The Drosophila Protein Mustard Tailors the Innate Immune Response Activated by the Immune Deficiency Pathway

Zhipeng Wang, Cristin D. Berkey and Paula I. Watnick
J Immunol April 15, 2012, 188 (8) 3993-4000; DOI: https://doi.org/10.4049/jimmunol.1103301

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    The mtdEY04695 mutant phenocopies IMD pathway mutants. (A) Fractional survival during oral V. cholerae infection of the control strain yw (WT), the transposon insertion line mtdEY04695, and the IMD pathway mutants, dreddB118 and key1. Log-rank analysis demonstrated a statistically significant difference between survival of control flies and mtdEY04695 mutants (p < 0.001), key1 mutants (p < 0.001), and dreddB118 mutants (p < 0.05). (B) Bacterial burden of yw, mtdEY04695, dreddB118, and key1. flies after 48 h infection. There was a statistically significant difference in bacterial burden between control flies and mtdEY04695 (p = 0.0022) or key1 (p = 0.0022) mutant flies. However, the difference in bacterial burden between control flies and dreddB118 mutant flies was not statistically significant (p = 0.0931).

  • FIGURE 2.
    FIGURE 2.

    dipt transcription is decreased in the mtdEY04695 mutant. qRT-PCR analysis of dipt levels in (A) uninfected and (B) systemically and (C) orally infected yw (WT) and mtdEY04695 mutant flies. ***p < 0.001

  • FIGURE 3.
    FIGURE 3.

    Proteolytic cleavage of Relish is similar in control flies and the mtdEY04695 mutant. Western analysis of Relish cleavage fragments in uninfected or infected w1118 (WT), mtdEY04695, or relE20 flies by probing with anti-Rel49 (A) and anti-Rel68 Abs (B).

  • FIGURE 4.
    FIGURE 4.

    Transcript abundance of genes encoding antimicrobial peptides in control flies, an IMD pathway mutant, and a mtdEY04695 mutant orally infected with V. cholerae. qRT-PCR analysis of (A) attacin A (AttA), (B) cecropin A (CecA), (C) defensin (Def), (D) drososin (Drc), (E) metchnikowin (Mtk), and (F) drosomycin (Drom) transcription in control Drosophila melanogaster (WT), a mtdEY04695 mutant (mtd), and an IMD pathway mutant (key). *p < 0.05.

  • FIGURE 5.
    FIGURE 5.

    Additional genes that are similarly regulated in an IMD pathway mutant and a mtdEY04695 mutant as compared with control flies orally infected with V. cholerae. qRT-PCR analysis of (A) edin, (B) CG12498, (C) CG10725, and (D) CG13659. *p < 0.05, ***p < 0.001.

  • FIGURE 6.
    FIGURE 6.

    Impact of the mtdEY04695 insertion on transcription of representative Mtd transcripts. (A) Diagram of the TLDc-only Mtd transcripts mtd-RH and mtd-RC, the LysM-only transcript mtd-RD, and a representative LysM/TLDc transcript mtd-RB encoded in the mtd locus. Small rectangles represent exons. The exons containing the conserved LysM and TLDc domains are labeled and filled in white and black, respectively. Triangles indicate the sites of transposon insertions studied. The box below contains the locations of the amplicons generated in qRT-PCR experiments shown in (B)–(E) and the corresponding transcript detected. (B–E) Relative transcript levels of mtd-RC, mtd-RH, mtd-RD, and all long transcripts (TLDc + LysM) in the mtdEY04695 mutant as compared with yw flies (WT). ***p < 0.001.

  • FIGURE 7.
    FIGURE 7.

