La démonstration du rôle de la CDT (« Cytolethal Distending Toxin ») de Helicobacterhepaticus dans le développement de l’hépatocarcinome murin fait de cette toxine un candidatpertinent dans l'activation de processus pro-cancéreux. Comme la toxine CagA de Helicobacterpylori, la sous-unité active CdtB de la CDT pourrait être une oncoprotéine. Nous avons étudié lerôle de la CdtB des Hélicobacters dans l’inflammation et la carcinogenèse digestive via unestratégie lentivirale d’expression constitutive ou conditionnelle de la CdtB ou de son mutant pourl’activité DNase. Nous avons réalisé une étude du transcriptome et montré que la CdtB deH. hepaticus induisait une réponse inflammatoire en surexprimant des cytokines, chimiokines,peptides antimicrobiens et en activant la voie du NF-κB des cellules épithéliales. La CdtB réguleégalement l’expression et la localisation nucléaire du facteur de transcription et oncogène MafB.Ces résultats ont été confirmés pour la CdtB de Helicobacter pullorum. Des expériencesd'infection des cellules avec des souches sauvages et mutées pour la CDT (deH. hepaticus & H. pullorum) ont permis de valider les résultats obtenus et de les attribuer à laCdtB et notamment à son activité DNase. Nous avons aussi développé un nouveau modèle dexénogreffes de cellules épithéliales inductibles pour l’expression de la CdtB de H. hepaticus.Dans ce modèle, la CdtB, en plus de ses effets déjà connus, retarde la croissance tumorale,induit l’apoptose, la sénescence et la surexpression du marqueur nucléaire de prolifération,Ki-67, suggérant la survie cellulaire. L’ensemble de ces résultats fournit de nouveaux argumentsen faveur du potentiel oncogénique de la CDT. / The demonstration of the role of the Cytolethal Distending Toxin (CDT) of Helicobacter hepaticusin the development of hepatocarcinoma in mice, makes this toxin a relevant candidate in theactivation of precancerous processes. As in the case of the CagA toxin of Helicobacter pylori, theCdtB active subunit of CDT could be an oncoprotein. We studied the role of Helicobacter CdtB ininflammation and digestive carcinogenesis using a lentiviral strategy for constitutive or conditionalexpression of the CdtB subunit or its corresponding DNase mutant. We conducted a study of thetranscriptome and showed that CdtB induced an inflammatory response by overexpressingcytokines, chemokines, antimicrobial peptides and activating the NF-kB pathway in epithelialcells. The CdtB also regulated the expression and nuclear localization of the transcription factorand oncogene MafB. These results were confirmed for the CdtB of Helicobacter pullorum.Infection of cells with wild type strains and the corresponding CDT-mutant strains (of H. hepaticus& H. pullorum) were used to validate the results and to attribute the effects to the CdtB and, inparticular, to its DNase activity. We also developed a novel epithelial cell xenograft model toevaluate the inducible expression of H. hepaticus CdtB. In this model, the CdtB, in addition to itspreviously well-known effects, delayed tumor growth, induced apoptosis, senescence and theoverexpression of nuclear proliferation marker, Ki-67, suggesting cell survival. All of these resultsprovide new arguments in favor of the oncogenic potential of the CDT.

A imunidade inata que o mosquito apresenta tenta combater os patógenos dentro do organismo do mosquito impedindo que este seja transmitido para outros hospedeiros. A resposta celular apresenta três diferentes processos: fagocitose, encapsulamento e formação nodular, todos estes processos buscam eliminar os patógenos. Peptídeos antimicrobianos fazem parte da resposta humoral do mosquito, sendo codificados por genes e secretados por diversos tipos celulares. Um peptídeo novo pouco conhecido descoberto em Anopheles gambiae, a gambicina, demonstrou bons resultados no combate de parasitas. Na infecção por Plasmodium galleceum, não há diferença significativa na expressão deste peptídeo entre o grupo controle e infectado nos intervalos analisados. Na infecção por vírus dengue, sorotipo 2, a gambicina não apresenta diferença significativa no intervalo de 24 horas após a infecção, quando comparamos grupo controle e infectado, nos intervalos de 7 dias e 14 dias após a infecção, a expressão da gambicina é maior no grupo controle quando comparemos com o grupo infectado. / Innate immunity presents a mosquito tries to combat the pathogens inside the body of the mosquito preventing it from being transmitted to other hosts. The cellular response has three different processes: phagocytosis, encapsulation and nodule formation, all these processes seek to eliminate pathogens. Antimicrobial peptides are part of the humoral response mosquito being encoded by genes and secreted by several cell types. A little known new peptide discovered in Anopheles gambiae, the gambicina showed good results in controlling pests. In Plasmodium infection galleceum, there is no significant difference in the expression of this peptide between the control group and infected in the analyzed intervals. In infection with dengue virus serotype 2, the gambicina no significant difference within 24 hours after infection when comparing the control group and infected at intervals of 7 days and 14 days after infection, the expression is higher in gambicina control group when compare to the infected group.

