Drosophila anti-nematode and antibacterial immune regulators revealed by RNA-Seq

BMC Genomics

Ioannis Eleftherianos²  Luke J. Tallon¹  Upasana Shokal²  Amol Shetty¹  Priti Kumari¹  Todd Creasy¹  Julio C. Castillo³ 

[1] Institute for Genome Sciences, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore 21201, MD, USA;Insect Infection and Immunity Lab, Department of Biological Sciences, Institute for Biomedical Sciences, The George Washington University, Washington DC 20052, USA;Laboratory of Malaria and Vector Research, National Institutes of Health, Rockville 20852, MD, USA

关键词: Parasitism;    Infection;    Immunity;    Transcriptomics;    RNA-sequencing;    Heterorhabditis;    Photorhabdus;    Drosophila;   

Others  :  1222467
DOI  :  10.1186/s12864-015-1690-2

 received in 2014-10-16, accepted in 2015-06-05,  发布年份 2015

【 摘 要 】

Background

Drosophila melanogaster activates a variety of immune responses against microbial infections. However, information on the Drosophila immune response to entomopathogenic nematode infections is currently limited. The nematode Heterorhabditis bacteriophora is an insect parasite that forms a mutualistic relationship with the gram-negative bacteria Photorhabdus luminescens. Following infection, the nematodes release the bacteria that quickly multiply within the insect and produce several toxins that eventually kill the host. Although we currently know that the insect immune system interacts with Photorhabdus, information on interaction with the nematode vector is scarce.

Results

Here we have used next generation RNA-sequencing to analyze the transcriptional profile of wild-type adult flies infected by axenic Heterorhabditis nematodes (lacking Photorhabdus bacteria), symbiotic Heterorhabditis nematodes (carrying Photorhabdus bacteria), and Photorhabdus bacteria alone. We have obtained approximately 54 million reads from the different infection treatments. Bioinformatic analysis shows that infection with Photorhabdus alters the transcription of a large number of Drosophila genes involved in translational repression as well in response to stress. However, Heterorhabditis infection alters the transcription of several genes that participate in lipidhomeostasis and metabolism, stress responses, DNA/protein sythesis and neuronal functions. We have also identified genes in the fly with potential roles in nematode recognition, anti-nematode activity and nociception.

Conclusions

These findings provide fundamental information on the molecular events that take place in Drosophila upon infection with the two pathogens, either separately or together. Such large-scale transcriptomic analyses set the stage for future functional studies aimed at identifying the exact role of key factors in the Drosophila immune response against nematode-bacteria complexes.

