Mise en evidence d'une nouvelle famille de peptides antibacteriens chez un insecte diptere: Phormia terranovae

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Insect immunity

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

A new diterpene enone from the soldier beetle Cantharis livida (Coleoptera: Cantharidae)

Cantharenone 1, a new diterpene structurally related to the prenylbisabolane skeleton, has been isolated from the beetle Cantharis livida (Cantharidae). Its structure has been determined on the basis of its spectral properties. This is the first report of such a type of diterpenoid from an insect. ©ARKAT-USA, Inc.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

A new diterpene enone from the soldier beetle Cantharis livida (Coleoptera : Cantharidae)

Cantharenone, a new diterpene structurally related to the prenylbisabolane skeleton, has been isolated from the beetle Cantharis livida (Cantharidae). Its structure has been determined on the basis of its spectral properties. This is the first report of such a type of diterpenoid from an insect

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

Insect immunity

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 Freund's 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)

Determination of disulfide bridges in natural and recombinant insect defensin A

The primary-structure comparison of natural insect defensin A from Phormia terranovae and recombinant insect defensin A from Saccharomyces cerevisiae has been accomplished using a combination of Edman degradation and liquid secondary ion mass spectrometry. The natural and recombinant proteins have the same primary structure with identical disulfide-bond designations (formula; see text) as determined from the peptides obtained after thermolysin digestion. The combined use of Edman degradation and mass spectometry allowed the disulfide-bridge structure to be determined with a total of only 40 micrograms (9.9 nmol) natural peptide. Mass spectrometry provides a rapid means of disulfide-bridge verification, requiring not more than 20 micrograms recombinant insect defensin A, which is compatible with use in batch analysis

Digital transformation in healthcare. The challenges of translating knowledge in a primary research, educational and clinical centre

The global healthcare system is currently facing an increasing technological swift, also driven by the need to engage the patients and other stakeholders in developing healthcare products and services. Knowledge Translation (KT) is getting increasing attention both from scholars as well as managers and professionals, due to its crucial role in fostering innovation, when most actors share different backgrounds, competencies, and skills. Such features represent severe challenges to the sharing and transfer of knowledge. The various actors need effective KT enablers to share, transfer, and create new knowledge. This paper aims to investigate such dynamics through a qualitative case, analyzing how KT is managed at IHU Strasbourg, the Institute of Image-Guided Surgery, one primary research, clinical, and educational centre, together with the Research Institute Against Digestive Cancers (IRCAD), the surgical minimally-invasive training centre. At such Institutes, several digital technologies are implemented and adopted to foster innovation in new surgical tools and methodologies. Findings demonstrate that KT at IHU Strasbourg/IRCAD can be defined at four different levels, corresponding to the Institutes’ aims: education, innovation, technological transfer, and care. For each dimension, a galaxy of stakeholders, KT processes and flows, and instruments emerge. KT enablers vary, from the ones more linked to digital technologies and procedures to the ones that are more creative and based on the soft and interpersonal skills of the professionals involved. Results suggest a mix of different methods, and the search for always new techniques