    Overexpression of mtd-RH but not mtd-RC blocks activation of dipt transcription. (A) qRT-PCR measurement of mtd-RC transcript levels in a strain with ubiquitous overexpression of mtd-RC (UAS-Mtd-RC/Da-Gal4) and a driver-only control (+/Da-Gal4). (B) qRT-PCR measurement of dipt transcript levels in a strain with ubiquitous overexpression of mtd-RC (UAS-Mtd-RC/Da-Gal4) and a driver-only control (+/Da-Gal4). (C) Same experiment as (B) performed with flies infected with V. cholerae by septic injury. (D) qRT-PCR measurement of mtd-RH transcript levels in a strain with ubiquitous overexpression of mtd-RH (UAS-Mtd-RH/Da-Gal4) and a driver-only control (+/Da-Gal4). (E) qRT-PCR measurement of dipt transcript levels in a strain with ubiquitous overexpression of mtd-RH (UAS-Mtd-RH/Da-Gal4) and a driver-only control (+/Da-Gal4). (F) Same experiment as (D) performed with flies infected with V. cholerae by septic injury. *p < 0.05, **p < 0.01, ***p < 0.001.

  • FIGURE 8.
    FIGURE 8.

    Overexpression of mtd-RH but not mtd-RC increases tolerance to oral V. cholerae infection. Fractional survival of flies with (A) ubiquitous expression of mtd-RC (UAS-Mtd-RC/Da-Gal4) or mtd-RH (UAS-Mtd-RH/Da-Gal4) compared with driver-only control flies (+/Da-Gal4) and (B) midgut-only expression of mtd-RC (UAS-Mtd-RC/NP1-Gal4) or mtd-RH (UAS-Mtd-RH/NP1-Gal4) compared with driver-only control flies (+/NP1-Gal4). Log-rank analysis demonstrated a statistically significant difference between survival of control flies and flies expressing mtd-RH either ubiquitously (p < 0.0001) or in the midgut alone (p < 0.0001) but not between survival of control flies and flies expressing mtd-RC ubiquitously (p > 0.05) or in the midgut alone (p > 0.05).

  • FIGURE 9.
    FIGURE 9.

    Mtd-PH is nuclearly localized, whereas Mtd-PC is excluded from the nucleus. Immunofluorescence of Mtd-PC-HA (A) and Mtd-PH-HA (B) in the adult midgut. Immunofluorescence of Mtd-PC-HA (C) and Mtd-PH-HA (D) in the larval fat body. In all cases, expression was driven by Da-Gal4. The tagged protein is visualized with Alexa 488 (green), and nuclear DNA is stained with DAPI (blue). The mosaic pattern of expression is a characteristic of the Da-Gal4 driver. Scale bars, 20 μm.

  • FIGURE 10.
    FIGURE 10.

    Overexpression of mtd-RH does not prevent nuclear localization of Rel68 in the larval fat body but does rescue the eclosion-lethal phenotype resulting from overexpression of rel68. Immunofluorescence of larval fat bodies overexpressing (A) rel68-FLAG (Rel68), (B) rel68-FLAG and mtd-RC-HA (Rel68 + Mtd-RC), or (C) rel68-FLAG and mtd-RH-HA (Rel68 + Mtd-RH) driven by Da-Gal4. Nuclei are stained with DAPI. Scale bars, 10 μm. (D) Images of the eclosion phenotypes in flies expressing rel68, rel68, and mtd-RC together (Rel68 + Mtd-RC), or rel68 and mtd-RH together (Rel68 + Mtd-RH) driven by Da-Gal4. The images were taken with a 2× objective. (E) Quantification of the fraction of abnormal pupa in flies overexpressing rel68 (Rel68), rel68, and mtd-RC (Rel68 + Mtd-RC), or rel68 and mtd-RH (Rel68 + Mtd-RH) driven by Da-Gal4. ***p < 0.001 as compared with rel68 overexpression alone.

Additional Files

  • Data Supplement

    Files in this Data Supplement:

    • Supplemental Table 1 and Supplemental Figures 1-3 (PDF, 294 Kb) - Description:
      Table S1: Microarray comparison of gene expression in control flies and mtdEY04695 or key1 mutants orally infected with V. cholerae.
      Figure S1. Mtd levels in the midgut and whole fly.
      Figure S2. Precise transposon excision rescues the mtdEY04695 mutant phenotype.
      Figure S3. The mtdEY04695 mutant phenotype is not recapitulated in mutants with decreased levels of mtd-RC.
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