Volume 23, Issue 12, Pages 3537-3550.e6 (June 2018) Analysis of Drosophila STING Reveals an Evolutionarily Conserved Antimicrobial Function  Marina Martin, Aoi Hiroyasu, R. Marena Guzman, Steven A. Roberts, Alan G. Goodman  Cell Reports  Volume 23, Issue 12, Pages 3537-3550.e6 (June 2018) DOI: 10.1016/j.celrep.2018.05.029 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 23, 3537-3550.e6DOI: (10.1016/j.celrep.2018.05.029) Copyright © 2018 The Authors Terms and Conditions

Figure 1 DmSTING Binds Cyclic Dinucleotides (A) Amino acid sequence alignment of the CDN-binding regions of human STING (hsSTING) with regions of dmSTING. Alpha helix and β sheet regions for hsSTING are indicated with a coil or arrow, respectively. Amino acid residues that interact with c-di-GMP directly or through water are highlighted, and the atoms with which they interact are indicated by a dotted line. Residues highlighted in red exhibit identity between hsSTING and dmSTING, while lighter shades of red represent similarity or nonsimilarity. (B) In-vitro-translated hsSTING, dmSTING, and C- or N-terminal deletion mutants (dST1–133, dST134–343) are pulled down with streptavidin beads coated with c-di-GMP-biotin. (C) In-vitro-translated deletion mutants lacking CDN-binding domains are pulled down with streptavidin beads coated with c-di-GMP-biotin. (D) Coomassie blue stain of 50 ng purified dmSTING 147–343 and deletion mutants lacking CDN-binding domains. (E) Purified dmSTING 147–343 is pulled down with streptavidin beads coated with c-di-GMP-biotin, but is reduced in the presence of competing unlabeled c-di-GMP. (F) Purified dmSTING 147-343 and a CDN2 deletion mutant is pulled-down by c-di-GMP-biotin, but proteins lacking domains 1 and 3 are not. Blots are representative of duplicate experiments. Cell Reports 2018 23, 3537-3550.e6DOI: (10.1016/j.celrep.2018.05.029) Copyright © 2018 The Authors Terms and Conditions

Figure 2 DmSTING Mediates an Immune Response to c-Di-GMP Stimulation and Listeria monocytogenes Infection (A) Drosophila S2 cells in biological duplicate were transfected with dsRNA for dmSTING RNAi or scrambled RNAi then mock transfected or transfected with c-di-GMP for 24 hr for microarray analysis. The heatmap shows those genes that are at least two-fold (Asymptotic ANOVA, p < 0.05) upregulated in scrambled RNAi cells as compared to dmSTING RNAi cells following c-di-GMP transfection and normalization to mock-transfected cells. (B–E) S2 cells in biological triplicate were treated as in (A), and levels of dmSTING (B), AttA (C), CecA2 (D), and Drs (E) were determined at 6 and 24 hr (Student’s t test; ∗p < 0.05). Error bars represent SDs. (F) Biological duplicates of adult flies knocked down for dmSTING (dmSTING RNAi) or not (control) were mock infected or infected with Listeria for 24 hr for microarray analysis. The heatmap shows the same gene set derived in (A), and genes highlighted in red are at least 1.5-fold upregulated in control flies compared to dmSTING RNAi flies. (G and H) Gene set enrichment analysis (GSEA) using the entire gene expression datasets from c-di-GMP-transfected cells (G) and Listeria-infected flies (H). Cell Reports 2018 23, 3537-3550.e6DOI: (10.1016/j.celrep.2018.05.029) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Flies Knocked Down for DmSTING Are More Susceptible to Listeria Infection (A–F and I) Adult flies knocked down for dmSTING (dmSTING RNAi) or not (control) were mock infected or infected with Listeria. Mortality was monitored for each genotype (control, n = 112; RNAi, n = 130), and percent survival from three separate infection experiments is shown (A). Dotted lines represent genotypes mock infected with saline (control, n = 82; RNAi, n = 88). At the indicated days post-infection (p.i.), at least eight sets of five flies from three separate infection experiments were homogenized in 50 μL PBS and bacterial titer was determined (B). At 24 hr p.i., levels of dmSTING (C), AttA (D), CecA2 (E), and Drs (F) were determined in biological triplicates. At 24 hr p.i., five flies were homogenized in RIPA buffer, and levels of Relish cleavage were determined by western blot analysis (I). Levels of dmSTING and actin are also shown. Blots are representative of duplicate experiments. (G–H) Adult flies knocked down for dmSTING or Relish in the fat body were infected as in (A)–(F) for 24 hr. Levels of CecA2 (G) and Drs (H) were determined in biological triplicates (Student’s t test; ∗p < 0.05). Error bars represent SDs. Cell Reports 2018 23, 3537-3550.e6DOI: (10.1016/j.celrep.2018.05.029) Copyright © 2018 The Authors Terms and Conditions

