Jean-Luc Dimarcq

Antimicrobial peptides in insects; structure and function

Antimicrobial peptides appear to be ubiquitous and multipotent components of the innate immune defense arsenal used by both prokaryotic and eukaryotic organisms. During the past 15 years a multitude of these peptides have been isolated largely from insects. In spite of great differences in size, amino acid composition and structure, most of the antimicrobial peptides from insects can be grouped into one of three categories. The largest category in number contains peptides with intramolecular disulfide bonds forming hairpin-like beta-sheets or alpha-helical-beta-sheet mixed structures. The second most important group is composed of peptides forming amphipathic alpha-helices. The third group comprises peptides with an overrepresentation in proline and/or glycine residues. In general, the insect antimicrobial peptides have a broad range of activity and are not cytotoxic. Despite a wealth of information on structural requirements for their antimicrobial activity, the mode of action of these peptides is not yet fully understood. However, some data suggest the existence of two types of mode of action: 1. through peptide-lipid interaction or 2. through receptor-mediated recognition processes. This review presents the main results obtained during the last four years in the field of antimicrobial peptides from insects with a special focus on the proline-rich and cysteine-rich peptides.

Insect immunity: developmental and inducible activity of the Drosophila diptericin promoter.

Diptericins are 9 kDa inducible antibacterial peptides initially isolated from immune haemolymph of Phormia (Diptera). Following the isolation of a Drosophila cDNA encoding a diptericin homologue, we have now cloned a genomic fragment containing the Drosophila diptericin gene. To dissect the regulation of this gene, we have transformed flies with a fusion gene in which the reporter beta‐galactosidase gene is under the control of 2.2 kb upstream sequences of the diptericin gene. We show that such a fusion gene is inducible by injection of live bacteria or complete Freunds adjuvant and respects the tissue specific expression pattern of the resident diptericin gene. Our analysis reveals at least four distinct phases in the regulation of this gene: young larvae, late third instar larvae, pupae and adults. This complexity may be related to the presence in the upstream sequences of multiple copies of response elements previously characterized in genes encoding acute phase response proteins in mammals (e.g. NK‐kappa B, NF‐kappa B related, NF‐IL6 response elements).

Treatment of l(2)mbn Drosophila tumorous blood cells with the steroid hormone ecdysone amplifies the inducibility of antimicrobial peptide gene expression

Insects rely on both humoral and cellular mechanisms to defend themselves against microbial infections. The humoral response involves synthesis of a battery of potent antimicrobial peptides by the fat body and, to a lesser extent, by blood cells. The cellular response on the other hand consists of phagocytosis of small microorganisms and melanization and encapsulation of larger parasites. The l(2)mbn cell line, established from tumorous larval hemocytes, represents a system of choice to dissect the molecular events controlling cellular immunity. We report here that l(2)mbn cells can be efficiently induced to differentiate in adherent, macrophage-like cells by treatment with 20-hydroxyecdysone. Ecdysone treatment increases both the phagocytic capacity of l(2)mbn cells and their competence to express antimicrobial genes in response to immune challenge. We also report that expression of several regulatory molecules thought to be involved in the immune response is up-regulated by ecdysone in l(2)mbn cells.

Insect immunity: expression of the two major inducible antibacterial peptides, defensin and diptericin, in Phormia terranovae.

Injections of low doses of bacteria into larvae of Phormia terranovae induce the appearance of potent bactericidal peptides in the blood, among which predominate the anti‐Gram positive insect defensins and the anti‐Gram negative diptericins. Insect defensins show significant homologies to mammalian (including human) microbicidal peptides present in polymorphonuclear leukocytes and macrophages. We report the molecular cloning of cDNAs and primer extension studies which indicate that insect defensin is produced as a prepro‐peptide yielding mature defensin A (40 residues) after cleavage of a putative signal peptide (23 residues) and a prosequence (34 residues). Previous studies have established that diptericin (82 residues) is matured from a pre‐peptide by cleavage of a putative signal peptide (19 residues) and C‐terminal amidation. Using oligonucleotide probes complementary to the sequences of the mRNAs for defensin and diptericin, we show by in situ hybridization that both antibacterial peptides are concomitantly synthesized by the same cells: thrombocytoids, a specialized blood cell type, and adipocytes. Transcriptional studies based on hybridization of RNAs to cDNAs of defensin and diptericin indicate that the transcription of both genes is induced regardless of the nature of the stimulus (injection of Gram positive or Gram negative bacteria, lipopolysaccharides). Even a sterile injury applied to axenically raised larvae is efficient in inducing the transcription of both genes suggesting that the local disruption of the integument aspecifically initiates a signalling mechanism which the thrombocytoids and the adipocytes are able to interpret. The transcription of immune genes is relatively short lived and a second challenge yields a response similar to that of the first stimulus, indicating that the experimental insects do not keep a ‘memory’ of their first injection.

Conversion of a radiolabelled ecdysone precursor, 2,22,25-trideoxyecdysone, by embryonic and larval tissues of Locusta migratoria

A high specific activity tritiated ecdysone precursor, 2,22,25-trideoxyecdysone, was used to probe the capacity of various embryonic and larval tissues to perform the last 3 hydroxylation steps in ecdysone biosynthesis. Embryos at early stages of development, prior to the differentiation of their endocrine glands and embryonic heads, thoraces and abdomens of later stages, were found to have the capacity to hydroxylate the precursor to ecdysone. Larval epidermis and fat body are also able to transform 2,22,25-trideoxyecdysone into ecdysone; Malpighian tubules and midgut hydroxylate the precursor at C-2 but are apparently unable to hydroxylate both at C-22 and C-25. Larval prothoracic glands convert the precursor to ecdysone at a very efficient rate, which is 1-2 magnitudes higher than that of the other tissues investigated; several data argue for the existence of a privileged sequence of hydroxylations, C-25, C-22, C-2, in the larval prothoracic glands.

