Peptidomic and proteomic analyses of the systemic immune response of Drosophila

Insects have developed an efficient host defense against microorganisms, which involves humoral and cellular mechanisms. Numerous data highlight similarities between defense responses of insects and innate immunity of mammals. The fruit fly, Drosophila melanogaster, is a favorable model system for the analysis of the first line defense against microorganisms. Taking advantages of improvements in mass spectrometry (MS), two-dimensional (2D) gel electrophoresis and bioinformatics, differential analyses of blood content (hemolymph) from immune-challenged versus control Drosophila were performed. Two strategies were developed: (i) peptidomic analyses through matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and high performance liquid chromatography for molecules below 15 kDa, and (ii) proteomic studies based on 2D gel electrophoresis, MALDI-TOF fingerprinting and database searches, for compounds of greater molecular masses. The peptidomic strategy led to the detection of a large number of peptides induced in the hemolymph of challenged flies as compared to controls. Of these, 28 were characterized, amongst which were antimicrobial peptides. The 2D gel electrophoresis strategy led to the detection of 70 spots differentially regulated by at least fivefold after microbial infection. This approach yielded the identity of a series of proteins that were related to the Drosophila immune response, such as proteases, protease inhibitors, prophenoloxydase-activating enzymes, serpins and a Gram-negative binding protein-like protein. This strategy also brought to light new candidates with a potential function in the immune response (odorant-binding protein, peptidylglycine alpha-hydroxylating monooxygenase and transferrin). Interestingly, several molecules resulting from the cleavage of proteins were detected after a fungal infection. Together, peptidomic and proteomic analyses represent new tools to characterize molecules involved in the innate immune reactions of Drosophila.     

Nematodes, bacteria, and flies: a tripartite model for nematode parasitism

More than a quarter of the world's population is infected with nematode parasites, and more than a hundred species of nematodes are parasites of humans [1-3]. Despite extensive morbidity and mortality caused by nematode parasites, the biological mechanisms of host-parasite interactions are poorly understood, largely because of the lack of genetically tractable model systems. We have demonstrated that the insect parasitic nematode Heterorhabditis bacteriophora, its bacterial symbiont Photorhabdus luminescens, and the fruit fly Drosophila melanogaster constitute a tripartite model for nematode parasitism and parasitic infection. We find that infective juveniles (IJs) of Heterorhabditis, which contain Photorhabdus in their gut, can infect and kill Drosophila larvae. We show that infection activates an immune response in Drosophila that results in the temporally dynamic expression of a subset of antimicrobial peptide (AMP) genes, and that this immune response is induced specifically by Photorhabdus. We also investigated the cellular and molecular mechanisms underlying IJ recovery, the developmental process that occurs in parasitic nematodes upon host invasion and that is necessary for successful parasitism. We find that the chemosensory neurons and signaling pathways that control dauer recovery in Caenorhabditis elegans also control IJ recovery in Heterorhabditis, suggesting conservation of these developmental processes across free-living and parasitic nematodes.     

Immunopeptides in the defense reactions of Glossina morsitans to bacterial and Trypanosoma brucei brucei infections

Several dipteran insects are vectors of parasites causing major human infectious diseases. Among these, the tsetse fly, Glossina spp., is responsible for the transmission of trypanosomes, the pathogens responsible for sleeping sickness in Africa. A better understanding of insect-parasite interactions will help establish new strategies to fight this important often fatal disease. Antimicrobial peptides (AMPs) are part of the humoral immune response in insects during bacterial, fungal and parasitic infections. Here, we studied the immune response of Glossina morsitans to bacteria and to Trypanosoma brucei brucei by analyzing the synthesis of AMPs as markers of the humoral immune response. By reversed-phase chromatography, mass spectrometry analysis, Edman degradation and in vitro antimicrobial assays of the hemolymph of immune-challenged adults of G. morsitans, we identified three AMPs: a cecropin, an attacin and a defensin. These three AMPs were found to be induced upon systemic bacterial infection and also after per os infections by bacteria and parasites.     

The evolution of antifungal peptides in Drosophila

An essential component of the immune system of animals is the production of antimicrobial peptides (AMPs). In vertebrates and termites the protein sequence of some AMPs evolves rapidly under positive selection, suggesting that they may be coevolving with pathogens. However, antibacterial peptides in Drosophila tend to be highly conserved. We have inferred the selection pressures acting on Drosophila antifungal peptides (drosomycins) from both the divergence of drosomycin genes within and between five species of Drosophila and polymorphism data from Drosophila simulans and D. melanogaster. In common with Drosophila antibacterial peptides, there is no evidence of adaptive protein evolution in any of the drosomycin genes, suggesting that they do not coevolve with pathogens. It is possible that this reflects a lack of specific fungal and bacterial parasites in Drosophila populations. The polymorphism data from both species differed from neutrality at one locus, but this was not associated with changes in the protein sequence. The synonymous site diversity was greater in D. simulans than in D. melanogaster, but the diversity both upstream of the genes and at nonsynonymous sites was similar. This can be explained if both upstream and nonsynonymous mutations are slightly deleterious and are removed more effectively from D. simulans due to its larger effective population size.     

