Big Defensins, a Diverse Family of Antimicrobial Peptides That Follows Different Patterns of Expression in Hemocytes of the Oyster Crassostrea gigas

Background: Big defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections. Findings: Using the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate beta-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CS alpha beta-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa) were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid. Conclusions: We provide here the first report showing that big defensins form a family of antimicrobial peptides diverse not only in terms of sequences but also in terms of genomic organization and regulation of gene expression

The COVID-19 pandemic and global environmental change: Emerging research needs.

The outbreak of COVID-19 raised numerous questions on the interactions between the occurrence of new infections, the environment, climate and health. The European Union requested the H2020 HERA project which aims at setting priorities in research on environment, climate and health, to identify relevant research needs regarding Covid-19. The emergence and spread of SARS-CoV-2 appears to be related to urbanization, habitat destruction, live animal trade, intensive livestock farming and global travel. The contribution of climate and air pollution requires additional studies. Importantly, the severity of COVID-19 depends on the interactions between the viral infection, ageing and chronic diseases such as metabolic, respiratory and cardiovascular diseases and obesity which are themselves influenced by environmental stressors. The mechanisms of these interactions deserve additional scrutiny. Both the pandemic and the social response to the disease have elicited an array of behavioural and societal changes that may remain long after the pandemic and that may have long term health effects including on mental health. Recovery plans are currently being discussed or implemented and the environmental and health impacts of those plans are not clearly foreseen. Clearly, COVID-19 will have a long-lasting impact on the environmental health field and will open new research perspectives and policy needs

Caractérisation et analyse de l'expression des pénaeidines, peptides antimicrobiens isolés chez la crevette pénéide Penaeus vannamei (Crustacea, Decapoda)

Haemocytes are the key elements of a complex defence system on which crustacean immunity is largely based. We report here for the first time in a crustacean that haemocytes are involved in the synthesis of antimicrobial peptides similar to those described in other arthropod classes. These molecules isolated from the penaeid shrimp Penaeus vannamei are members of an homogeneous farnily which differ from the antimicrobial peptides previously described in that they combine in a single molecule a proline-rich arninoterminal domain, and a cyclic carboxy-terminal domain containing three intramolecular disulphide bridges. In addition, penaeidins bear post-translational modifications at their arnino- and carboxy-termini. Five molecules of the penaeidin family were isolated by a biochemical approach and by cDNA cloning. They are characterised by antibacterial properties essentially directed against Gram positive strains et by antifungal activities against filamentous fungi. Penaeidins are mainly synthesised by haemocytes where they are stored within the cytoplasmic granules of the so-called granular and semigranular cell types. They are also present in haemocyte containing tissues (heart, lymphoid organ), in intestinal epithelium, and in gonads. Lower  expression levels were detected in hepatopancreas. From our results, and in opposition to the insect immune system, it seems that penaeidin expression is not transcriptionnally regulated upon infection, but that the bioactive peptides are released by haemocyte exocytosis in response to bacterial challenge. These data strongly suggest that antimicrobial peptide expression in crustacean is regulated in a similar manner to that described in chelicerata.L'immunité des crustacés repose sur un système de défense complexe dont les principaux effecteurs sont les hémocytes. Nous avons montré pour la première fois chez un crustacé que les hémocytes participaient à la synthèse de peptides antimicrobiens comparables à ceux décrits chez les autres arthropodes. Ces molécules isolées chez la crevette pénéide Penaeus vannamei forment une famille homogène qui se distingue des peptides antimicrobiens précédemment décrits dans d'autres groupes par la présence conjointe d'un domaine amino-terminal riche en résidus proline et d'un domaine carboxy-terminal comportant trois ponts disulfure intramoléculaires. Les pénaeidines portent par ailleurs des modifications post-traductionnelles à leurs deux extrémités. Au total, cinq molécules ont été isolées par voie biochimique et par le clonage de leurs ADN complémentaires. Elles présentent des propriétés antibactériennes essentiellement dirigées contre les germes à Gram positif et des activités antifongiques sur les champignons filamenteux. Les pénaeidines sont majoritairement synthétisées dans les hémocytes où elles sont stockées dans les granules cytoplasmiques des cellules granuleuses et semi-granuleuses. Elles sont par ailleurs retrouvées dans les tissus riches en hémocytes (coeur, organe lymphoïde), dans l'épithélium intestinal et les gonades, de plus faibles niveaux d'expression étant observés dans l'hépatopancréas. Il semble que l'expression des pénaeidines ne soit pas régulée transcriptionnellement comme cela a été décrit pour les peptides antimicrobiens d'insectes, mais que les peptides biologiquement actifs soient libérés par exocytose des hémocytes producteurs en réponse aux stimulations bactériennes. Ce mode de régulation rapproche le système immunitaire des crustacés de celui des chélicérates

Effecteurs moléculaires de lassociation Crassostrea gigas / Vibrio splendidus. Rôle de la porine OmpU dans les mécanismes de résistance et déchappement à la réponse immunitaire de lhôte.

