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

Topoisomer Differentiation of Molecular Knots by FTICR MS: Lessons from Class II Lasso Peptides

Lasso peptides constitute a class of bioactive peptides sharing a knotted structure where the C-terminal tail of the peptide is threaded through and trapped within an N-terminalmacrolactamring. The structural characterization of lasso structures and differentiation from their unthreaded topoisomers is not trivial and generally requires the use of complementary biochemical and spectroscopic methods. Here we investigated two antimicrobial peptides belonging to the class II lasso peptide family and their corresponding unthreaded topoisomers: microcin J25 (MccJ25), which is known to yield two-peptide product ions specific of the lasso structure under collisioninduced dissociation (CID), and capistruin, for which CID does not permit to unambiguously assign the lasso structure. The two pairs of topoisomers were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) upon CID, infrared multiple photon dissociation (IRMPD), and electron capture dissociation (ECD). CID and ECDspectra clearly permitted to differentiate MccJ25 from its non-lasso topoisomer MccJ25-Icm, while for capistruin, only ECD was informative and showed different extent of hydrogen migration (formation of c\bullet/z from c/z\bullet) for the threaded and unthreaded topoisomers. The ECD spectra of the triply-charged MccJ25 and MccJ25-lcm showed a series of radical b-type product ions {\eth}b0In{\TH}. We proposed that these ions are specific of cyclic-branched peptides and result from a dual c/z\bullet and y/b dissociation, in the ring and in the tail, respectively. This work shows the potentiality of ECD for structural characterization of peptide topoisomers, as well as the effect of conformation on hydrogen migration subsequent to electron capture

Can Insects Develop Resistance to Insect Pathogenic Fungi?

This paper presents new, important information on the microevolution of insect resistance to the insect pathogenic fungus Beauveria bassiana which will have far-reaching implications for the development of insect pathogenic fungi as biological control agents. We placed successive generations of a melanic population of the Greater wax moth, Galleria mellonella, under constant selective pressure from the insect pathogenic fungus, Beauveria bassiana. Enhanced fungal resistance was observed and larvae from the 25th generation were studied in detail to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. There are 3 novel, core findings from the study:1.Antifungal resistance in these insects is pathogen species-specific, and probably arises through trans-generational immune priming. The resistance was less obvious in earlier generations, suggesting subtle cumulative changes that are only fully apparent in the 25th generation. 2.The insect’s fecundity is already pushed close to minimum by its melanic phenotype. Therefore, the additional drain on resources required to boost antifungal defence still more, comes not from further compromising life history traits but via a re-allocation of the insect’s immune defences. Specifically during B. bassiana infection, systemic (fat body and hemocoel) responses, particularly the expression of antimicrobial peptides, are damped down in favour of a tailored repertoire of enhanced responses in the integument (cuticle and epidermis) – the foremost and most important barrier to natural fungal infection. 3.A previously-overlooked range of putative stress-management factors are activated during the specific response of selected insects to B. bassiana. This too occurs primarily in the integument, and contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host’s own immune responses during the battle between host and pathogen.No other study to date has examined so many genes in this context. Indeed, we show that the epidermis has a great capacity to express defense and stress-management genes as well as the fat body (which is the main tissue producing antimicrobial peptides and has been the traditional focus of attention). We therefore propose a “be specific / fight locally / de-stress” model to explain resource allocation and defence priorities for insects selected for superior resistance to insect-pathogenic fungi. However, we also show that these insects are less fecund and probably at no evolutionary advantage in the wild, implying that the risk is small of biological control agents failing in the field

Chemical and Biological Aspects of Nutritional Immunity - Perspectives for New Anti-infectives Targeting Iron Uptake Systems : Perspectives for New Anti-infectives Targeting Iron Uptake Systems

Upon bacterial infection, one of the defense mechanisms of the host is the withdrawal of essential metal ions, in particular iron, which leads to "nutritional immunity". However, bacteria have evolved strategies to overcome iron starvation, for example, by stealing iron from the host or other bacteria through specific iron chelators with high binding affinity. Fortunately, these complex interactions between the host and pathogen that lead to metal homeostasis provide several opportunities for interception and, thus, allow the development of novel antibacterial compounds. This Review focuses on iron, discusses recent highlights, and gives some future perspectives which are relevant in the fight against antibiotic resistance

Siderophore-microcins, antimicrobial peptides with potent activity against Enterobacteriaceae: structure, biosynthesis and recognition