Insect inhibitors of metalloproteinases

Two types of peptidic metalloproteinase inhibitors have recently been identified in insects. A homologue of vertebrate tissue inhibitors of metalloproteinases (TIMPs) was found in the fruitfly Drosophila melanogaster which may contributes to regulation of a corresponding matrix metalloproteinase (MMP). The first member of MMPs from insects which shares similarity with vertebrate MMPs has also been cloned and characterized from Drosophila, suggesting conserved evolution of both MMPs and TIMPs. The first insect inhibitor of metalloproteinases (IMPI), which was identified in larvae of the greater wax moth, Galleria mellonella, shares no sequence similarity with known vertebrate or invertebrate proteins and represents the first non-TIMP-like inhibitor of metalloproteinases reported to date. In contrast to TIMPs, the IMPI is not active against MMPs but inhibits microbial metalloproteinases such as bacterial thermolysin. Insects may recognize such toxic metalloproteinases associated with invading pathogens by particular peptidic fragments that result from their nonregulated activity within the hemolymph. Metalloproteinases induce expression of the IMPI along with other antimicrobial proteins in course of humoral immune response of G. mellonella, thereby mediating regulation of metalloproteinase activity released within the hemolymph and inhibition of pathogen development as well.     

Primary structure and in vitro antibacterial properties of the Drosophila melanogaster attacin C Pro-domain

In Drosophila melanogaster, seven distinct families of antimicrobial peptides with different structures and specificities are synthesized by the fat body and released into the hemolymph during the immune response. Using microscale high performance liquid chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and Edman degradation, we have isolated and characterized from immune-challenged Drosophila two novel induced molecules, under the control of the Imd pathway, that correspond to post-translationally modified antimicrobial peptides or peptide fragments. The first molecule is a doubly glycosylated form of drosocin, an O-glycosylated peptide that kills Gram-negative organisms. The second molecule represents a truncated form of the pro-domain of the Drosophila attacin C carrying two post-translational modifications and has significant structural similarities to proline-rich antibacterial peptides including drosocin. We have synthesized this peptide and found that it is active against Gram-negative bacteria. Furthermore, this activity is potentiated when the peptide is used in combination with the Drosophila antimicrobial peptide cecropin A. The synergistic action observed between these two molecules suggests that the truncated post-translationally modified pro-domain of attacin C by itself may play an important role in the antimicrobial defense of Drosophila.     

A drosomycin-GFP reporter transgene reveals a local immune response in Drosophila that is not dependent on the Toll pathway.

A hallmark of the systemic antimicrobial response of Drosophila is the synthesis by the fat body of several antimicrobial peptides which are released into the hemolymph in response to a septic injury. One of these peptides, drosomycin, is active primarily against fungi. Using a drosomycin-green fluorescent protein (GFP) reporter gene, we now show that in addition to the fat body, a variety of epithelial tissues that are in direct contact with the external environment, including those of the respiratory, digestive and reproductive tracts, can express the antifungal peptide, suggesting a local response to infections affecting these barrier tissues. As is the case for vertebrate epithelia, insect epithelia appear to be more than passive physical barriers and are likely to constitute an active component of innate immunity. We also show that, in contrast to the systemic antifungal response, this local immune response is independent of the Toll pathway.     

Differential Expression of Immune Defences Is Associated with Specific Host-Parasite Interactions in Insects

Recent ecological studies in invertebrates show that the outcome of an infection is dependent on the specific pairing of host and parasite. Such specificity contrasts the long-held view that invertebrate innate immunity depends on a broad-spectrum recognition system. An important question is whether this specificity is due to the immune response rather than some other interplay between host and parasite genotypes. By measuring the expression of putative bumblebee homologues of antimicrobial peptides in response to infection by their gut trypanosome Crithidia bombi, we demonstrate that expression differences are associated with the specific interactions.     

A model of bacterial intestinal infections in Drosophila melanogaster

Serratia marcescens is an entomopathogenic bacterium that opportunistically infects a wide range of hosts, including humans. In a model of septic injury, if directly introduced into the body cavity of Drosophila, this pathogen is insensitive to the host's systemic immune response and kills flies in a day. We find that S. marcescens resistance to the Drosophila immune deficiency (imd)-mediated humoral response requires the bacterial lipopolysaccharide O-antigen. If ingested by Drosophila, bacteria cross the gut and penetrate the body cavity. During this passage, the bacteria can be observed within the cells of the intestinal epithelium. In such an oral infection model, the flies succumb to infection only after 6 days. We demonstrate that two complementary host defense mechanisms act together against such food-borne infection: an antimicrobial response in the intestine that is regulated by the imd pathway and phagocytosis by hemocytes of bacteria that have escaped into the hemolymph. Interestingly, bacteria present in the hemolymph elicit a systemic immune response only when phagocytosis is blocked. Our observations support a model wherein peptidoglycan fragments released during bacterial growth activate the imd pathway and do not back a proposed role for phagocytosis in the immune activation of the fat body. Thanks to the genetic tools available in both host and pathogen, the molecular dissection of the interactions between S. marcescens and Drosophila will provide a useful paradigm for deciphering intestinal pathogenesis.     