【 授权许可】

   
2015 Castillo et al.

【 图 表 】

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【 参考文献 】

• [1]Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature. 2007; 449(7164):819-26.
• [2]Glavis-Bloom J, Muhammed M, Mylonakis E. Of model hosts and man: using Caenorhabditis elegans, Drosophila melanogaster and Galleria mellonella as model hosts for infectious disease research. Adv Exp Med Biol. 2012; 710:11-7.
• [3]Brivio MF, Mastore M, Pagani M. Parasite-host relationship: a lesson from a professional killer. Invertebr Surv J. 2005; 2:41-53.
• [4]Dionne MS, Schneider DS. Models of infectious diseases in the fruit fly Drosophila melanogaster. Dis Model Mech. 2008; 1(1):43-9.
• [5]Limmer S, Quintin J, Hetru C, Ferrandon D. Virulence on the fly: Drosophila melanogaster as a model genetic organism to decipher host-pathogen interactions. Curr Drug Targets. 2011; 12(7):978-99.
• [6]Rämet M. The fruit fly Drosophila melanogaster unfolds the secrets of innate immunity. Acta Paediatr. 2012; 101(9):900-5.
• [7]Castillo JC, Reynolds SE, Eleftherianos I. Insect immune responses to nematode parasites. Trends Parasitol. 2011; 27(12):537-47.
• [8]Dillman AR, Chaston JM, Adams BJ, Ciche TA, Goodrich-Blair H, Stock SP et al.. An entomopathogenic nematode by any other name. PLoS Pathog. 2012; 8(3):e1002527.
• [9]Ciche TA, Darby C, Ehlers RU, Forst S, Goodrich-Blair H. Dangerous liaisons: The symbiosis of entomopathogenic nematodes and bacteria. Biol Control. 2006; 38(1):22-46.
• [10]Ciche T. The biology and genome of Heterorhabditis bacteriophora. WormBook. 2007;1–9.
• [11]Ffrench-Constant RH, Dowling A, Waterfield NR. Insecticidal toxins from Photorhabdus bacteria and their potential use in agriculture. Toxicon. 2007; 49(4):436-51.
• [12]Bode HB. Entomopathogenic bacteria as a source of secondary metabolites. Curr Opin Chem Biol. 2009; 13(2):224-30.
• [13]Ciche TA, Ensign JC. For the insect pathogen Photorhabdus luminescens, which end of a nematode is out? Appl Environ Microbiol. 2003; 69(4):1890-7.
• [14]Hallem EA, Rengarajan M, Ciche TA, Sternberg PW. Nematodes, bacteria, and flies: a tripartite model for nematode parasitism. Curr Biol. 2007; 17(10):898-904.
• [15]Wang Z, Wilhelmsson C, Hyrsl P, Loof TG, Dobes P, Klupp M et al.. Pathogen entrapment by transglutaminase–a conserved early innate immune mechanism. PLOS Pathog. 2010; 6(2):e1000763.
• [16]Hyrsl P, Dobes P, Wang Z, Hauling T, Wilhelmsson C, Theopold U. Clotting factors and eicosanoids protect against nematode infections. J Innate Immun. 2011; 3(1):65-70.
• [17]Castillo JC, Shokal U, Eleftherianos I. Immune gene transcription in Drosophila adult flies infected by entomopathogenic nematodes and their mutualistic bacteria. J Insect Physiol. 2013; 59(2):179-85.
• [18]Arefin B, Kucerova L, Dobes P, Markus R, Strnad H, Wang Z et al.. Genome-wide transcriptional analysis of Drosophila larvae infected by entomopathogenic nematodes shows involvement of complement, recognition and extracellular matrix proteins. J Innate Immun. 2014; 6(2):192-204.
• [19]Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009; 10(1):57-63.
• [20]Ozsolak F, Milos PM. RNA sequencing: advances, challenges and opportunities. Nat Rev Genet. 2011; 12(2):87-98.
• [21]de Klerk E, den Dunnen JT, 't Hoen PA. RNA sequencing: from tag-based profiling to resolving complete transcript structure. Cell Mol Life Sci. 2014; 71(18):3537-51.
• [22]Xuan J, Yu Y, Qing T, Guo L, Shi L. Next-generation sequencing in the clinic: promises and challenges. Cancer Lett. 2013; 340(2):284-95.
• [23]Costa V, Aprile M, Esposito R, Ciccodicola A. RNA-Seq and human complex diseases: recent accomplishments and future perspectives. Eur J Hum Genet. 2013; 21(2):134-42.
• [24]Daines B, Wang H, Wang L, Li Y, Han Y, Emmert D et al.. The Drosophila melanogaster transcriptome by paired-end RNA sequencing. Genome Res. 2011; 21(2):315-24.
• [25]Ekblom R, Galindo J. Applications of next generation sequencing in molecular ecology of non-model organisms. Heredity. 2011; 107(1):1-15.
