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. 2017 Feb 14;8:14401.
doi: 10.1038/ncomms14401.

Infection-derived Lipids Elicit an Immune Deficiency Circuit in Arthropods

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Free PMC article

Infection-derived Lipids Elicit an Immune Deficiency Circuit in Arthropods

Dana K Shaw et al. Nat Commun. .
Free PMC article

Abstract

The insect immune deficiency (IMD) pathway resembles the tumour necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether unidentified chemical moieties activate the IMD signalling cascade remains unknown. Here, we show that infection-derived lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) stimulate the IMD pathway of ticks. The tick IMD network protects against colonization by three distinct bacteria, that is the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale. Cell signalling ensues in the absence of transmembrane peptidoglycan recognition proteins and the adaptor molecules Fas-associated protein with a death domain (FADD) and IMD. Conversely, biochemical interactions occur between x-linked inhibitor of apoptosis protein (XIAP), an E3 ubiquitin ligase, and the E2 conjugating enzyme Bendless. We propose the existence of two functionally distinct IMD networks, one in insects and another in ticks.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. The I. scapularis XIAP interactome.
Top 50 related genes interacting with the human (a) XIAP and (b) ML-IAP were compiled in a network according to GeneMANIA. Interactomes were visualized by using Cytoscape. Networks and biological pathways were integrated based on previously observed protein and genetic interactions, pathway, and co-localization assays. Proteins were grouped according to the GO functional categories available at DAVID. Size corresponds to the score given to each node. Edge type is based on the interaction map. Width is determined by scored evidence of interaction. (c) I. scapularis homologues were obtained with PSI-BLAST and GeneCard searches based on the human XIAP or the ML-IAP interactome. Sixty-eight candidates were grouped according to the GO functional categories available at DAVID. Immunity genes are represented in green and all candidates highlighted in red are predicted to be involved in the arthropod IMD pathway. See also Supplementary Figs 1–4 and Supplementary Table 1.
Figure 2
Figure 2. Bendless-XIAP molecular interactions.
(a) Protein differential expression on A. phagocytophilum infection of I. scapularis ticks based on iTRAQ proteomics data deposited on the Dryad repository database. (b) Structural docking model demonstrating the interaction between I. scapularis XIAP and the UbcH13 homologue, Bendless. (c,d) Native gel and ELISA analysis of a fixed amount of recombinant (0.2 μg) XIAP incubated with increasing amounts of recombinant Bendless. The analysis shown is one of two biological replicates. (e) XIAP-Bendless binding inhibition with a monoclonal antibody against the human homologue of Bendless (UbcH13). (d,e) The average of two technical replicates are plotted. (f) XIAP polyubiquitylation assay with recombinant Bendless (lane 1). Control conditions were performed in the absence of an E1 enzyme—Ube1 (lanes 2 and 6), XIAP (lanes 3, 5 and 6), wild-type ubiquitin (lanes 4, 11 and 12), Bendless (lanes 6, 7 and 9) and Uev1a (lanes 6, 8 and 9). Immunoblots were probed with antibodies specific for K63- (lanes 1–9) and K48- (lane 10) polyubiquitin chains or with a pan-ubiquitin antibody (lanes 11 and 12). GST was used as a negative control. The Western blot (WB) shown is one of two biological replicates. (g) Immunoprecipitation (IP) analysis followed by WB showing the interaction between XIAP and Bendless within HEK293 T cells transfected with the indicated vectors. Input indicates normalizing amounts. The WB shown is one of two biological replicates. (h) ELISA with protein extracts from unfed I. scapularis nymphs that were microinjected with either a scrambled control (scBendless) or siRNA specific for bendless (siBendless) and incubated with increasing amounts of recombinant XIAP. Points are the average of 10 biological replicates with two technical replicates each. See also Supplementary Figs 1–3 and 8–10.
Figure 3
Figure 3. The I. scapularis IMD pathway responds to A. phagocytophilum infection.
(ad) ISE6 (1 × 105) cells were infected with A. phagocytophilum (MOI 50) following targeted gene silencing of (a) xiap, (b) bendless/uev1a, (c) relish and (d) caspar. Gene silencing and A. phagocytophilum load (16 s rDNA) were measured by quantitative reverse transcriptase–PCR at 18 h post-infection in ISE6 cells. Replicates of 5 were expressed as means±s.e.m. All experiments shown are representative of five biological replicates with two technical replicates each. Student's t test. *P<0.05. scRNA, scrambled RNA; siRNA, small interference RNA. See also Supplementary Table 2.