Vibrio splendidus LGP32 est une bactérie pathogène associée aux épisodes de mortalités estivales qui affectent la production d'huître Crassostrea gigas depuis des décennies. Nous avons montré ici que la porine OmpU était un effecteur majeur de l'interaction V. splendidus / C. gigas. Nous avons pour cela construit un mutant ompU de V. splendidus. Celui-ci nous a permis de mo ntrer l'implication de OmpU (i) dans la résistance de V. splendidus aux antimicrobiens, incluant ceux de l'huître, (ii) dans la fitness chez l'huître, et (iii) dans la virulence en infections expérimentales (mortalités de 56 % pour le sauvage versus pour le 11% mutant). En accord avec ces résultats, nous avons montré que la délétion de ompU modifiait la sécrétion de protéines dont l'expression est contrôlée par les voies de régulation de la virulence (ToxR) et de l'intégrité membranaire (SigmaE). Par ailleurs, nous avons montré que OmpU jouait un rôle essentiel dans la reconnaissance par les hémocytes. En effet, (i) in vivo, les gènes hémocytaires répondent différemment à l'infection par le Vibrio sauvage ou ompU, et (ii) in vitro, OmpU est nécessaire à l'invasion hémocytaire par V. splendidus. Cette invasion utilise la phagocytose dépendante de l'intégrine b et la SOD extracellulaire du plasma d'huître comme opsonine qui lie OmpU. Ainsi, OmpU est un facteur de virulence majeur qui permet l'infection des hémocytes dans lesquels il est capable de survivre en inhibant la formation de radicaux oxygénés et de vacuoles acides. La résistance du Vibrio aux antimicrobiens hémocytaires de l'huître, elle-même dépendante de OmpU, est probablement un élément supplémentaire favorable à la survie intra-cellulaire.Vibrio splendidus LGP32 is a bacterial pathogen associated to the summer mortality outbreaks that have affected the production of Crassostrea gigas oysters over the past decades. We showed here that the OmpU porin is a major effector of the V. splendidus / C. gigas interaction. For that, we have constructed a ompU mutant of V. splendidus, and shown that the OmpU porin is implicated (i) in the resistance of V. splendidus to antimicrobials, including those of oyster, (ii) in its in vivo fitness, and (iii) in its virulence in oyster experimental infections (mortalities have been reduced from 56 % to 11 % upon mutation). In agreement, we have shown that the ompU deletion modified the expression of secreted proteins controlled by the virulence (ToxR) and the membrane integrity (SigmaE) regulation pathways. Furthermore, we have shown that OmpU has a major role in the recognition of V. splendidus by oyster hemocytes. Indeed, (i) in vivo, hemocyt e genes displayed differential responses to an infection with the wild-type or the ompU mutant, and (ii) in vitro, OmpU was necessary for hemocyte invasion by V. splendidus. This invasion process required the hemocyte b-integrin and the oyster plasma extracellular SOD, which was found to act as an opsonin recognizing OmpU. Thus, OmpU is a major virulence factor that allows infection of hemocytes in which V. splendidus is able to survive by inhibiting the production of reactive oxygen species and the formation of acidic vacuoles. Resistance of V. splendidus to hemocyte antimicrobials, which is also OmpU-dependant, is probably an additional determinant of V. splendidus intracellular survival.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes

Antimicrobial peptides and proteins (AMPs) are widespread in the living kingdom. They are key effectors of defense reactions and mediators of competitions between organisms. They are often cationic and amphiphilic, which favors their interactions with the anionic membranes of microorganisms. Several AMP families do not directly alter membrane integrity but rather target conserved components of the bacterial membranes in a process that provides them with potent and specific antimicrobial activities. Thus, lipopolysaccharides (LPS), lipoteichoic acids (LTA) or the peptidoglycan precursor Lipid II are targeted by a broad series of AMPs. Studying the functional diversity of immune effectors tells us about the essential residues involved in AMP mechanism of action. Marine invertebrates have been found to produce a remarkable diversity of AMPs. Molluscan defensins and crustacean anti-LPS factors (ALF) are diverse in terms of amino acid sequence and show contrasted phenotypes in terms of antimicrobial activity. Their activity is directed essentially against Gram-positive or Gram-negative bacteria due their specific interactions with Lipid II or Lipid A, respectively. Through those interesting examples, we discuss here how sequence diversity generated throughout evolution informs us on residues required for essential molecular interaction at the bacterial membranes and subsequent antibacterial activity. Through the analysis of molecular variants having lost antibacterial activity or shaped novel functions, we also discuss the molecular bases of functional divergence in AMPs