Les peptides antimicrobiens (PAMs) représentent des composants importants de l`immunité innée. Ils sont présents dans la plupart des organismes multicellulaires et constituent la première ligne de défense contre les infections. Ils possèdent un large éventail d`activités, une non-toxicité contre les cellules de l`hôte et des effets synergiques avec les antibiotiques conventionnels. Par conséquent, ils peuvent être d`excellents candidats dans le développement de nouveaux antibiotiques pour lutter contre la résistance de microorganismes. Concernant les PAMs dérivés de la chromogranine A (CgA), la cateslytine (Ctl) présente des activités antimicrobiennes directes et des propriétés immunomodulatrices. Dans ma thèse, j`ai cherché à caractériser l`épipeptide D-Ctl, où tous les résidus en conformation-L ont été remplacés par des résidus en conformation-D. Tout d`abord, la stabilité dans les surnageants bactériens et des dosages de l`activité antimicrobienne ont été réalisés, ainsi que l`analyse de viabilité des cellules et des dosages des cytokines libérées par les cellules immunitaires. L`efficacité de D-Ctl a été comparée à celle de L-Ctl contre des souches bactériennes, puis les CMIs ont été déterminées et comparées dans le cas de combinaisons avec des antibiotiques conventionnels, afin de montrer un effet synergique et/ou additif. De plus, D-Ctl ne déclenche pas de résistance chez E. coli. Des tests de cytotoxicité ont été effectués sur plusieurs types de lignées cellulaires et de PBMCs. Les effets inflammatoires aussi ont été testés. Ensuite, le modèle bactérien E. coli MDR a été utilisé pour des analyses physico-chimiques, telles que la microscopie à épifluorescence, la spectroscopie ATR-FTIR et la microscopie à force atomique. Enfin, le brevet D-Ctl a été déposé en 2016 sous le numéro EP 16306539.4 « Nouveau peptide de cateslytine en conformation D ». En conclusion, D-Ctl est capable de tuer rapidement un large spectre de micro-organismes, et il pourrait potentialiser l`effet antimicrobien de plusieurs antibiotiques. / Antimicrobial peptides (AMPs) represent important components of innate immunity. They are present in most multicellular organisms and constitute the first line of defense against infections. They exhibit a large spectrum of activities, a non-toxicity against host cells and synergistic effects with conventional antibiotics. Therefore, they can be as excellent candidates in the development of new antibiotics to fight pathogens resistance. Concerning to AMPs derived from chromogranin A (CgA), Cateslytin (Ctl) represents a new antibiotic, which displays direct antimicrobial activities and immunomodulatory properties. In my thesis, I aimed to characterize the epipeptide D-Ctl, where all (L-conformation) residues were replaced by (D-conformation) residues. Firstly, antimicrobial assays were performed, cells viability, immune assays, and the stability in bacterial supernatant was tested. The efficiency of D-Ctl was compared with L-Ctl against bacterial strains, then MICs were determined and compared with combinations in presence of classical antibiotics in order to show synergistic or/and additive effect. Moreover, D-Ctl does not trigger resistance in E. coli. Also, cytotoxicity assays were performed on several types of cell line and PBMCs. Inflammatory effects were tested too. Then, bacterial model E. coli MDR was used for physicochemical analysis such as epifluorescence microscopy, ATR-FTIR spectroscopy and atomic force microscopy. Finally, D-Ctl patent has been deposited in 2016 under the number EP 16306539.4 “New D-configured cateslytin peptide”. To conclude: D-Ctl is able to rapidly kill a broad spectrum of microorganisms, and it could potentiate the antimicrobial effect of several 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