• [26]Westermann AJ, Gorski SA, Vogel J. Dual RNA-seq of pathogen and host. Nat Rev Microbiol. 2012; 10(9):618-30.
• [27]Castillo JC, Shokal U, Eleftherianos I. A novel method for infecting Drosophila adult flies with insect pathogenic nematodes. Virulence. 2012; 3(3):339-47.
• [28]Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR et al.. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012; 7(3):562-78.
• [29]Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M. The KEGG resource for deciphering the genome. Nucleic Acids Res. 2004; 32:D277-80.
• [30]Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nat Protoc. 2009; 4(1):44-57.
• [31]Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009; 37(1):1-13.
• [32]Mi H, Thomas P. PANTHER pathway: an ontology-based pathway database coupled with data analysis tools. Methods Mol Biol. 2009; 563:123-40.
• [33]Thomas PD, Mi H, Lewis S. Ontology annotation: mapping genomic regions to biological function. Curr Opin Chem Biol. 2007; 11(1):4-11.
• [34]Ayres JS, Freitag N, Schneider DS. Identification of Drosophila mutants altering defense of and endurance to Listeria monocytogenes infection. Genetics. 2008; 178(3):1807-15.
• [35]Özkan E, Carrillo RA, Eastman CL, Weiszmann R, Waghray D, Johnson KG et al.. An extracellular interactome of immunoglobulin and LRR proteins reveals receptor-ligand networks. Cell. 2013; 154(1):228-39.
• [36]Gummalla M, Maeda RK, Castro Alvarez JJ, Gyurkovics H, Singari S, Edwards KA et al.. abd-A regulation by the iab-8 noncoding RNA. PLoS Genet. 2012; 8(5):e1002720.
• [37]Wright VM, Vogt KL, Smythe E, Zeidler MP. Differential activities of the Drosophila JAK/STAT pathway ligands Upd, Upd2 and Upd3. Cell Signal. 2011; 23(5):920-7.
• [38]Ekengren S, Hultmark D. A family of Turandot-related genes in the humoral stress response of Drosophila. Biochem Biophys Res Commun. 2001; 284(4):998-1003.
• [39]Brun S, Vidal S, Spellman P, Takahashi K, Tricoire H, Lemaitre B. The MAPKKK Mekk1 regulates the expression of Turandot stress genes in response to septic injury in Drosophila. Genes Cells. 2006; 11(4):397-407.
• [40]Gordon MD, Ayres JS, Schneider DS, Nusse R. Pathogenesis of listeria-infected Drosophila wntD mutants is associated with elevated levels of the novel immunity gene edin. PLoS Pathog. 2008; 4(7):e1000111.
• [41]Mellroth P, Karlsson J, Steiner HA. Scavenger function for a Drosophila peptidoglycan recognition protein. J Biol Chem. 2003; 278(9):7059-64.
• [42]Tanji T, Ohashi-Kobayashi A, Natori S. Participation of a galactose-specific C-type lectin in Drosophila immunity. Biochem J. 2006; 396(1):127-38.
• [43]Stroschein-Stevenson SL, Foley E, O'Farrell PH, Johnson AD. Identification of Drosophila gene products required for phagocytosis of Candida albicans. PLoS Biol. 2006; 4(1):e4.
• [44]Karouzou MV, Spyropoulos Y, Iconomidou VA, Cornman RS, Hamodrakas SJ, Willis JH. Drosophila cuticular proteins with the R&R Consensus: annotation and classification with a new tool for discriminating RR-1 and RR-2 sequences. Insect Biochem Mol Biol. 2007; 37(8):754-60.
• [45]Perrimon N, Smouse D, Miklos GLG. Developmental genetics of loci at the base of the X chromosome of Drosophila melanogaster. Genetics. 1989; 121:313-31.
• [46]Kambris Z, Brun S, Jang IH, Nam HJ, Romeo Y, Takahashi K et al.. Drosophila immunity: a large-scale in vivo RNAi screen identifies five serine proteases required for Toll activation. Curr Biol. 2006; 16(8):808-13.
• [47]Chakrabarti S, Poidevin M, Lemaitre B. The Drosophila MAPK p38c Regulates Oxidative Stress and Lipid Homeostasis in the Intestine. PLoS Genet. 2014; 10(9):e1004659.
• [48]Varghese J, Lim SF, Cohen SM. Drosophila miR-14 regulates insulin production and metabolism through its target, sugarbabe. Genes Dev. 2010; 24(24):2748-53.
• [49]Tang AH, Tu CPD. Biochemical characterization of Drosophila glutathione S-transferases D1 and D21. J Biol Chem. 1994; 269(45):27876-84.