Figure 4 DmSTING Null Flies Are More Susceptible to Listeria Infection and Exhibit Reduced Antimicrobial Peptide Induction (A–E) Adult flies homozygous for a CRISPR/Cas9 deletion in dmSTING (ΔRG3 or ΔRG5) were mock infected or infected with Listeria. Control flies are the parental nos-Cas9 strain. Mortality was monitored for each genotype (control, n = 219; ΔRG3, n = 233; ΔRG5, n = 206), and percent survival from five separate infection experiments is shown (A). Dotted lines represent genotypes mock infected with saline (control, n = 109; ΔRG3, n = 98; ΔRG5, n = 77). At 24 hr p.i., levels of dmSTING (B), AttA (C), CecA2 (D), and Drs (E) were determined in biological triplicates. (F–I) Adult flies heterozygous for BSC133 and the dmSTING deletion (ΔRG3 or ΔRG5) were mock infected or infected with Listeria. At 24 hr p.i., levels of dmSTING (F), AttA (G), CecA2 (H), and Drs (I) were determined in biological triplicates (Student’s t test; ∗p < 0.05). Error bars represent SDs. Cell Reports 2018 23, 3537-3550.e6DOI: (10.1016/j.celrep.2018.05.029) Copyright © 2018 The Authors Terms and Conditions

Figure 5 Flies Overexpressing DmSTING Are More Resistant to Pathogen Infection due to Increased Antimicrobial Peptide Induction and Relish Activation Adult flies overexpressing (OE) dmSTING or not (control) were mock infected or infected with Listeria. (A) Mortality was monitored for each genotype (control, n = 144; OE, n = 110), and percent survival from three separate infection experiments is shown. Dotted lines represent genotypes mock infected with saline (control, n = 92; OE, n = 80). (B) At the indicated days p.i., at least three sets of five flies from three separate infection experiments were homogenized in PBS, and bacterial titer was determined. (C–F) At 24 hr p.i., levels of dmSTING (C), AttA (D), CecA2 (E), and Drs (F) were determined in biological triplicates. (G and H) Adult flies overexpressing dmSTING in the fat body were infected as in (A)–(F) for 24 hr. Levels of CecA2 (G) and Drs (H) were determined in biological triplicates (Student’s t test; ∗p < 0.05). Error bars represent SDs. (I) At 24 hr p.i., five flies were homogenized in RIPA buffer, and levels of Relish cleavage were determined by western blot analysis. Levels of dmSTING and actin are also shown. Blots are representative of duplicate experiments. Cell Reports 2018 23, 3537-3550.e6DOI: (10.1016/j.celrep.2018.05.029) Copyright © 2018 The Authors Terms and Conditions

Figure 6 DmSTING Activates NF-κB (A) Schematic of STING constructs used. From top to bottom: full-length human STING (hsSTING); hsSTING 1-338 lacking the C-terminal tail; full-length dmSTING (dmSTING); and chimeraSTING, full-length dmSTING followed by the hsSTING C-terminal tail. (B–E) 293T cells in biological triplicate were transfected with firefly luciferase vectors encoding the promoters for IFN-β (B), ISRE (C), PRD3-1 (D), or NF-κB (E) and the vectors shown in (A). Promoter activity was determined at 24 hr. (F) 293T cells in biological triplicate were transfected with NF-κB-luc along with full-length dmSTING or the CDN deletion mutants from Figure 1C. NF-κB activity was determined at 24 hr (Student’s t test; ∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.0005). Error bars represent SDs. (G–I) STING−/− MEFs were stably transformed with retrovirus expressing hsSTING or dmSTING and then mock transfected or transfected with c-di-GMP. At 12 hr post-transfection, IRF3 (G) and NF-κB (H) localization was imaged, or levels of the indicated proteins were analyzed by western blot (I). Results are representative of duplicate experiments. Cell Reports 2018 23, 3537-3550.e6DOI: (10.1016/j.celrep.2018.05.029) Copyright © 2018 The Authors Terms and Conditions

Figure 7 Relish and IMD Are Necessary for Proper dmSTING Signaling (A) Outline of Toll and IMD signaling pathways and color-coded schematic of dmSTING overexpression (OE) with knockdown of signaling components. (B–D) Adult flies overexpressing dmSTING and knocked down for luciferase (luc, non-specific control) (yellow dots), Relish (red dots), IMD (gray dots), PGRP-LC (pink dots), DIF (blue dots), or dMyD88 (orange dots) were mock infected or infected with Listeria for 24 hr. The presence (+) or absence (−) of the GAL4 driver indicates OE and RNAi or not. Levels of AttA (B), CecA2 (C), or Drs (D) were determined in four biological replicates (Student’s t test: ∗p < 0.05 as compared to Relish RNAi; #p < 0.05 as compared to IMD RNAi). (E–G) Adult flies overexpressing dmSTING and a wild-type allele (+, control) (yellow dots), a mutation in IMD (imd1) (gray dots), or a mutation in DIF (Dif2) (blue dots) were mock infected or infected with Listeria for 24 hr. Levels of AttA (E), CecA2 (F), or Drs (G) were determined in biological triplicates (Student’s t test; ∗p < 0.05). (H and I) S2∗ cells in biological triplicate were treated with 20E for 40 hr and then mock transfected (white dots), transfected with a dmSTING OE plasmid (gray dots) or c-di-GMP (red dots) or treated with TCT (light red dots). 6 hr post-treatment, levels of AttA (H) and Drs (I) were determined (Student’s t test; ∗p < 0.05). (I) Error bars represent SDs. Cell Reports 2018 23, 3537-3550.e6DOI: (10.1016/j.celrep.2018.05.029) Copyright © 2018 The Authors Terms and Conditions