The growing resistance of bacteria to traditional antibiotics promotes the interest in finding new substances for their production. Antimicrobial peptides have comparable effect to conventional antibiotics, but a different mechanism of action and they do not provoke bacterial resistance. These peptides were characterized in all forms of multicellular organisms. Hymenoptera venom contains many biologically active substances including antimicrobial peptides. For this reason, this thesis focuses on the acquisition of antimicrobial peptide sequences from selected species of bumblebees (Bombus terrestris, B. hortorum, B. hypnorum, B. pratorum, B. lucorum, B. lapidarius, B. humilis and B. bohemicus). The isolation from the venom glands was performed by high performance liquid chromatography with reversed phases. Subsequent analysis was performed using the methods of mass spectrometry, matrix-assisted laser desorption/ionization with time of flight analyzer and electrospray ionization connected with hybrid linear ion trap analyzer with orbitrap. The sequences for the found peptides were determined by tandem mass spectrometry methods "de novo" and Edman degradation. In this work we characterized 17 sequences of peptides extracted from bumblebee venom glands for which antimicrobial activity was determined...

With increasing incidence rates aligned with poor prognosis; sepsis represents one of the biggest challenges in modern health care. It is a multifactorial syndrome defined as organ dysfunction caused by disturbed systemic response to an infection. Most of the inpatient sepsis are caused by Gram positive bacteria and one of its major constituents of the cell envelope: lipoteichoic acid (LTA). An adjuvant treatment that has gained prominence recently is extracorporeal blood removal therapies i.e., hemoperfusions. The concept is to remove the bacterial virulence factors that triggers immune responses and therefor stabilize the hemodynamic parameters of the patient. The dominating research of this method centres around adsorption of the Gram negative bacterias’ endotoxin lipopolysaccharide (LPS) but not LTA, whose biochemical and physiological properties resembles each other. The aim of this study was to determine whether LTA can be adsorbed using immobilized truncated derivates of antimicrobial peptides (AMPs). LTA was quantified using ELISA comparing before and after passage through columns with immobilized peptides. Further, the absorption abilities of LTA from two different solid phases with distinctive surfaces were investigated. This was of interest to elucidate the nature of the mechanisms behind LTA extractions. All results generated inconclusive data, except for one trial which demonstrated that peptide KEF-19 adsorbed most LTA and that the electrostatic force had the greatest influence of the adsorption. Future studies should however be carried out to validate these statements as well as feasibility and safety estimations for KEF-19 as the sorbent in hemoperfusions for Gram positive bacteria and LTA.

<p>Pathogenic bacteria present a critical threat to modern medicine. Therapeutic strategies to target and eliminate resilient bacteria are not advancing at the same rate as the emergence of bacterial resistance. An associated urgent concern regarding antibiotic resistance is the existence and proliferation of intracellular bacteria, which find refuge from bactericidal mechanisms by hiding within mammalian cells. Therefore, many once-successful antibiotics become ineffective through the development of resistance, or through failure to reach intracellular locations in therapeutic concentration. To overcome these challenges, the covalent combination of a conventional antibiotic with an antibiotic, cell-penetrating peptide was explored to develop dual-action antibiotic conjugates. </p> <p>Herein, we utilized a strategy in conjugating the antibiotics by a cleavable linkage to cationic amphiphilic polyproline helices (CAPHs) to improve vancomycin and linezolid antibiotics. This approach enables the conjugate to penetrate cells and deliver two potent monomeric antimicrobial drugs. The vancomycin-CAPH conjugate, <b>VanP14S</b>, showed enhanced mammalian cell uptake compared to vancomycin, a poor mammalian cell-penetrating agent; and <b>VanP14S</b> was capable of cleaving and releasing two antibiotics under mimicked physiological conditions. Enhanced antibacterial activity was observed against a spectrum of Gram-positive and Gram-negative pathogens, including drug-resistant strains. Further investigation revealed that this conjugate’s bactericidal activity was not entirely the result of significant membrane perturbation such as a lytic mode of action. Mammalian cell toxicity and red blood cell lysis were insignificant at relevant bactericidal concentrations below 20 µM. The current results suggest an enhanced binding to the peptidoglycan of bacteria, the target of vancomycin, although more work is needed to justify this claim. Preliminary results on <b>VanP14GAPS</b>, a conjugate with a more rigid CAPH, convey similar activity to <b>VanP14S; </b>however,<b> </b>moderate increases in red blood cell lysis and cytotoxicity were observed. </p> <p>Regarding the <b>LnzP14</b> conjugate, preliminary data reveal that the conjugate has Gram-negative activity against <i>Escherichia coli</i>, whereas linezolid is ineffective in killing Gram-negative bacteria. This conjugate showed significant enhancement in cellular uptake compared to the CAPH, and the release of linezolid and CAPH in physiological conditions was confirmed. Overall, arming a conventional antibiotic with an antimicrobial, cell-penetrating peptide appears to be a powerful strategy in providing novel antibiotic conjugates with the propensity to overcome the limitations in treating challenging pathogens.</p>