Functional Insights From the Evolutionary Diversification of Big Defensins

Big defensins are antimicrobial polypeptides believed to be the ancestors of \u3b2-defensins, the most evolutionary conserved family of host defense peptides (HDPs) in vertebrates. Nevertheless, big defensins underwent several independent gene loss events during animal evolution, being only retained in a limited number of phylogenetically distant invertebrates. Here, we explore the evolutionary history of this fascinating HDP family and investigate its patchy distribution in extant metazoans. We highlight the presence of big defensins in various classes of lophotrochozoans, as well as in a few arthropods and basal chordates (amphioxus), mostly adapted to life in marine environments. Bivalve mollusks often display an expanded repertoire of big defensin sequences, which appear to be the product of independent lineage-specific gene tandem duplications, followed by a rapid molecular diversification of newly acquired gene copies. This ongoing evolutionary process could underpin the simultaneous presence of canonical big defensins and non-canonical (\u3b2-defensin-like) sequences in some species. The big defensin genes of mussels and oysters, two species target of in-depth studies, are subjected to gene presence/absence variation (PAV), i.e., they can be present or absent in the genomes of different individuals. Moreover, big defensins follow different patterns of gene expression within a given species and respond differently to microbial challenges, suggesting functional divergence. Consistently, current structural data show that big defensin sequence diversity affects the 3D structure and biophysical properties of these polypeptides. We discuss here the role of the N-terminal hydrophobic domain, lost during evolution toward \u3b2-defensins, in the big defensin stability to high salt concentrations and its mechanism of action. Finally, we discuss the potential of big defensins as markers for animal health and for the nature-based design of novel therapeutics active at high salt concentrations

Field enhanced bacterial sample stacking in isotachophoresis using wide-bore capillaries

International audienc

Resistance to Antimicrobial Peptides in Vibrios

Vibrios are associated with a broad diversity of hosts that produce antimicrobial peptides (AMPs) as part of their defense against microbial infections. In particular, vibrios colonize epithelia, which function as protective barriers and express AMPs as a first line of chemical defense against pathogens. Recent studies have shown they can also colonize phagocytes, key components of the animal immune system. Phagocytes infiltrate infected tissues and use AMPs to kill the phagocytosed microorganisms intracellularly, or deliver their antimicrobial content extracellularly to circumvent tissue infection. We review here the mechanisms by which vibrios have evolved the capacity to evade or resist the potent antimicrobial defenses of the immune cells or tissues they colonize. Among their strategies to resist killing by AMPs, primarily vibrios use membrane remodeling mechanisms. In particular, some highly resistant strains substitute hexaacylated Lipid A with a diglycine residue to reduce their negative surface charge, thereby lowering their electrostatic interactions with cationic AMPs. As a response to envelope stress, which can be induced by membrane-active agents including AMPs, vibrios also release outer membrane vesicles to create a protective membranous shield that traps extracellular AMPs and prevents interaction of the peptides with their own membranes. Finally, once AMPs have breached the bacterial membrane barriers, vibrios use RND efflux pumps, similar to those of other species, to transport AMPs out of their cytoplasmic space

Taking advantage of electric field induced bacterial aggregation for the study of interactions between bacteria and macromolecules by capillary electrophoresis

International audienceThe quantification of interaction stoechiometry and binding constant between bacteria (or other microorganism) and(macro)molecules remains a challenging issue for which only a few adapted methods are available. In this paper, a new methodologywas developed for the determination of the interaction stoichiometry and binding constant between bacteria and (macro)molecules. The originality of this work is to take advantage of the bacterial aggregation phenomenon to directly quantify the freeligand concentration in equilibrated bacteria-ligand mixtures using frontal analysis continuous capillary electrophoresis. The describedmethodology does not require any sample preparation such as filtration step or centrifugation. It was applied to the study ofinteractions between Erwinia carotovora and different generations of dendrigraft poly-L-lysines leading to quantitative informations(i.e. stoichiometry and binding site constant). High stoichiometries in the order of 106-107 were determined between nanometricdendrimer-like ligands and the rod-shaped micrometric bacteria. The effect of the dendrimer generation on the binding constantand the stoichiometry is discussed. Stoichiometries were compared with those obtained by replacing the bacteria by polystyrenemicrobeads to demonstrate the internalization of the ligands inside the bacteria and the increase of the specific surface via theformation of vesicles