Les problèmes de santé publique causés par des bactéries comme Mycobacterium tuberculosis et Salmonella enterica sont amplifiés par l’émergence de souches multi-résistantes aux antibiotiques, d’où la nécessité de mettre au point rapidement de nouvelles stratégies antimicrobiennes. La découverte récente de peptides membranaires bactériens qui ont la propriété de réguler des partenaires protéiques membranaires impliqués dans la virulence bactérienne permet d’envisager des stratégies « anti-virulence » innovantes.Mon travail a porté sur l’étude de deux peptides membranaires, KdpF et MgtR, chez M. bovis BCG et S. typhimurium, qui sont des pathogènes intracellulaires capable de survivre dans les macrophages. La surexpression de KdpF chez M. bovis BCG et de MgtR chez S. typhimurium entraîne une diminution de la survie bactérienne dans les macrophages et je me suis attachée à en comprendre la mécanistique et à étudier l’effet biologique de peptides KdpF et MgtR synthétiques.Ces travaux m’ont permis de mettre en évidence que les peptides KdpF endogène et synthétique augmentent la sensibilité des bactéries au stress azoté. Nous proposons que cela soit en lien avec la déstabilisation de la nitrate réductase. De plus et pour la première fois, j’ai démontré que le peptide synthétique KdpF est biologiquement actif et mime les propriétés anti-virulence du peptide endogène KdpF dans les macrophages.J’ai montré que le peptide synthétique MgtR réduit la survie intra-macrophagique de S. typhimurium, et j’ai identifié un peptide variant ayant une action encore plus efficace. Ces deux peptides entraînent une diminution du taux de plusieurs protéines de la membrane interne, dont certaines sont des facteurs de virulence reconnus.De plus, j’ai contribué à une étude visant à tester l’effet anti-virulence du peptide MgtR de Salmonella chez les mycobactéries. En utilisant un système hétérologue, j’ai montré que l’ajout du peptide synthétique MgtR peut empêcher la dimérisation de la protéine MgtC de M. tuberculosis et également favoriser sa déstabilisation.En conclusion, l’ensemble de mes résultats permet de mieux comprendre le mécanisme d’action des peptides membranaires KdpF et MgtR et montre que les peptides synthétiques KdpF et MgtR possèdent des propriétés anti-virulence prometteuses, point essentiel pour valider la pertinence de ces molécules dans le cadre de stratégies innovantes alternatives ou complémentaires aux antibiotiques. / The public health problems caused by bacteria such as Mycobacterium tuberculosis and Salmonella enterica are amplified by the emergence of multi-drug resistant strains, leading to the demand of new antimicrobial strategies. The recent discovery of bacterial membrane peptides that have regulatory effects on membrane protein involved in bacterial virulence makes it possible to envisage innovative "anti-virulence" strategies.My work was focused on the study of two membrane peptides, KdpF and MgtR, in M. bovis BCG and S. typhimurium respectively, which are intracellular pathogens capable of surviving in macrophages. Overexpression of KdpF in M. bovis BCG and MgtR in S. typhimurium, has already been reported to decrease bacterial survival in macrophages. Therefore, I focused on the study of mechanism and biological effect of synthetic KdpF and MgtR peptides.This work allowed me to conclude that endogenous and synthetic KdpF peptides increase the sensitivity of bacteria to nitrosative stress. I propose that this is related to the destabilization of the enzyme nitrate reductase. In addition and for the first time, I have demonstrated that the synthetic KdpF peptide is biologically active and mimics the anti-virulence properties of the endogenous KdpF peptide in macrophages.In the next part of my study, I have shown that the synthetic MgtR peptide reduces the intramacrophage survival of S. typhimurium and I have identified a variant peptide with even stronger action. These two peptides cause a decrease in the level of several proteins of the inner membrane, some of which are recognized as virulence factors.In addition to that, I tested the anti-virulence effect of Salmonella MgtR peptide in mycobacteria. Using a heterologous system, I have shown that the addition of the synthetic MgtR peptide can prevent the dimerization of M. tuberculosis MgtC protein and also promote its destabilization.In conclusion, all my results provide a better understanding of the mechanism of action of KdpF and MgtR membrane peptides and show that the synthetic peptides KdpF and MgtR have promising anti-virulence properties. This is an essential factor to validate the relevance of these molecules as part of innovative, alternative or complementary strategies to antibiotics.