• [50]Han Q, Fang J, Ding H, Johnson JK, Christensen BM, Li J. Identification of Drosophila melanogaster yellow-f and yellow-f2 proteins as dopachrome-conversion enzymes. Biochem J. 2002; 368(1):333-40.
• [51]Clyne PJ, Warr CG, Carlson JR. Candidate taste receptors in Drosophila. Science. 2000; 287(5459):1830-4.
• [52]Andrés M, Turiégano E, Göpfert MC, Canal I, Torroja L. The extracellular matrix protein artichoke is required for integrity of ciliated mechanosensory and chemosensory organs in Drosophila embryos. Genetics. 2014; 196(4):1091-02.
• [53]Sieber MH, Thummel CS. Coordination of Triacylglycerol and Cholesterol Homeostasis by DHR96 and the Drosophila LipA Homolog magro. Cell Metab. 2012; 15(1):122-7.
• [54]Kleino A, Silverman N. The Drosophila IMD pathway in the activation of the humoral immune response. Dev Comp Immunol. 2014; 42(1):25-35.
• [55]Lindsay SA, Wasserman SA. Conventional and non-conventional Drosophila Toll signaling. Dev Comp Immunol. 2014; 42(1):16-24.
• [56]Myllymäki H, Rämet M. Jak/STAT pathway in Drosophila immunity. Scand J Immunol. 2014; 79(6):377-85.
• [57]Blandin S, Levashina EA. Thioester-containing proteins and insect immunity. Mol Immunol. 2004; 40(12):903-8.
• [58]Delaney JR, Stoven S, Uvell H, Anderson KV, Engstrom Y, Mlodzik M. Cooperative control of Drosophila immune responses by the JNK and NF-kappaB signaling pathways. EMBO J. 2006; 25(13):3068-77.
• [59]Ríos-Barrera LD, Riesgo-Escovar JR. Regulating cell morphogenesis: the Drosophila Jun N-terminal kinase pathway. Genesis. 2013; 51(3):147-62.
• [60]Hamilton PT, Leong JS, Koop BF, Perlman SJ. Transcriptional responses in a Drosophila defensive symbiosis. Mol Ecol. 2014; 23(6):1558-70.
• [61]Krishnan N, Dickman MB, Becker DF. Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress. Free Radic Biol Med. 2008; 44(4):671-81.
• [62]Phang JM, Liu W. Proline metabolism and cancer. Front Biosci. 2012; 17:1835-45.
• [63]McIntire CR, Yeretssian G, Saleh M. Inflammasomes in infection and inflammation. Apoptosis. 2009; 14(4):522-35.
• [64]Ffrench-Constant R, Waterfield N, Daborn P, Joyce S, Bennett H, Au C et al.. Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen. FEMS Microbiol Rev. 2003; 26(5):433-56.
• [65]Irving P, Troxler L, Heuer TS, Belvin M, Kopczynski C, Reichhart JM et al.. A genome-wide analysis of immune responses in Drosophila. Proc Natl Acad Sci USA. 2001; 98(26):15119-24.
• [66]De Gregorio E, Spellman PT, Tzou P, Rubin GM, Lemaitre B. The Toll and Imd pathways are the major regulators of the immune response in Drosophila. EMBO J. 2002; 21(11):2568-79.
• [67]Apidianakis Y, Mindrinos MN, Xiao W, Lau GW, Baldini RL, Davis RW et al.. Profiling early infection responses: Pseudomonas aeruginosa eludes host defenses by suppressing antimicrobial peptide gene expression. Proc Natl Acad Sci USA. 2005; 102(7):2573-8.
• [68]Sonnleitner E, Valentini M, Wenner N, Haichar FZ, Haas D, Lapouge K. Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa. PLoS One. 2012; 7(10):e44637.
• [69]Dhur A, Galan P, Hercberg S. Folate status and the immune system. Prog Food Nutr Sci. 1991; 15(1-2):43-60.
• [70]Courtemanche C, Elson-Schwab I, Mashiyama ST, Kerry N, Ames BN. Folate deficiency inhibits the proliferation of primary human CD8+ T lymphocytes in vitro. J Immunol. 2004; 173(5):3186-92.
• [71]Silva CP, Waterfield NR, Daborn PJ, Dean P, Chilver T, Au CP et al.. Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta. Cell Microbiol. 2002; 4(6):329-39.
• [72]Eleftherianos I, Ffrench-Constant RH, Clarke DJ, Dowling AJ, Reynolds SE. Dissecting the immune response to the entomopathogen Photorhabdus. Trends Microbiol. 2010; 18(12):552-60.
• [73]Royet J. Epithelial homeostasis and the underlying molecular mechanisms in the gut of the insect model Drosophila melanogaster. Cell Mol Life Sci. 2011; 68(22):3651-60.
• [74]Davis MM, Engström Y. Immune response in the barrier epithelia: lessons from the fruit fly Drosophila melanogaster. J Innate Immun. 2012; 4(3):273-83.
• [75]Kuraishi T, Hori A, Kurata S. Host-microbe interactions in the gut of Drosophila melanogaster. Front Physiol. 2013; 4:375.