Supported lipid bilayers have been used as an artificial model of biological membranes and their interaction with 5 selected antimicrobial peptides was studied by several experimental techniques, mainly ellipsometry, laser scanning microscopy and fluorescence correlation spectroscopy. The thesis explains basic principles of the applied techniques focusing on their aspects relevant to characterization of lipid bilayers. The biological significance of antimicrobial peptides, their modes of interaction with membranes and the basic characteristics of the selected peptides are briefly discussed. The following text describes the main types of experimental studies performed and the interpretation of their results. Peptide-induced changes in lipid bilayer morphology were characterized by ellipsometry and laser scanning microscopy. Most interesting effects were observed in the case of melittin, which induced formation of long lipid tubules protruding from the bilayer. Lipid lateral diffusion measured by fluorescence correlation spectroscopy can provide information on bilayer organization on length-scales below resolution of optical microscopy.

Previously melittin, the α-helical basic honey bee venom peptide, was shown to inhibit F1-ATPase by binding at the β-subunit DELSEED motif of F1Fo-ATP synthase. Herein, we present the inhibitory effects of the basic α-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F1 and membrane bound F1Fo Escherichia coli ATP synthase. We found that the extent of inhibition by amphibian peptides is variable. Whereas MRP-amide inhibited ATPase essentially completely (∼96% inhibition), carein 1.8 did not inhibit at all (0% inhibition). Inhibition by other peptides was partial with a range of ∼13-70%. MRP-amide was also the most potent inhibitor on molar scale (IC50 ∼3.25 μM). Presence of an amide group at the c-terminal of peptides was found to be critical in exerting potent inhibition of ATP synthase (∼20-40% additional inhibition). Inhibition was fully reversible and found to be identical in both F1Fo membrane preparations as well as in isolated purified F1. Interestingly, growth of E. coli was abrogated in the presence of ascaphin-8, aurein 2.2, aurein 2.3, citropin 1.1, dermaseptin, magainin II-amide, MRP, MRP-amide, melittin, or melittin-amide but was unaffected in the presence of carein 1.8, carein 1.9, maculatin 1.1, magainin II, or XT-7. Hence inhibition of F1-ATPase and E. coli cell growth by amphibian antimicrobial peptides suggests that their antimicrobial/anticancer properties are in part linked to their actions on ATP synthase.

In this review we discuss the inhibitory effects of dietary polyphenols and amphibian antimicrobial/antitumor peptides on ATP synthase. In the beginning general structural features highlighting catalytic and motor functions of ATP synthase will be described. Some details on the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it an interesting drug target with respect to dietary polyphenols and amphibian antimicrobial peptides will also be reviewed. ATP synthase is known to have distinct polyphenol and peptide binding sites at the interface of α/β subunits. Molecular interaction of polyphenols and peptides with ATP synthase at their respective binding sites will be discussed. Binding and inhibition of other proteins or enzymes will also be covered so as to understand the therapeutic roles of both types of molecules. Lastly, the effects of polyphenols and peptides on the inhibition of Escherichia coli cell growth through their action on ATP synthase will also be presented.