Orientador: Reinaldo Marchetto / Coorientador: Edson Crusca Junior / Banca: Saulo Santesso Garrido / Banca: José Luiz de Souza Lopes / Resumo: O peptídeo antimicrobiano Pantinina-3 (P3) possui 13 resíduos e é derivado do veneno do escorpião africano Pandinus imperator. Ele apresenta alta atividade antimicrobiana contra bactérias Gram-positivas, principalmente Enterococcus resistente à vancomicina (VRE-S13) in vitro. Entretanto, este peptídeo também possui atividade hemolítica. Neste contexto, foram desenhados e sintetizados cinco análogos de P3 a partir de substituições pontuais de resíduos específicos por um resíduo de lisina, os quais foram denominados L2K, I5K, N7K, I9K e L13K. O objetivo da criação destes análogos foi estudar a relação de estrutura e atividade do peptídeo P3, a fim de investigar o efeito da modificação de suas características biofísicas em sua atividade biológica. Para isso, os peptídeos foram sintetizados por meio da técnica de síntese em fase sólida (SPFS), purificados por cromatografia líquida de alta eficiência em coluna de fase reversa (CLAE-FR) e identificados por espectrometria de massas (SM). Os peptídeos purificados foram testados quanto à sua atividade biológica e foi verificado que o análogo L2K apresentou atividade somente para a bactéria Gram-negativa testada, o análogo N7K mostrou ser mais ativo que P3 tanto para Gram-positivas quanto para Gram-negativas e os outros análogos não apresentaram atividade antimicrobiana. Foi avaliada também a atividade hemolítica dos peptídeos, e foi verificado que o único análogo que apresentou atividade contra eritrócitos foi N7K. A hidrofobicidade d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The antimicrobial peptide Pantinin-3 (P3) has 13 residues and is derived from the venom of the African scorpion Pandinus imperator. It has high antimicrobial activity against Gram-positive bacteria, mainly vancomycin-resistant Enterococcus (VRE-S13) in vitro. However, this peptide also has hemolytic activity. In this context, five P3 analogs were designed and synthesized from point substitutions of specific residues by a lysine residue, which were named L2K, I5K, N7K, I9K and L13K. The objective of the creation of these analogues was to study the structure and activity relationship of the P3 peptide in order to investigate the effect of the modification of its biophysical characteristics on its biological activity. For this, the peptides were synthesized using the solid-phase peptide synthesis technique (SPPS), purified by high performance liquid chromatography on a reverse phase column (HPLC-RF) and identified by mass spectrometry (MS). The purified peptides were tested for their biological activity and it was found that the L2K analog showed activity only for the Gram-negative bacteria tested, the N7K analog showed to be more active than P3 for both Gram-positive and Gram-negative and the others did not present antimicrobial activity. The hemolytic activity of the peptides was also evaluated, and it was verified that the only analog that showed activity against erythrocytes was N7K. The hydrophobicity of the peptides was analyzed by the retention time obtained by HPLC-RF an... (Complete abstract click electronic access below) / Mestre

As técnicas espectroscópicas utilizam a interação entre luz e matéria como forma de obter informações das características moleculares de um sistema. Os diversos níveis de energia manifestam-se em bandas espectrais que ao serem interpretadas fornecem características sobre estrutura, orientação e interações a nível molecular. O presente trabalho explorou as diversas facetas espectroscópicas, aliadas a técnicas biofísicas/bioquímicas, no intuito de compreender melhor dois tipos de biomoléculas importantes para a maquinaria dos seres vivos: proteínas e peptídeos. O citocromo P450 é uma enzima do tipo monooxigenase e constitui uma das maiores superfamílias de proteínas. Essa hemoproteína foi assim nomeada devido à sua característica absorção na região da Banda de Soret (450 nm), sendo esta uma particularidade