre intégré par différentes interactions indépendantes conduit à une reconnaissance très sensible et spécifique qui est peu affectée par des mutations ponctuelles de l’effecteur. En résumé, cette étude a produit des connaissances nouvelles sur la fonction des récepteurs des plantes de type NLRs et sur leur capacité à reconnaitre des effecteurs. Ceci contribue à une meilleure compréhension du système immunitaire des plantes, ce qui est un élément important pour l’obtention de cultures durablement résistantes aux maladies / Plant diseases caused by fungi constitute a worldwide threat to food security and disease resistance (R) genes that allow to breed resistant crops are key elements for efficient disease control. The vast majority of R genes code for NLR multi domain proteins characterized by nucleotide-binding and leucine-rich repeat domains and acting as intracellular immune receptors for pathogen-secreted virulence factors termed effectors. Phytopathogenic fungi possess huge effector repertoires that are dominated by hundreds of sequence-unrelated small secreted proteins. The first question I addressed in my PhD thesis is: how is the tremendous diversity of fungal effectors generated? A structural analysis had identified the family of sequence-unrelated but structurally conserved MAX-effectors (Magnaporthe Avrs and ToxB like) that has expanded specifically in Magnaporthe oryzae the causal agent of rice blast disease. By expression analysis, I confirmed that the majority of M. oryzae MAX-effectors are expressed specifically during early infection suggesting important functions during host colonization. MAX effectors are the first structurally defined family of effectors in fungi and this study gives therefore news clues for the identification of candidate effectors in fungi and constitutes a crucial step towards a better understanding of effector evolution. In fact, the scenario observed for MAX-effectors leads to the hypothesis that the enormous number of sequence-unrelated fungal effectors belong in fact to a restricted set of structurally conserved effector families.The second question I investigated in my PhD thesis is: what are the molecular mechanisms of effector recognition by NLR immune receptors? I addressed this question by studying recognition of the M. oryzae effector AVR-Pia by the rice NLR pair RGA4/RGA5. Previous work has shown that RGA5 acts as a receptor that binds directly to AVR-Pia while RGA4 acts as a constitutively active signaling protein that is, in the absence of pathogen, repressed by RGA5. This functional interaction involves formation of an RGA4/RGA5 receptor complex. By protein-protein interaction studies, I showed that complex formation involves interactions between the RA4 and RGA5 NB-ARC and LRR domains, in addition to previously identified interactions between the coiled-coil domains. AVR-Pia recognition seems not to induce dissociation of the RGA4/RGA5 complex but a ternary RGA4/RGA5/AVR-Pia complex could also not be detected consistently. How effector recognition is translated into receptor complex activation remains therefore to be elucidated in more detail in the future. Previous work has shown that a domain of RGA5 normally not present in NLRs and related to the copper chaperone ATX1 (RATX1 domain) interacts physically with AVR-Pia and may be crucial for effector recognition. The RATX1 domain was hypothesized to mimic the true host targets of AVR-Pia leading to the development of the ‘integrated decoy’ model that states that unconventional domains in NLRs act as decoys in the recognition of effector proteins. By detailed structure-informed analysis of AVR-Pia, I could confirm the pivotal role of the AVR-Pia-RATX1 interaction for effector recognition lending important support to the integrated decoy model. In addition, I could precisely characterize the interaction surface with which AVR-Pia binds to the RGA5 RATX1 domain. Finally, I detected interactions of AVR-Pia with other parts of RGA5, in particular the NB-ARC and the LRR domains. Based on these results, I developed a model that explains how such binding to several independent sites in NLRs leads to high overall affinity and robust effector recognition that is resilient to effector mutations. Taken together, this study provides important novel insight into NLR function and effector recognition and contributes by this to a better understanding of plant immunity which is crucial for generating durable disease resistance in crops.