果蝇先天性免疫研究进展

果蝇是生命科学与人类疾病研究的重要模式生物,虽然不具有人类高度专一的获得性免疫,但也有对病原微生物感染作出快速有效反应的先天性免疫应答系统,主要包括体液免疫,细胞免疫和黑化反应。文章结合国外最新研究,详细介绍果蝇体液免疫中控制抗菌肽合成的Toll信号通路和Imd信号通路中涉及的蛋白及其相互作用,并对果蝇细胞免疫中的吞噬、包埋功能和黑化反应作简要阐述。研究表明,果蝇的Toll和Imd信号通路分别与人类的TLR4和TNRF-1信号通路存在着惊人的相似之处,说明果蝇与人类在免疫调控通路方面可能存在着共同的进化起源。     

Pefabloc – A sulfonyl fluoride serine protease inhibitor blocks induction of Diptericin in Drosophila l(2)mbn cells

Abstract Insects protect themselves against microbial infection by an efficient innate immune system that is activated by recognition of invariant microbial surface molecules. In the fruit fly Drosophila melanogaster the presence of bacteria is associated with expression of antimicrobial peptides in host immune‐competent tissues. Host receptors detect infection and relay the signal to mount the appropriate immune response. In Drosophila hemocyte‐like l(2)mbn cells pre‐infection treatment with Pefabloc, a commonly used serine protease inhibitor, induced two major effects: it blocked expression of the antibacterial peptide Diptericin in response to live Gram‐negative bacteria and bacterial surface molecules (crude lipopolysaccharide contaminated by peptidoglycans) and it induced morphological changes.     

Interactions between the cellular and humoral immune responses in Drosophila

Drosophila has highly efficient defenses against infection. These include both cellular immune responses, such as the phagocytosis of invading microorganisms, and humoral immune responses, such as the secretion of antimicrobial peptides into the hemolymph [1] [2]. These defense systems are thought to interact, but the nature and extent of these interactions is not known. Here we describe a method for inhibiting phagocytosis in Drosophila blood cells (hemocytes) by injecting polystyrene beads into the body cavity. This treatment does not in itself make a fly susceptible to Escherichia coli infection. However, when performed on flies carrying the mutation immune deficiency (imd), which affects the humoral immune response [3], the treatment results in a striking decrease in resistance to infection. We therefore carried out a sensitized genetic screen to identify immunocompromised mutants by co-injecting beads and E. coli. From this screen, we identified a new gene we have named red shirt and identified the caspase Dredd as a regulator of the Drosophila immune response. The observation that mutants with defects in the humoral immune response are further immunocompromised by blocking phagocytosis, and thus inhibiting the cellular immune response, shows that the Drosophila cellular and humoral immune responses act in concert to fight infection.     

Proteomics of immune-challenged Drosophila melanogaster larvae hemolymph

In the last decade, the fruit fly Drosophila melanogaster has emerged as a promising invertebrate model for the investigation of innate immunity, in part because of its well characterised genetics. The information provided by the innumerous reports on Drosophila's immune response indicates that a large number of genes, in addition to the well-known antimicrobial peptide genes, are both up- and down-regulated upon immune challenge. Nevertheless, their contribution to fighting off infection has not been seriously addressed. With the application of recent advances in proteomics, the effects of an immune challenge in the overall modification of Drosophila 2-DE protein patterns were investigated. The aim of this study was to investigate hemolymph proteins differentially expressed between control and immunised larvae sets, which could be related solely to the Drosophila immune response. The list of immune-related protein spots included heat shock proteins and other proteins with chaperone properties, serine proteases, phenol oxidase, and Drosophila antioxidant system components, which accounted for 21% of the total of 70 identified proteins, metabolic enzymes implicated in pathways such as cellular respiration, fatty-acid oxidation, protein biosynthesis, and structural proteins.     

Galleria mellonella infected with Bacillus thuringiensis involves Hsp90

Insects are good models for studying the innate immune response. We report that Galleria mellonella larvae infected with entomopathogenic bacteria Bacillus thuringiensis kurstaki show changes in the level of Hsp90. Our experimental approach was to pre-treat larvae with the Hsp90-binding compound, 17-DMAG, before infection with B. thuringiensis. We show that pre-treated animals display a higher level of immune response. This was mainly manifested by enhanced action of their hemolymph directed toward living bacteria as well as lysozyme activity digesting bacterial peptidoglycan. The observed phenomenon was due to the higher activity of antimicrobial peptides which, in contrast to healthy animals, was detected in the hemolymph of the immunestimulated larvae. Finally, the physiological significance of our observation was highlighted by the fact that G. mellonella pre-treated with 17-DMAG showed a prolonged survival rate after infection with B. thuringiensis than the control animals. Our report points to a role for Hsp90 in the immune response of G. mellonella after infection with B. thuringiensis at the optimal growth temperature.