natural muito utilizada em estudos por espectroscopia. Além disso, existe interesse de estudar esta família de proteínas em virtude de suas variadas funções metabólicas e biossintéticas, nos mais diversos organismos, como catalisação de esteróides, ácidos graxos, fármacos, carcinógenos químicos e metabólitos de plantas. Em especial, para a P450 de Streptomyces clavuligerus (P450CLA), ainda não se sabe ao certo como e com quais moléculas interage, e como funciona o mecanismo utilizado pela proteína para atuar em vias de síntese como a do ácido clavulânico, importante composto terapêutico. Paralelo ao paradigma de interação de uma proteína e potenciais ligantes, o entendimento dos mecanismos de interação entre peptídeos e membranas lipídicas também são de suma importância para uma melhor compreensão dos sistemas biomoleculares. Peptídeos ativos na membrana desempenham funções fundamentais no sistema de defesa de diversos organismos e decifrar os mecanismos de como essas biomoléculas agem quando inseridas em bicamadas lipídicas pode auxiliar, por exemplo, no desenvolvimento de terapias seguras e eficientes contra doenças degenerativas. Desta forma, aproveitamos as características espectroscópicas naturais de ambas as moléculas para serem empregadas em técnicas de absorção UV/vis, Dicroísmo Circular Eletrônico e Magnético, Ressonância Paramagnética Eletrônica e Ressonância Magnética Nuclear, auxiliados por técnicas termodinâmicas e de fluorescência, de modo a explorar a interação da luz com a matéria, sem interferências de sondas externas, dando enfoque às alterações de estrutura e orientação, nas mais variadas formas de interação entre moléculas de sistemas biológicos. / Spectroscopic techniques use the interaction between light and matter as a way for obtaining information about molecular characteristics of a system. The many energy levels manifest themselves in spectral bands, which when are interpreted, it provides characteristics about structure, orientation and interactions at the molecular level. The present study explored the various spectroscopic facets, allied to biophysical/biochemical techniques, in order to understand better two important biomolecules types for living beings machinery: proteins and peptides. Cytochrome P450 is a monooxygenase-like enzyme and it belongs to one of the largest proteins superfamilies. The hemoprotein received this name due its unique spectral absorption in Soret Band region (450 nm), a natural particularity widely used in spectroscopic studies. In addition, there is interest in studying this protein family by virtue of their several metabolic and biosynthetic functions in the most diverse organisms, such as steroids, fatty acids, drugs, chemical carcinogens and plant metabolites. In particular, regarding P450 from Streptomyces clavuligerus (P450CLA), it is still unclear how and which molecules it interacts with, and how the mechanism used by the protein to act in synthesis pathways such as clavulanic acid, an important therapeutic compound. Parallel to the interaction paradigm between proteins and potential ligands, the interaction mechanisms understanding between peptides and lipid membranes are also of paramount importance for a better understanding of the biomolecular systems. Active membrane peptides play key roles in the defense system of various organisms and to decipher the mechanisms how these biomolecules act when inserted into lipid bilayers, for example in the development of safe and efficient therapies against degenerative diseases. This way, we take advantage of the natural spectroscopic characteristics of both molecules to be used in UV/vis absorption techniques, Electronic and Magnetic Circular Dichroism, Electronic Paramagnetic Resonance and Nuclear Magnetic Resonance, aided by thermodynamic and fluorescence techniques, in order to explore the interaction of light with matter, without interference from external probes, focusing on changes in structure and orientation, in the most varied forms of interaction between biological systems molecules.

