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The Drosophila protein mustard tailors the innate immune response activated by the immune deficiency pathway.

ABSTRACT: In this study, we describe a Drosophila melanogaster transposon insertion mutant with tolerance to Vibrio cholerae infection and markedly decreased transcription of diptericin as well as other genes regulated by the immune deficiency innate immunity signaling pathway. We present genetic evidence that this insertion affects a locus previously implicated in pupal eclosion. This genetic locus, which we have named mustard (mtd), contains a LysM domain, often involved in carbohydrate recognition, and a TLDc domain of unknown function. More than 20 Mtd isoforms containing one or both of these conserved domains are predicted. We establish that the mutant phenotype represents a gain of function and can be replicated by increased expression of a short, nuclearly localized Mtd isoform comprised almost entirely of the TLDc domain. We show that this Mtd isoform does not block Relish cleavage or translocation into the nucleus. Lastly, we present evidence suggesting that the eclosion defect previously attributed to the Mtd locus may be the result of the unopposed action of the NF-?B homolog, Relish. Mtd homologs have been implicated in resistance to oxidative stress. However, to our knowledge this is the first evidence that Mtd or its homologs alter the output of an innate immunity signaling cascade from within the nucleus.

SUBMITTER: Wang Z

PROVIDER: S-EPMC3324637 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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The Drosophila protein mustard tailors the innate immune response activated by the immune deficiency pathway.

Wang Zhipeng Z Berkey Cristin D CD Watnick Paula I PI

Journal of immunology (Baltimore, Md. : 1950) 20120316 8

In this study, we describe a Drosophila melanogaster transposon insertion mutant with tolerance to Vibrio cholerae infection and markedly decreased transcription of diptericin as well as other genes regulated by the immune deficiency innate immunity signaling pathway. We present genetic evidence that this insertion affects a locus previously implicated in pupal eclosion. This genetic locus, which we have named mustard (mtd), contains a LysM domain, often involved in carbohydrate recognition, and ...[more]

PMID: 22427641

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Project description:The insect immune deficiency (IMD) pathway is a defense mechanism that senses and responds to Gram-negative bacteria. Ticks lack genes encoding upstream components that initiate the IMD pathway. Despite this deficiency, core signaling molecules are present and functionally restrict tick-borne pathogens. The molecular events preceding activation remain undefined. Here, we show that the unfolded-protein response (UPR) initiates the IMD network. The endoplasmic reticulum (ER) stress receptor IRE1α is phosphorylated in response to tick-borne bacteria but does not splice the mRNA encoding XBP1. Instead, through protein modeling and reciprocal pulldowns, we show that Ixodes IRE1α complexes with TRAF2. Disrupting IRE1α-TRAF2 signaling blocks IMD pathway activation and diminishes the production of reactive oxygen species. Through in vitro, in vivo, and ex vivo techniques, we demonstrate that the UPR-IMD pathway circuitry limits the Lyme disease-causing spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale (anaplasmosis). Altogether, our study uncovers a novel linkage between the UPR and the IMD pathway in arthropods. IMPORTANCE The ability of an arthropod to harbor and transmit pathogens is termed "vector competency." Many factors influence vector competency, including how arthropod immune processes respond to the microbe. Divergences in innate immunity between arthropods are increasingly being reported. For instance, although ticks lack genes encoding key upstream molecules of the immune deficiency (IMD) pathway, it is still functional and restricts causative agents of Lyme disease (Borrelia burgdorferi) and anaplasmosis (Anaplasma phagocytophilum). How the IMD pathway is activated in ticks without classically defined pathway initiators is not known. Here, we found that a cellular stress response network, the unfolded-protein response (UPR), functions upstream to induce the IMD pathway and restrict transmissible pathogens. Collectively, this explains how the IMD pathway can be activated in the absence of canonical pathway initiators. Given that the UPR is highly conserved, UPR-initiated immunity may be a fundamental principle impacting vector competency across arthropods.

