INITIAL INSIGHTS INTO THE STRUCTURE-ACTIVITY RELATIONSHIPS OF AVIAN DEFENSINS.

Numerous β-defensins have been identified in birds and the potential use of these peptides as alternatives to antibiotics has been proposed, in particular to fight antibiotic-resistant and zoonotic bacterial species. Little is known about the mechanism of antibacterial activity of avian β-defensins (AvBDs), and the present work was carried out to obtain initial insights into the involvement of structural features or specific residues in the antimicrobial activity of chicken AvBD2. Chicken AvBD2 and its enantiomeric counterpart were chemically synthesized. Peptide elongation and oxidative folding were both optimized. The similar antimicrobial activity measured for both L- and D- proteins clearly indicates that there is no chiral partner. Therefore the bacterial membrane is in all likelihood the primary target. Moreover, this work evidences that the three-dimensional fold is required for an optimal antimicrobial activity, in particular for Gram-positive bacterial strains. The three-dimensional NMR structure of chicken AvBD2 defensin displays the structural 3-stranded antiparallel β-sheet characteristic of β-defensins. The surface of the molecule does not display any amphipathic character. In light of this new structure and of the king penguin AvBD103b defensin structure, the consensus sequence of avian β-defensin's family was analyzed. Well conserved residues were highlighted and the potential strategic role of the lysine 31 residue of AvBD2 emphasized. The synthetic AvBD2-K31A variant displayed substantial N-terminal structural modifications and a dramatic decrease in activity. Taken together, these results demonstrate the structural as well as the functional role of the critical lysine 31 residue in antimicrobial activity

Role of avian beta-defensins in chicken resistance to intestinal salmonella enteridis infection

