The Drosophila Protein Mustard Tailors the Innate Immune Response Activated by the Immune Deficiency Pathway This information is current as Zhipeng Wang, Cristin D. Berkey and Paula I. Watnick of October 2, 2021. J Immunol 2012; 188:3993-4000; Prepublished online 16 March 2012; doi: 10.4049/jimmunol.1103301 http://www.jimmunol.org/content/188/8/3993 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2012/03/16/jimmunol.110330 Material 1.DC1 References This article cites 45 articles, 17 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/188/8/3993.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 2, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Drosophila Protein Mustard Tailors the Innate Immune Response Activated by the Immune Deficiency Pathway Zhipeng Wang, Cristin D. Berkey, and Paula I. Watnick 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-kB homolog, Relish. Mtd homologs Downloaded from 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. The Journal of Immunology, 2012, 188: 3993– 4000. he epithelia of most animals are continually exposed to protein recognition of meso-diaminopimelic acid-type peptido- microorganisms. By necessity, these animals have evolved glycan. Through a series of intermediates, this leads to activation http://www.jimmunol.org/ T signaling pathways that enable symbiotic interactions with of the caspase 8 homolog Dredd and the IkK complex consisting beneficial microbes and activate an innate immune defense against of the catalytic subunit IkKb, encoded by ird5, and the regulatory invading pathogens. subunit IkKg, encoded by Kenny (key). Phosphorylation of Relish The common fruit fly Drosophila melanogaster harbors two by the IkK complex at residues 528 and 529 is required for ac- distinct innate immune signaling pathways, namely the immune tivation of Relish targets. Dredd is required for cleavage of the deficiency (Imd) pathway, which responds primarily to Gram- Relish C-terminal inhibitory ankyrin repeat domain, allowing negative bacteria, and the Toll pathway, which responds primar- nuclear translocation of a 68 kDa N-terminal RHD-containing ily to Gram-positive bacteria and fungi (1, 2). Both of these fragment and activation of an immune response against Gram- by guest on October 2, 2021 pathways regulate nuclear translocation of NF-kB homologs, negative and some Gram-positive bacterial pathogens (4–7). which activate the transcription of many genes, including those Many studies have demonstrated that, in addition to generation that encode small cationic antimicrobial peptides (AMPs). of reactive oxygen species by the host (8, 9), IMD pathway sig- Three NF-kB homologs are encoded in the Drosophila genome. naling plays a role in the Drosophila innate immune response to Two of these, Dorsal and Dorsal-related immunity factor, respond both commensal and pathogenic intestinal bacteria (10–12). To to signaling through the Toll pathway (3). The third NF-kB ho- protect the host against its own immune response, signaling molog, Relish, comprised of an N-terminal Rel homology domain through this pathway is tightly regulated (12–18). (RHD) and a C-terminal ankyrin repeat domain, responds to sig- Vibrio cholerae, a Gram-negative, halophilic bacterium, is re- naling through the IMD pathway, which is initiated by receptor sponsible for the severe diarrheal disease cholera (19). Further- more, it has been associated with both marine and terrestrial Division of Infectious Diseases, Children’s Hospital Boston, Boston, MA 02115 arthropods in the environment (20–24), and arthropods have been Received for publication November 30, 2011. Accepted for publication February 11, proposed as both reservoirs and vectors of V. cholerae. We de- 2012. veloped D. melanogaster as a model in which to study the inter- This work was supported by National Institutes of Health Grant AI071147 (to P.I.W.). action of V. cholerae with the arthropod intestine (25) and The Intellectual and Developmental Disabilities Research Center Imaging Core, previously reported that IMD pathway mutants have increased Children’s Hospital Boston is supported by National Institutes of Health Grant P30-HD-18655. The Children’s Hospital Boston Molecular Genetics Core Facility tolerance to oral V. cholerae infection (26). is supported by National Institutes of Health Grants P50-NS40828 and P30- As part of a genetic screen for host susceptibility factors, we HD18655. identified a mutant with increased tolerance to oral V. cholerae The microarray data presented in this article have been deposited in the Gene Ex- infection and a transcriptional profile similar to that of an IMD pression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under accession no. GSE35439. pathway mutant. This mutant, which we have named Mustard (Mtd), carries a P-element insertion in a complex genetic locus Address correspondence and reprint requests to Dr. Paula I. Watnick, Division of Infectious Diseases, Children’s Hospital Boston, 300 Longwood Avenue, Boston, encoding conserved LysM and TLDc domains. LysM domains are MA 02115. E-mail address: [email protected] often involved in carbohydrate recognition (27). Although the The online version of this article contains supplemental material. TLDc domain has been associated with resistance to oxidative Abbreviations used in this article: AMP, antimicrobial peptide; HA, hemagglutinin; stress, its mechanism of action is unknown (28). The Mtd locus is IMD, immune deficiency; key, Kenny; LB, Luria–Bertani; Mtd, Mustard; qRT-PCR, predicted to give rise to 21 transcripts containing one or both of quantitative RT-PCR; RHD, Rel homology domain; ROS, reactive oxygen species. these conserved domains (29). We show that the phenotype of the Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 Mtd mutant represents a gain of function and can be reproduced www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103301 3994 MUSTARD TAILORS IMD PATHWAY OUTPUT by expression of a nuclearly localized isoform containing only collected and homogenized in 200 ml PBS. Dilutions of the suspension the TLDc domain. TLDc domains are widespread in eukaryotes. were plated on LB agar supplemented with streptomycin. CFUs per fly In this study, to our knowledge we present the first evidence for were enumerated. No colonies were cultured from flies fed LB broth alone, suggesting that all bacteria retrieved from LB agar plates supplemented modulation of the IMD innate immune signaling pathway by with streptomycin were V. cholerae. Statistical significance was calculated a TLDc domain-containing protein. using a Mann–Whitney U test. Materials and Methods Quantitative RT-PCR Bacterial strains, fly stocks, and growth media Flies were homogenized in TRIzol reagent (Invitrogen). Total RNA was extracted once, treated with DNase I, and then extracted a second time. MO10, a V. cholerae O139 clinical isolate, was used in all Drosophila Purified RNA (1 mg) was used as a template for synthesis of cDNA using infections (30). Either w1118 or yw stocks obtained from the Bloomington a QuantiTect reverse transcription system (Qiagen). The resulting cDNA Drosophila stock center at Indiana University were used as controls. The was used for quantitative PCR with iTaq SYBR Green supermix with details of other fly lines used in these experiments are available upon re- carboxy-X-rhodamine (ROX; Bio-Rad) and 2 pmol relevant primers in quest. Other than the decreased rates of eclosion observed for insertion a 20-ml reaction volume. The sequence of primers used for quantitative mutants with global defects in Mtd transcription, no developmental defects RT-PCR (qRT-PCR) is available on request. The experiments were con- were noted for the fly lines used in these experiments. D. melanogaster ducted with a StepOnePlus PCR system (Applied Biosystems). The tran- strains were reared at 24˚C on standard Drosophila medium, and bacterial script level of each gene was quantified by comparison with a standard strains were propagated in Luria–Bertani (LB) broth supplemented with curve and normalized using the level of the reference gene rp49. Ten to streptomycin (100 mg/ml) at 27˚C.