Figure 4
Figure 4. The I. scapularis IMD pathway affects bacterial colonization in vivo.
(a) RNAi silencing of bendless/uev1a, relish and caspar in I. scapularis nymphs following tick feeding on (ac) A. phagocytophilum-infected or (df) B. burgdorferi-infected mice. Silencing levels and bacterial load were measured six days post-infection in whole I. scapularis nymphs. Samples represent the mean of 5–10 individual ticks, two technical replicates each,±standard errors of the means (SEM). Student's t test. *P<0.05. scRNA, scrambled RNA; siRNA, small interference RNA. See also Supplementary Table 2.
Figure 5
Figure 5. Infection-derived lipids stimulate the IMD pathway.
(a) Reference structures for the three lipids used in stimulation studies: (1) POPG, (2) PODAG and (3) MPPC. (b,c) Triplicate samples of 1 × 106 S2* cells were primed with 20-hydroxyecdysone (1 μM) and stimulated with 0.01–1 ng of indicated lipids, A. phagocytophilum (MOI 50) and positive controls for the Toll pathway (S. aureus) and the IMD pathway (E. coli peptidoglycan). Quantitative PCR (qPCR) quantifying diptericin and im1 transcripts are shown. (d) Triplicate samples of 1 × 106 S2* cells or (e) Five replicates of 1x105 ISE6 cells were incubated with 1 ng of indicated lipids before A. phagocytophilum infection at MOI 50. Bacterial load was quantified by qPCR and normalized to either rp49 (Drosophila) or β-actin (ISE6 tick cells). Data are represented as the mean±s.e.m. Analysis of variance-Dunnet. *P<0.05. NS, not significant. (−), non-stimulated. ISE6 (1 × 106) cells were stimulated with (f) diaminopimelic-type peptidoglycan (PGN) (10 μg/ml), A. phagocytophilum (MOI 50) and indicated lipids (1 ng) at indicated times or (g) the indicated ranges of A. phagocytophilum and lipids for 15 min. Lysates were probed against an I. scapularis Relish polyclonal antibody (Rel-N 41 kDa). β-Actin (45 kDa) was used as a loading control. (be) Data are representative of 3–5 biological replicates, as indicated, and two technical replicates. (fg) Western blots (WBs) shown represent one of three biological replicates. See also Supplementary Figs 5, 11 and 12 and Supplementary Tables 2 and 3.
Figure 6
Figure 6. Lipid priming is protective against bacterial colonization of ticks.
(a–h) Five replicates of 1 × 105 ISE6 cells were transfected with siRNA molecules targeting components of the I. scapularis immune system. (a,c,e,g) Silencing efficiency or (b,d,f,h) A. phagocytophilum load was measured for the components of the (ad) IMD, (ef) Toll and (gh) JAK-STAT pathways. Transfected cells were incubated with 1 ng of indicated lipids for 6 h and infected with A. phagocytophilum at MOI 50. Bacterial burden was quantified and normalized against β-actin. Data are represented as the mean±standard errors of the means (SEM). Analysis of variance (ANOVA)-Dunnet; Student's t-test. *P<0.05. NS, not significant. (−), non-stimulated. Data are representative of 5 biological replicates and two technical replicates. (i) D. andersoni ticks were mock- or lipid-injected (1 ng). Ticks were allowed to feed in individual group patches on a splenectomized, acute, A. marginale-infected calf for six days. Midguts from individual ticks were assessed for A. marginale infection levels by quantitative reverse transcriptase–PCR. Bacterial burden was quantified and normalized against β-actin. Samples represent the mean of 15-20 individual ticks±s.e.m. ANOVA-Dunnet. *P<0.05. NS, not significant. (−), non-primed. See also Supplementary Fig. 6 and Supplementary Tables 2 and 3.
Figure 7
Figure 7. The atypical IMD pathway in Chelicerates/Myriapods.
(a) BLAST was used to survey arthropod sequences. The Drosophila IMD was used as a query sequence. Confounding factors, such as sparsely populated data matrices, sequence misalignment and biased statistical confidence were removed. *Trilobites are an extinct subphylum. (b) The Rel homology domain sequence from D. melanogaster Relish was used to search arthropod transcripts for relish (class I) and other Rel domain-containing targets (dorsal and dif; class II) with tBLASTn. Two human NF-κB molecules served as outgroups. (c) I. scapularis PGRP sequences include PGRP-1: XM_002411731.1, PGRP-2: XM_002433644.1, PGRP-3: XM_002410377.1 and PGRP-4: XM_002413046.1. PGRP-1 was used to search chelicerate proteomes for PGRPs. Bootstrap values greater than or equal to 70 are shown. Yellow shading indicates chelicerate PGRP sequences, with I. scapularis PGRPs highlighted in red. Insect PGRPs are coloured gray and light blue. Blue labels and asterisks denote probable amidase activity based on the residues HHC in the active site. (b,c) MUSCLE was used to generate the multiple sequence alignment. The maximum likelihood phylogenetic tree was calculated with RAxML and re-sampled 100 times to assess clade support. See also Supplementary Figs 6 and 7.

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