Les entérobactéries du genre Salmonella représentent une préoccupation majeure de santé publique car elles sont responsables de la majorité des cas de toxi-infections alimentaires dans le monde. La persistance de Salmonella Enteritidis dans les élevages de volailles, sous forme de portage bactérien intestinal, constitue une source de contamination importante pour les produits alimentaires qui en sont issus. Parmi, les nombreuses solutions mises en place, l’amélioration de l’immunité de la muqueuse digestive des oiseaux faisant barrière à la colonisation bactérienne représente une stratégie de lutte alternative à l’utilisation controversée des antibiotiques. Au cours de la réponse de l’hôte contre l’infection des salmonelles, il avait été montré que les gènes de ß-défensines aviaires, AvBD1 et AvBD2, sont fortement exprimés dans l’intestin des poulets résistants à la colonisation bactérienne. Les ß-défensines sont des peptides cationiques capables de détruire les bactéries mais aussi, chez les mammifères, d’attirer les cellules immunitaires. Dans ce contexte, la compréhension du mode d’action des deux ß-défensines aviaires contre l’infection intestinale des poulets par les salmonelles a constitué l’objectif principal du travail de thèse. Tandis que les AvBD1 et AvBD2 ont été initialement identifiées à partir de cellules hétérophiles, nous avons cherché à savoir si les cellules épithéliales intestinales, constituant la première barrière rencontrée par les bactéries, pouvaient les exprimer. Une procédure de culture primaire de cellules épithéliales intestinales isolées d’embryons d’oiseaux a été développée. Les cellules ont montré les caractéristiques de cellules épithéliales confirmées par l’expression de protéines du cytosquelette. Elles ont été capables d’exprimer les gènes d’AvBD1 et d’AvBD2, avec des taux différents selon la lignée d’animaux dont elles sont issues. De plus, cette expression s’est avérée modulable par les salmonelles qui sont capables d’inhiber l’expression d’AvBD2 dans les cellules épithéliales intestinales de la lignée la plus sensible. En parallèle, nous avons extrait trois ß-défensines aviaires de la moelle osseuse, AvBD1, AvBD2 et AvBD7, afin d’évaluer leur activité antimicrobienne. La séquence primaire de ces trois ß-défensines caractérisée par spectrométrie de masse a permis de mettre en évidence des modifications post-traductionnelles dans la partie C-terminale d’AvBD1 et dans la partie N-terminale d’AvBD7. L’activité antimicrobienne mesurée avec les trois ß-défensines a révélé des activités élevées contre des bactéries Gram + et Gram -, avec cependant une moindre efficacité d’AvBD2 contre les bactéries Gram -. La spécififcité d’AvBD2 contre les Gram + est retrouvée chez la ß-défensine du manchot empereur, AvBD103b de structure tridimensionnelle connue. Nous avons alors cherché à résoudre la structure de la ß-défensine 2 de poulet par résonance magnétique nucléaire afin d’analyser les relations entre la structure et l’activité des peptides AvBD2 et AvBD103b. La comparaison de leurs structures nous a permis de proposer un mode d’action de ces deux ß-défensines et plus particulièrement d’AvBD2. Finalement, l’ensemble des résultats obtenus permet de préciser le rôle des ß-défensines aviaires dans le phénotype de résistance/sensibilité à l’invasion intestinale des salmonelles. Outre leur effet antibactérien qui peut être contrecarré par les salmonelles, il semblerait que ces défensines aviaires puissent participer à d’autres fonctions comme le chimiotactisme permettant le recrutement de cellules immunocompétentes. Le spectre d’activité antibactérienne des ß-défensines aviaires caractérisées ouvre de plus des perspectives d’application thérapeutique dans un domaine où les antibiotiques sont plus que sujets à caution.Salmonella is a bacterial enteropathogen representing the main cause of food poisoning worldwide. Persistence of Salmonella Enteritidis in poultry flocks in the form of intestinal carriage represents an important source of contamination of poultry derived food products. Improving immunity of the mucosal barrier of the bird intestine to fight bacterial colonisation is a strategy alternative to the controversial use of antibiotics. During the study of chicken response to Salmonella infection, it had been shown that avian ß-defensin genes (AvBD1 and 2) were highly expressed in intestinal tissue of birds resistant to Salmonella colonisation. ß-defensins are cationic peptides able to kill bacteria and also, in mammalian species, to attract cells of the animal immune defence. In this context, understanding the mechanism of action of these two avian ß-defensins against intestinal infection of birds by Salmonella represented the main objective of the thesis work. While AvBD1 and 2 were initially identified from heterophils, we were interested in determining whether the intestinal epithelial cells, which constitute the first barrier encountered by the bacteria, were able to express them. A primary culture of intestinal epithelial cells has been developed from chicken embryos of divergent inbred lines either resistant or susceptible to Salmonella intestinal carriage. The cells exhibited characteristics of epithelial type according to morphology and expression of specific cytoskeletal proteins. They were shown to express the two ß-defensin genes AvBD1 and AvBD2 according to the chicken line. Furthermore, S. Enteritidis interfered with AvBD2 expression only in the cells from the susceptible line. In parallel, we were interested in preparing these ß-defensins in order to assess their antibacterial activity. Chicken bone marrow was used to isolate ß-defensins by acetic acid extraction followed by chromatographic separation. Three purified ß-defensins, AvBD1, AvBD2 and AvBD7 were characterized by mass spectrometry, revealing post-translational modifications in AvBD1 C-terminal end and AvBD7 N-terminal end. Antimicrobial activity measured by radial diffusion assay has shown a high efficiency against Gram + and Gram – except for AvBD2 with a lower potency against Gram – bacteria. Antibacterial spectrum of AvBD2 showed specificity towards Gram + bacteria as well as the king penguin AvBD103b whose three-dimensional structure has been described. This prompted us to resolve the structure of AvBD2 by nuclear magnetic resonance in order to analyze structure-activity relationships by comparing these two avian ß-defensins. This approach has led us to propose a mechanism of action for these two ß-defensins and particularly for AvBD2 in the intestinal mucosa. Finally, taken together the results allow to further define the role of avian ß-defensins in the phenotype of resistance/susceptibility to intestinal invasion by Salmonella. In addition to their antibacterial activity, which can be overcome to some extend by Salmonella, they could display other functions such as chemotaxis allowing recruitment of immune competent cells. The characterization of the activity spectrum of these avian ß-defensins opens perspectives of therapeutic application in a domain where antibiotics use should be avoided