As técnicas espectroscópicas utilizam a interação entre luz e matéria como forma de obter informações das características moleculares de um sistema. Os diversos níveis de energia manifestam-se em bandas espectrais que ao serem interpretadas fornecem características sobre estrutura, orientação e interações a nível molecular. O presente trabalho explorou as diversas facetas espectroscópicas, aliadas a técnicas biofísicas/bioquímicas, no intuito de compreender melhor dois tipos de biomoléculas importantes para a maquinaria dos seres vivos: proteínas e peptídeos. O citocromo P450 é uma enzima do tipo monooxigenase e constitui uma das maiores superfamílias de proteínas. Essa hemoproteína foi assim nomeada devido à sua característica absorção na região da Banda de Soret (450 nm), sendo esta uma particularidade natural muito utilizada em estudos por espectroscopia. Além disso, existe interesse de estudar esta família de proteínas em virtude de suas variadas funções metabólicas e biossintéticas, nos mais diversos organismos, como catalisação de esteróides, ácidos graxos, fármacos, carcinógenos químicos e metabólitos de plantas. Em especial, para a P450 de Streptomyces clavuligerus (P450CLA), ainda não se sabe ao certo como e com quais moléculas interage, e como funciona o mecanismo utilizado pela proteína para atuar em vias de síntese como a do ácido clavulânico, importante composto terapêutico. Paralelo ao paradigma de interação de uma proteína e potenciais ligantes, o entendimento dos mecanismos de interação entre peptídeos e membranas lipídicas também são de suma importância para uma melhor compreensão dos sistemas biomoleculares. Peptídeos ativos na membrana desempenham funções fundamentais no sistema de defesa de diversos organismos e decifrar os mecanismos de como essas biomoléculas agem quando inseridas em bicamadas lipídicas pode auxiliar, por exemplo, no desenvolvimento de terapias seguras e eficientes contra doenças degenerativas. Desta forma, aproveitamos as características espectroscópicas naturais de ambas as moléculas para serem empregadas em técnicas de absorção UV/vis, Dicroísmo Circular Eletrônico e Magnético, Ressonância Paramagnética Eletrônica e Ressonância Magnética Nuclear, auxiliados por técnicas termodinâmicas e de fluorescência, de modo a explorar a interação da luz com a matéria, sem interferências de sondas externas, dando enfoque às alterações de estrutura e orientação, nas mais variadas formas de interação entre moléculas de sistemas biológicos. / Spectroscopic techniques use the interaction between light and matter as a way for obtaining information about molecular characteristics of a system. The many energy levels manifest themselves in spectral bands, which when are interpreted, it provides characteristics about structure, orientation and interactions at the molecular level. The present study explored the various spectroscopic facets, allied to biophysical/biochemical techniques, in order to understand better two important biomolecules types for living beings machinery: proteins and peptides. Cytochrome P450 is a monooxygenase-like enzyme and it belongs to one of the largest proteins superfamilies. The hemoprotein received this name due its unique spectral absorption in Soret Band region (450 nm), a natural particularity widely used in spectroscopic studies. In addition, there is interest in studying this protein family by virtue of their several metabolic and biosynthetic functions in the most diverse organisms, such as steroids, fatty acids, drugs, chemical carcinogens and plant metabolites. In particular, regarding P450 from Streptomyces clavuligerus (P450CLA), it is still unclear how and which molecules it interacts with, and how the mechanism used by the protein to act in synthesis pathways such as clavulanic acid, an important therapeutic compound. Parallel to the interaction paradigm between proteins and potential ligands, the interaction mechanisms understanding between peptides and lipid membranes are also of paramount importance for a better understanding of the biomolecular systems. Active membrane peptides play key roles in the defense system of various organisms and to decipher the mechanisms how these biomolecules act when inserted into lipid bilayers, for example in the development of safe and efficient therapies against degenerative diseases. This way, we take advantage of the natural spectroscopic characteristics of both molecules to be used in UV/vis absorption techniques, Electronic and Magnetic Circular Dichroism, Electronic Paramagnetic Resonance and Nuclear Magnetic Resonance, aided by thermodynamic and fluorescence techniques, in order to explore the interaction of light with matter, without interference from external probes, focusing on changes in structure and orientation, in the most varied forms of interaction between biological systems molecules.