• [76]Imler JL, Bulet P. Antimicrobial peptides in Drosophila: structures, activities and gene regulation. Chem Immunol Allergy. 2005; 86:1-21.
• [77]Lemaitre B, Hoffmann J. The host defense of Drosophila melanogaster. Ann Rev Immunol. 2007; 25:697-743.
• [78]Blandin SA, Marois E, Levashina EA. Antimalarial responses in Anopheles gambiae: from a complement-like protein to a complement-like pathway. Cell Host Microbe. 2008; 3(6):364-74.
• [79]Bou Aoun R, Hetru C, Troxler L, Doucet D, Ferrandon D, Matt N. Analysis of thioester-containing proteins during the innate immune response of Drosophila melanogaster. J Innate Immun. 2011; 3(1):52-64.
• [80]Thompson AA, Binham J, Plant T, Whyte MK, Walmsley SR. Hypoxia, the HIF pathway and neutrophilic inflammatory responses. Biol Chem. 2013; 394(4):471-7.
• [81]Gorr TA, Wichmann D, Hu J, Hermes-Lima M, Welker AF, Terwilliger N et al.. Hypoxia tolerance in animals: biology and application. Physiol Biochem Zool. 2010; 83(5):733-52.
• [82]Hasnain SZ, Gallagher AL, Grencis RK, Thornton DJ. A new role for mucins in immunity: insights from gastrointestinal nematode infection. Int J Biochem Cell Biol. 2012; 45(2):364-74.
• [83]Fundytus ME. Glutamate receptors and nociception: implications for the drug treatment of pain. CNS Drugs. 2001; 15(1):29-58.
• [84]Szekely JI, Torok K, Mate G. The role of ionotropic glutamate receptors in nociception with special regard to the AMPA binding sites. Curr Pharm Des. 2002; 8(10):887-912.
• [85]Numazaki M, Tominaga M. Nociception and TRP Channels. Curr Drug Targets CNS Neurol Disord. 2004; 3(6):479-85.
• [86]Benton R, Vannice KS, Gomez-Diaz C, Vosshall LB. Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell. 2009; 136(1):149-62.
• [87]Robertson JL, Tsubouchi A, Tracey WD. Larval defense against attack from parasitoid wasps requires nociceptive neurons. PLoS One. 2013; 8(10):e78704.
• [88]AbuHatab M, Selvan S, Gaugler R. Role of proteases in penetration of insect gut by the entomopathogenic nematode Steinernema glaseri (Nematoda: Steinernematidae). J Invert Path. 1995; 66(2):125-30.
• [89]McKerrow JH, Caffrey C, Kelly B, Loke P, Sajid M. Proteases in parasitic diseases. Annu Rev Pathol. 2006; 1:497-536.
• [90]Bai X, Adams BJ, Ciche TA, Clifton S, Gaugler R, Kim KS et al.. A lover and a fighter: the genome sequence of an entomopathogenic nematode Heterorhabditis bacteriophora. PLoS One. 2013; 8(7):e69618.
• [91]Babusyte A, Kotthoff M, Fiedler J, Krautwurst D. Biogenic amines activate blood leukocytes via trace amine-associated receptors TAAR1 and TAAR2. J Leukoc Biol. 2013; 93(3):387-94.
• [92]Ligoxygakis P. Genetics of immune recognition and response in Drosophila host defense. Adv Genet. 2013; 83:71-97.
• [93]Cornman RS. Molecular evolution of Drosophila cuticular protein genes. PLoS One. 2009; 4(12):e8345.
• [94]Rebers JE, Willis JH. A conserved domain in arthropod cuticular proteins binds chitin. Insect Biochem Mol Biol. 2001; 31(11):1083-93.
• [95]Cornman RS, Willis JH. Annotation and analysis of low-complexity protein families of Anopheles gambiae that are associated with cuticle. Insect Mol Biol. 2009; 18(5):607-22.
• [96]Tang L, Liang J, Zhan Z, Xiang Z, He N. Identification of the chitin-binding proteins from the larval proteins of silkworm, Bombyx mori. Insect Biochem Mol Biol. 2010; 40(3):228-34.
• [97]Gendrin M, Zaidman-Rémy A, Broderick NA, Paredes J, Poidevin M, Roussel A et al.. Functional analysis of PGRP-LA in Drosophila immunity. PLoS One. 2013; 8(7):e69742.
• [98]White GFR. A method for obtaining infective nematode larvae from cultures. Science. 1927; 66(1709):302-3.
• [99]Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009; 25(9):1105-11.
• [100]Langmead B. Aligning short sequencing reads with Bowtie. Curr Protoc Bioinformatics. 2010, Chapter 11:Unit 11.7.
• [101]Aanes H, Winata C, Moen LF, Østrup O, Mathavan S, Collas P et al.. Normalization of RNA-sequencing data from samples with varying mRNA levels. PLoS One. 2014; 9(2):e89158.