Analyse intégrative des biomarqueurs par ICM-MS et top down

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Crystal structure of greglin, a novel non-classical Kazal inhibitor, in complex with subtilisin.

International audienceGreglin is an 83-residue serine protease inhibitor purified from the ovaries of the locust Schistocerca gregaria. Greglin is a strong inhibitor of subtilisin and human neutrophil elastase, acting at sub-nanomolar and nanomolar concentrations, respectively; it also inhibits neutrophil cathepsin G, α-chymotrypsin and porcine pancreatic elastase, but to a lesser extent. In the present study, we show that greglin resists denaturation at high temperature (95 °C) and after exposure to acetonitrile and acidic or basic pH. Greglin is composed of two domains consisting of residues 1-20 and 21-83. Mass spectrometry indicates that the N-terminal domain (1-20) is post-translationally modified by phosphorylations at three sites and probably contains a glycosylation site. The crystal structure of the region of greglin comprising residues 21-78 in complex with subtilisin was determined at 1.75 Å resolution. Greglin represents a novel member of the non-classical Kazal inhibitors, as it has a unique additional C-terminal region (70-83) connected to the core of the molecule via a supplementary disulfide bond. The stability of greglin was compared with that of an ovomucoid inhibitor. The thermostability and inhibitory specificity of greglin are discussed in light of its structure. In particular, we propose that the C-terminal region is responsible for non-favourable interactions with the autolysis loop (140-loop) of serine proteases of the chymotrypsin family, and thus governs specificity. DATABASE: The atomic coordinates and structure factors for the greglin-subtilisin complex have been deposited with the RCSB Protein Data Bank under accession number 4GI3. STRUCTURED DIGITAL ABSTRACT: Greglin and Subtilisin Carlsberg bind by X-ray crystallography (View interaction)

Antibacterial spectrum of avian defensin 2: peculiarities in relationship with structural features

National audienceDefensins of birds belong to the large family of antimicrobial peptides that are key components of mucosal innate immunity. Among them, avian defensin 2 (AvBD2) has been pointed out for its antimicrobial activity more efficient against Gram+ than against Gram - bacterial strains, as determined by radial diffusion assay . To determine structural features that may be involved in the mode of action of AvBD2 and in its peculiar antibacterial spectrum, synthetic form of AvBD2 was prepared and characterized structurally by NMR 131. The beta-sheet 3D-structure of AvBD2, typical of beta-defensins, revealed a protruding K31 residue in a hydrophobic environment of the C-terminal part of the molecule, which may interact preferentially with bacterial surfaces. Synthetic enantiomers and point-mutated (K31A) variants of AvBD2, either folded or linear, were then synthesised and assayed on various bacterial strains. Both L- and D-AvBD2 exhibited similar antimicrobial activity, indicating that the bacterial molecular target is not chiral. The loss of the folding increased importantly the minimum inhibitory concentration of the defensin, in a more marked way towards Gram+ than towards Gram- bacterial species. Finally the point mutation of the K31A AvBD2 also reduced significantly the antibacterial activity. Taken together, the results reveal a different impact of structural features on antibacterial activity of this antimicrobial peptide according to the bacterial type, suggesting that the antimicrobial mechanism may differ between Gram+ and Gram- bacterial species

Avian beta-defensins in the control of bacterial pathogens of chicken digestive tract

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ICM-MS and Top down analysis to characterize biomarker