Les protéines membranaires sont particulièrement riches en acides aminés aromatiques, tels que le tryptophane (W). On retrouve cette originalité dans beaucoup de peptides antimicrobiens et dans les protéines de fusion virales. La glycoprotéine de l'enveloppe de HIV-1, gp41, en est un exemple. Manifestement, les résidus W sont impliqués dans la perturbation des membranes et la formation des pores. L'objectif de ce travail est d'étudier le rôle des résidus W dans de telles activités en utilisant l'optique non linéaire. Pour cela, nous avons préalablement déterminé l'hyperpolarisabilité (le potentiel non linéaire) du W par la diffusion Hyper Raleigh (HRS). Puis nous avons montré une évolution de la réponse non linéaire de petits peptides synthétiques en fonction du nombre croissant de leurs résidus W. Ces résultats ont permis de suivre l'implication des tryptophanes de deux peptides K3W et gp41W, lors de leurs interactions avec des monocouches lipidiques à l'interface air-eau par la génération de second harmonique (SHG). D'autre part, l'influence de telles interactions sur la structure secondaire et l'orientation des peptides a été déterminée par le PM-IRRAS. Nous avons ainsi montré la cohérence entre les modifications du signal SHG, liées à des changements d'orientation des tryptophanes et celles des spectres de PM-IRRAS, dues à des changements d'orientation de la structure secondaire de gp41W / Membrane proteins are extremely rich in aromatic amino acids, like tryptophan (W). This particularity is found in many antimicrobial peptides and in several virus fusion proteins. An example of these fusion proteins is the HIV-1 envelop glycoprotein, the gp41. It is clear that the W residues are implicated in membrane perturbation and pore formation. The aim of this work was the investigation of the W residue role in such activities, using the nonlinear optic. First, we determined the W hyperpolarizabilité (nonlinear potential) by the Hyper Rayleigh Scattering (HRS). Then, the evolution of the nonlinear signal of small synthetic peptides, as function of the increasing number of their W residues, was demonstrated. These results allowed us to follow the W residue involvement of two peptides, K3W4 and gp41W, in the interaction with lipids monolayer at the air-water interface, using the second harmonic generation (SHG). The influence of such interaction in the peptide structure and orientation was determined using the PM-IRRAS. In conclusion, we showed the coherence between the SHG signal variation, due to the W orientation changes, and the PMIRRAS spectra modification, due to the gp41W helix orientation changes

Un nouveau modèle de biomembrane de type liposome a été développé à partir de lipides synthétisés comportant une unité biphényle sur leur chaînes sn2 et une chaîne aliphatique sn1 de longueur et insaturation variables. L’anisotropie de susceptibilité magnétique positive de ces molécules induit une déformation en oblate de ces liposomes dits « biphényles » dans le champ magnétique B0. Cette déformation spécifique a été caractérisée par RMN des solides 31P et 2H en faisant varier différents paramètres : l’intensité de B0, l'élasticité membranaire, la température et la taille des liposomes (Helfrich, 1973). Ces vésicules déformées ont pu être observées par microscopies optiques et électroniques et la rémanence de la déformation en dehors de B0 a pu être analysée par diffusion des rayons X aux petits angles (SAXS). Enfin, les premières applications des liposomes biphényles comme nouveau modèle de biomembrane pour analyser la structure et l’orientation (par RMN des solides 15N) de peptides ou protéines membranaires, ont été étudiées. / A new model of biomembrane (liposome) was developed from synthesized lipids containing a biphenyl unit on the sn2 aliphatic chain and possessing a sn1 aliphatic chain which varies in length and unsaturation. The positive magnetic susceptibility anisotropy of these molecules induces an oblate deformation of these «biphenyls » liposomes under the magnetic field B0. This particular deformation has been characterized by 31P and 2H solid state NMR by varying different parameters: the intensity of B0, the membrane elasticity, the temperature and the size of the liposomes (Helfrich, 1973). These deformed vesicles were observed by optical and electron microscopy and the remanence of the deformation outside B0 has been analyzed by Small angles X-ray scattering (SAXS).Finally, the first applications of biphenyls liposomes as new biomembrane model to analyze the structure and orientation of membrane proteins or peptides were studied by 15N solid state NMR