Lead optimization of antifungal peptides with 3D NMR structures analysis.

Antimicrobial peptides are key components of the innate immune response in most multicellular organisms. These molecules are considered as one of the most innovative class of anti‐infective agents that have been discovered over the last two decades, and therefore, as a source of inspiration for novel drug design. Insect cystein‐rich antimicrobial peptides with the CSαβ scaffold (an α‐helix linked to a β‐sheet by two disulfide bridges) represent particularly attractive templates for the development of systemic agents owing to their remarkable resistance to protease degradation. We have selected heliomicin, a broad spectrum antifungal CSαβ peptide from Lepidoptera as the starting point of a lead optimization program based on phylogenic exploration and fine tuned mutagenesis. We report here the characterization, biological activity, and 3D structure of heliomicin improved analogs, namely the peptides ARD1, ETD‐135, and ETD‐151. The ARD1 peptide was initially purified from the immune hemolymph of the caterpillars of Archeoprepona demophoon. Although it differs from heliomicin by only two residues, it was found to be more active against the human pathogens Aspergillus fumigatus and Candida albicans. The peptides ETD‐135 and ETD‐151 were engineered by site‐directed mutagenesis of ARD1 in either cationic or hydrophobic regions. ETD‐135 and ETD‐151 demonstrated an improved antifungal activity over the native peptides, heliomicin and ARD1. A comparative analysis of the 3D structure of the four molecules highlighted the direct impact of the modification of the amphipathic properties on the molecule potency. In addition, it allowed to characterize an optimal organization of cationic and hydrophobic regions to achieve best antifungal activity.

Expression and secretion in yeast of active insect defensin, an inducible antibacterial peptide from the fleshfly Phormia terranovae

Summary Insect defensin A is an inducible antibacterial peptide of the fleshfly Phormia terra-novae and has been recently characterized as a 40 residue basic peptide with six cysteines engaged in three intramolecular disulfide bridges. We report the expression of this peptide in Saccharomyces cerevisiae as a fusion protein carrying at its N-terminus leader sequences which are derived from the precursor of the yeast pheromone mating factor α (MFαl). These sequences allow the biologically active peptide to be secreted at high levels in a correctly processed form. Thus heterologous production of defensin A circumvents laborious purification of minute amounts of the peptide from its natural source. The insect defensin pro peptide can substitute for the MFαl pro peptide, indicating that yeast can process an insect pro sequence.

Thanatin activity on multidrug resistant clinical isolates of Enterobacter aerogenes and Klebsiella pneumoniae

Efflux pumps protect bacterial cells by ejecting intracellular toxic molecules such as antibiotics, detergents and defensins that have penetrated the cell envelope. Some of these efflux pumps recognise structurally unrelated compounds (mdr pump) and account for the resistance of some organisms to two or more agents. It would be of interest to identify molecules that are able to circumvent the problems created by multidrug resistance phenotypes during antibiotic therapy. We have studied the activity of thanatin, a 21-residue cationic antimicrobial peptide produced by an insect, against three bacterial species. The antibacterial effect depended on the size of lipopolysaccharide side chains. In clinically resistant isolates of Enterobacter aerogenes and Klebsiella pneumoniae, the biological activity of thanatin is independent of the membrane permeability, possibly controlled by one or more porins, and/or the expression of drug efflux pumps, two mechanisms which confer high level antibiotic resistance. In addition, thanatin was able to improve the activity of structurally unrelated antibiotics (norfloxacin, chloramphenicol, tetracycline) on a multidrug- resistant E. aerogenes clinical isolate.

Conversion studies of tritiated 2-deoxyecdysone during the embryonic development on Locusta migratoria

Abstract Eggs of Locusta have been reported previously to contain high concentrations (> 100 μM) of 2-deoxyecdysone, predominantly in conjugated form. Using high specific activity [ 3 H]2-deoxyecdysone (Hetru et al. , 1983), we have investigated the conversions of this molecule by embryos and the yolk compartment of eggs at a stage prior to the differentiation of the embryonic prothoracic glands. The results indicate the presence of two major and mutually exclusive pathways: C-2 hydroxylation yielding ecdysone, and 3-epimerisation leading to the formation of 3-epi-2-deoxyecdysone which in turn is conjugated at C-3. Both pathways can occur in embryos and in the yolk compartment; it is however apparent that 3-epimerisation is the predominant pathway in the yolk compartment.

Lawrence's book review unfair to Hoffmann.

Peter Lawrence [1xRank, reinvention and the Nobel Prize. Lawrence, P. Curr. Biol. 2012; 22: R214–R216Abstract | Full Text | Full Text PDFSee all References[1], in his otherwise scholarly review of a book by Peter Pringle “Experiment Eleven”, is incorrect in asserting that Jules Hoffmann took unfair credit for discoveries made in his laboratory, like exemplars described in the book. As close witnesses to events we know that Hoffmann assembled and animated a group of researchers from various scientific backgrounds to decipher the mechanisms of innate immunity in insects, and has been impeccable in his assignment of credit and support for his co-workers both while they served in his laboratory and in their future independent careers.All signatories are either long-standing collaborators or co-workers of Professor Jules Hoffmann.