National audienceIntact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) is a well established differential and quantitative approach to display biomarkers on prokaryotic or eukaryotic cells. ICM-MS is a fast, reproducible and sensitive method for peptidomic and proteomic studies in 2-30 kDa mass range. However, by this approach the biomarker of interest remain to be identified. Here we demonstrate the application of ICM-MS profiling coupled to Top-down analysis to characterize three avian β-defensins : AvBD1, AvBD2 and a newly isolated β-defensin AvBD7. β-defensins are important components of chicken innate immunity in mucosal tissue, a major entry site for pathogens that may cause food poisoning. Chicken inbred lines diverge phenotypically with respect to levels of pathogens intestinal carriage and to level of gene expression of two β-defensin (AvBD1, AvBD2). In order to detect the known mature β-defensins and to discover other molecular species with a potential antimicrobial activity, ICM-MS was performed on intact heterophil granulocytes, which are known to infiltrate intestinal tissue early after pathogen colonization. The in silico predicted AvBD1 and AvBD2 sequences, taking into account the three characteristic β-defensins disulfide bonds, were compared to experimental peaks. Despite the lack of precision mass in linear MALDI-TOF spectra, two peaks may correspond to AvBD1 and AvBD2. To characterize finely molecular species observed on ICM-MS profile, we purified them by chromatography from bone marrow extract, in which defensin genes have been shown to be highly expressed and that contains the heterophils precursors. All size exclusion chromatography fractions were analysed by classic bottom-up proteomic in order to identify molecular species. AvBD1 and AVBD2 and a newly isolated AvBD7 were identified. To obtain structural information, a Top-down proteomic approach was applied on RP-HPLC purified intact molecular species. By a top-down MS strategy using nano-ESI–Q-TOF MS, the AvBDs were measured with a mass accuracy of 100 ppm. By comparison with the theoretical monoisotopic mass (with 3 disulfide bonds), bone marrow AvBD2 exhibited the expected mass but AVBD1 and AvBD7 presented respectively a delta mass = -1 Da and -17 Da. Through the topdown MS/MS strategy, we confirmed respectively the C and N terminal sequences for AvBD1 and AvBD7 and characterized post-translational modifications which correspond to an amidated C-terminal and to a Gln N-terminal cyclized as a pyroglutamic acid residue. In this study, a hybrid strategy combining «ICM-MS», «bottom-up» and «top-down» proteomic approaches enabled, for the first time, characterization of AvBD1, AvBD2, and AvBD7 as well as isoforms of these defensins. The use of complementary ICM-MS – Top-down technologies allowed us to follow these structures in their biological (or cellular) context

Primary Structure and Antibacterial Activity of Chicken Bone Marrow-Derived β-Defensins▿

Three biologically active β-defensins were purified by chromatography from chicken bone marrow extract: avian β-defensin 1 (AvBD1), AvBD2, and the newly isolated β-defensin AvBD7. Mass spectrometry analyses showed that bone marrow-derived AvBD1, -2, and -7 peptides were present as mature peptides and revealed posttranslational modifications for AvBD1 and AvBD7 in comparison to their in silico-predicted amino acid sequences. Tandem mass spectrometry analysis using the nanoelectrospray-quadrupole time of flight method showed N-terminal glutaminyl cyclization of mature AvBD7 and C-terminal amidation of mature AvBD1 peptide, while posttranslational modifications were absent in bone marrow-derived mature AvBD2 peptide. Furthermore, mass spectrometry analysis performed on intact cells confirmed the presence of these three peptides in mature heterophils. In addition, the antibacterial activities of the three β-defensins against a large panel of gram-positive and -negative bacteria were assessed. While the three defensins displayed similar antibacterial spectra of activity against gram-positive strains, AvBD1 and AvBD7 exhibited the strongest activity against gram-negative strains in comparison to AvBD2

Improvement of the chemical production of β-defensins including straighforward attribution of disulfide bridges

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Improvement of the chemical production of β-defensins including straightforward attribution of disulfide bridges

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