A proteomic approach for the analysis of instantly released wound and immune proteins in Drosophila melanogaster hemolymph

Abstract

Insects respond to microbial infection by the rapid and transient expression of several genes encoding antibacterial peptides. In this paper we describe a powerful technique, two-dimensional difference gel electrophoresis, that, when combined with mass spectrometry, can be used to study the immune response of Drosophila melanogaster at the protein level. By comparatively analyzing the hemolymph proteome of 2,000 third-instar Drosophila larvae, we identified 10 differential proteins that appear in the fruit fly hemolymph very early after an immune-challenge with lipopolysaccharides. These proteins can be assigned to the immune response, because they are not induced after sterile injury. Reduction of intergel variability or quantification problems related to conventional two-dimensional electrophoresis and improvement of image analysis were achieved by the use of two fluorescent dyes to label the two different protein samples. Some of the immune-induced proteins, such as thioestercontaining protein 2, can be assigned to specific aspects of the immune response; others were already reported as being involved in stress response. An immune-induced protein (CG18594) is homologous to a mammalian serine protease inhibitor that mediates the mitogen-activated protein kinase and the NF-B signaling pathways. In addition, a number of proteins that had not been associated with the immune response before were isolated and identified, and some of these were still present in the hemolymph 4 h after injury. Determining the function of all of these immune-induced proteins represents an exciting challenge for increasing our knowledge of insect immunity.immune response proteins ͉ two-dimensional difference gel electrophoresis ͉ stress response ͉ secretome ͉ innate immunity

Pre-exposure to yeast protects larvae of Galleria mellonella from a subsequent lethal infection by Candida albicans and is mediated by the increased expression of antimicrobial peptides

“…Induction of expression of immune relevant proteins of G. mellonella by RT PCR Larval RNA was extracted using TRI-reagent at 1,4,8,24 and 48 h post-infection. RNA (2 mg) was treated with DNase I prior to cDNA synthesis using the SuperScript Kit (Invitrogen) with oligo(dT) primers.…”

“…AMPs are released from a range of organs and cells [5,6] into the haemolymph of the insect where they diffuse to the site of infection and attack components of the bacterial or fungal cell wall [7]. Haemocytes, the fat body and the digestive tract secrete antimicrobial proteins and peptides into the insect haemolymph, which performs many functions analogous to mammalian serum [5,6,8]. The similarity of a range of insect immune responses with vertebrate innate immune responses to infection has been highlighted by the discovery of the Toll receptors in insects and their similarity with the toll like receptors (TLR) in mammals and 11 members of this family have been identified in humans [9].…”

Pre-exposure ofGalleria mellonellalarvae to different doses ofAspergillus fumigatusconidia causes differential activation of cellular and humoral immune responses

“…Ferritin contributes to cellular iron homeostasis by sequestering free iron and has been demonstrated to show increased expression following bacterial expression in Drosophila melanogaster. 26 The antimicrobial protein apolipophorin (spot 4, gi:50404098, mass: 167 kDa, score 132, peptides matched: 3, coverage: 28%) showed a 1.7-fold increase in larvae that received an inoculum of 1 x 10 4 conidia. Apolipophorin plays a role in the insect immune response and has been shown to display functionality in the response to bacterial challenge where coagulation of apolipophorin takes place to form a globular structure resulting from oligomeric interactions with lipopolysaccharide thus neutralizing LPS from the surrounding environment.…”

“…This balancing of iron availability has been demonstrated by others to play a key role in the immune system/fungal iron sequesterization balance. 26,28 It is possible that enhanced synthesis of ferritin acts as a means of sequestering iron from Aspergillus siderophores and may play a role in protecting the host against the higher fungal inoculum by depleting the fungus of its iron requirement. The increased expression of apolipophorin (apo 1) is interesting in the light of work of Ma et al where the oligomeric interactions with LPS initiate particle neutralization.…”

Proteomic Studies of Lipopolysaccharide-induced Polypeptides in the Silkworm,Bombyx mori

“…Very recently, the significance of the proteomic approach for the analysis of polypeptides induced with LPS in hemolymph of the fruit fly Drosophila melanogaster has been reported. 13) Although there might be a difference between dipteran and lepidopteran insects, proteomic knowledge of insect innate immunity is still very limited in comparison with genomic knowledge of it. Further proteomic work is in progress in our laboratory.…”