Project description:This project aims to define, by using a functional proteomic approach, the molecular partners associated with the canonical coupound of the IMD pathway. The Drosophila defense against pathogens largely relies on the activation of two major pathways, IMD and TOLL. The IMD pathway is activated mainly by Gram-negative bacteria, whereas the TOLL pathway responds predominantly to Gram-positive bacteria and fungi. The activation of this pathway leads to the rapid induction via NF-kB of numerous genes of the immune system, including various antimicrobial peptide genes. Recent evidence indicates that the IMD pathway is also involved in various other reactions in the absence of infection ("inflammatory-like" reactions). To gain a better understanding of the molecular machineries underlying the pleiotropic functions of this pathway, we have developed a proteomics analysis. Our first aim was to identify the proteins interacting with the 11 canonical members (PGRP-LC, PGRP-LE, IMD, BG4 (dFADD), DREDD, TAK1, TAB2, IAP2, IRD5 (DmIKKbeta), KEY (DmIKKgamma), and RELISH) previously shown to be involved in the activation of the IMD pathway. Each protein was fused N-terminally or C-terminally with a biotin tag and was stably expressed in S2 cells previously subjected to stable integration of the bacterial enzyme BirA to allow for biotinylation of the tags. In total, we established 22 stable transformant cell lines (11 genes x 2 tag locations), which were individually stimulated by heat-killed E.coli before harvest at 4 different time points (t=0 min, 10 min, 2 hour, and 8 hour). This procedure was followed by streptavidin-mediated affinity purification of 96 bait-protein complexes (22 transformants x 4 time points, plus 8 unstimulated controls without baits), on-bead trypsin digestions of the protein complexes and six LC-MS/MS analyses. We have identified 371 interactanting proteins in heat-killed E.coli stimulated Drosophila S2 cells, 90% of which have human orthologues. Comparative analysis of Gene Ontology (GO) annotation datasets from Human and Flies point to three significant common categories: (1) NuA4 Histone acetyltransferase complex, (2) SWI/SNF-type Chromatin remodeling complex, and (3) Transcription cofactor binding. Our Drosophila data point in particular to SUMOylation of the IkB-kinase homologue Ird5 in Drosophila, a level of regulation not described to date. Among numerous directions opened by our proteomics analysis, we have decided to first investigate this facet of the IMD pathway. We show nthat a highly conserved SUMOylation consensus site binds Drosophila SUMO (SMT3) in a challenge-dependent manner and that this process confers an increased level of activation of the antimicrobial peptide genes during bacterial challenge both in vitro and in flies. Our results demonstrate that SUMOylation of IKKbeta plays an important role in the induction of antimicrobial peptide genes. The MS analysis was performed on a FT ICR mass spectrometer (LTQ-FT Ultra, ThermoFisher Scientific, San Jose, CA) with the top 7 acquisition method: MS resolution 60,000, mass range 500-2000 Th, followed by 7 MS/MS (LTQ) on the 7 most intense peaks, with a dynamic exclusion for 90 s. The raw data were processed using Xcalibur 2.0.7 software. Each sample was first analyzed in triplicate then an exclusion list was added for three next runs. The database search was done on merged data using Mascot search engine (Matrix Science Mascot 2.3) on the 17D melanogaster databank (16535 sequences). Proteome Discoverer 1.3 (ThermoFisher Scientific) and Mascot were used to search the data and filter the results. The following parameters were used: up to 2 miss cleavages; MS tolerance 10 ppm; MSMS tolerance 1 Da; full tryptic peptides; partial modifications carbamidomethyla- tion (C), oxydation (M, H, W), Phosphorylation (Y).

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The Drosophila protein mustard tailors the innate immune response activated by the immune deficiency pathway.

Publications

The Drosophila protein mustard tailors the innate immune response activated by the immune deficiency pathway.

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