Microcins are gene-encoded antimicrobial peptides with low-molecular masses (< 10 kDa) secreted by Enteriobacteriaceae. They play a role in microbial competitions within the intestinal microflora by exerting potent antibacterial activity against phylogenetically-related bacteria, with minimal inhibitory concentrations in the nanomolar range. We have previously characterized the first siderophore-microcin, MccE492m, an 84-residue peptide that carries a linear trimer of dihydroxybenzoyl serine (DHBS), a catechol-type siderophore. MccE492m is the modified form of microcin E492 (MccE492). Escherichia coli Nissle 1917 (O6:K5:H1) is a non-pathogenic commensal faecal isolate forming the basis of the probiotic preparation Mutaflor® used for treatment of various gastrointestinal disorders and diseases. E. coli Nissle 1917 has been shown to produce two bactericidal activities identified as microcins H47 and M (MccM). The genetic system responsible for production and secretion of MccM and for immunity of the producing strain to the microcin has been sequenced previously. However, MccM had never been isolated until now. Here we describe the expression and purification of MccM and show that it carries such a posttranslational modification. We also characterize the recognition mechanisms and biosynthetic pathways of this novel class of potent antibacterial peptides

Microcin M, an antibacterial peptide from the probiotic bacterium Escherichia coli Nissle 1917

Escherichia coli Nissle 1917 (O6:K5:H1) is a non-pathogenic commensal faecal isolate forming the basis of the probiotic preparation Mutaflor® used for treatment of various gastrointestinal disorders and diseases. E. coli Nissle 1917 has been shown to produce two bactericidal activities identified as microcins H47 and M (MccM). Microcins are gene-encoded antibacterial peptides with molecular masses inferior to 10 kDa secreted by Enterobacteriaceae. They exert bactericidal activity directed against phylogenetically-related bacteria, using complex mechanisms that involve various bacterial targets. Previously, the genetic system responsible for production and secretion of MccM and for immunity of the producing strain to the microcin has been sequenced, but MccM has never been isolated until now. In the present work, we expressed, isolated and purified MccM. Mass spectrometry of MccM identified a molecular mass of 7282 Da and consequently the cleavage site of the precursor. These data also suggested that MccM could carry a posttranslational modification similar to that characterized for microcin E492. Thus, MccM belongs probably to the recently characterized siderophore-peptide family

Microcins, Gene-Encoded Antibacterial Peptides from Enterobacteria

Review covering 1982 to 2006International audienceMicrocins are gene-encoded antibacterial peptides, with molecular masses below 10 kDa, produced by enterobacteria. They are secreted under conditions of nutrient depletion and exert potent antibacterial activity against closely related species. Typical gene clusters encoding the microcin precursor, the self-immunity factor, the secretion proteins and frequently the post-translational modification enzymes are located either on plasmids or on the chromosome. Contrary to most of the antibiotics of microbial origin, which are synthesized by multimodular enzymes termed peptide synthetases, microcins are ribosomally-synthesized as precursors further modified enzymatically. They form a restricted class of potent antibacterial peptides. Fourteen microcins have been reported until now, among which only seven have been isolated and characterized. Despite the low number of known representatives, microcins exhibit a diversity of structures and antibacterial mechanisms. This review gives an updated overview of microcin structures, biosyntheses, antibacterial activities and mechanisms of action. It shows how fascinating, clever and complex the mechanisms used by microcins are to kill target bacteria. Covering 1982 to 2006

Les microcines-sidérophores, peptides antibactériens parasitant les voies d'import du fer

Les microcines sont des peptides antibactériens de faible masse moléculaire produits par les entérobactéries. Elles sont synthétisées par la voie ribosomique et présentent une activité antibiotique très puissante (CMI < 1 µM) contre des souches phylogénétiquement proches de la souche productrice. Leurs structures sont diverses et originales, et leurs modes d'action variés. La microcine E492 (MccE492) possède une modification post-traductionnelle dont la structure est étroitement apparentée aux sidérophores (molécules capables de complexer le fer) de type catécholate, tels que l'entérobactine. Elle constitue le premier peptide-sidérophore décrit à ce jour. Nous avons montré que deux gènes du système génétique de MccE492 sont impliqués dans la synthèse de la modification post-traductionnelle et que l'entérobactine est un précurseur de cette modification. Ces résultats ont permis d'établir un modèle de biosynthèse de ce peptide-sidérophore. D'autre part, le système génétique de MccE492 présente de grandes similitudes avec ceux de trois autres microcines, MccM, MccH47 et MccI47, suggérant fortement que celles-ci pourraient aussi être des microcines-sidérophores. MccM a été isolée pour la première fois au laboratoire et des expériences de spectrométrie de masse ont montré qu'elle acquiert effectivement une modification post-traductionnelle similaire à celle de MccE492