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Molecular cloning and characterization of gloverin from the diamondback moth, Plutella xylostella L. and its interaction with bacterial membrane

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Abstract

Gloverin restricted to Lepidoptera is known to be a glycine-rich and heat stable antimicrobial protein. The current research reports a 650 bp full-length cDNA encoding gloverin from Plutella xylostella (PxGlo) by reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. PxGlo transcript was detected in both developmental stages and several tissues of 4th instar naïve larvae of P. xylostella with higher levels in the fat bodies. The mRNA levels of PxGlo increased appreciably in fat bodies after injection of Escherichia coli K12. The recombinant PxGlo expressed in S2 cells was purified by Anti-V5 M2 agarose beads which showed high activity against E. coli K12, while low activity against Bacillus thuringiensis, Staphylococcus aureus and E. coli D31. The analysis of transmission electron microscope and scan electron microscope showed PxGlo to cause significant morphological alteration in the E. coli K12 cell surface. Knockdown of PxGlo expression by RNAi increased the larval susceptibility towards the pathogenic bacteria i.e., Serratia marcescens and B. thuringiensis. Our results showed that PxGlo is an inducible antibacterial peptide which exhibits high activity mainly against E. coli K12, and PxGlo performs vital roles against the infection of pathogenic bacteria.

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References

  • Åsling B, Dushay MS, Hultmark D (1995) Identification of early genes in the Drosophila immune response by PCR-based differential display: the Attacin A gene and the evolution of attacin-like proteins. Insect Biochem Mol Biol 25:511–518

    Article  Google Scholar 

  • Axen A, Carlsson A, Engström Å, Bennich H (1997) Gloverin, an antibacterial protein from the immune hemolymph of Hyalophora pupae. Eur J Biochem 247:614–619

    Article  CAS  Google Scholar 

  • Boman HG (1995) Peptide antibiotics and their role in innate immunity. Annu Rev Immunol 13:61–92

    Article  CAS  Google Scholar 

  • Boman H, Boman IA, Andreu D, Li Z, Merrifield R, Schlenstedt G, Zimmermann R (1989) Chemical synthesis and enzymic processing of precursor forms of cecropins A and B. J Biol Chem 264:5852–5860

    CAS  Google Scholar 

  • Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria. Nat Rev Microbiol 3:238–250

    Article  CAS  Google Scholar 

  • Bulet P, Hetru C, Dimarcq JL, Hoffmann D (1999) Antimicrobial peptides in insects; structure and function. Develop Comp Immunol 23:329–344

    Article  CAS  Google Scholar 

  • Bulet P, Cociancich S, Reuland M, Sauber F, Bischoff R, Hegy G, Van Dorsselaer A, Hetru C, Hoffmann JA (2005) A novel insect defensin mediates the inducible antibacterial activity in larvae of the dragonfly Aeschna cyanea (Paleoptera, Odonata). Eur J Biochem 209:977–984

    Article  Google Scholar 

  • Carlsson A, Engström P, Palva ET, Bennich H (1991) Attacin, an antibacterial protein from Hyalophora cecropia, inhibits synthesis of outer membrane proteins in Escherichia coli by interfering with omp gene transcription. Infect Immun 59:3040–3045

    CAS  Google Scholar 

  • Carlsson A, Nyström T, de Cock H, Bennich H (1998) Attacin-an insect immune protein-binds LPS and triggers the specific inhibition of bacterial outer-membrane protein synthesis. Microbiol 144:2179–2188

    Article  CAS  Google Scholar 

  • Costa A, Jan E, Sarnow P, Schneider D (2009) The Imd pathway is involved in antiviral immune responses in Drosophila. PLoS ONE 4:e7436

    Article  Google Scholar 

  • Ferrandon D, Imler JL, Hetru C, Hoffmann JA (2007) The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. Nat Rev Immunol 7:862–874

    Article  CAS  Google Scholar 

  • Ganesan S, Aggarwal K, Paquette N, Silverman N (2011) NF-κB/Rel Proteins and the Humoral Immune Responses of Drosophila melanogaster. Curr Topics Microbiol Immunol 349:25–60

    CAS  Google Scholar 

  • Gely IV, Lemaitre B, Boccard F (2008) Bacterial strategies to overcome insect defences. Nat Rev Microbiol 6:302–313

    Article  Google Scholar 

  • Gunne H, Hellers M, Steiner H (2005) Structure of preproattacin and its processing in insect cells infected with a recombinant baculovirus. Eur J Biochem 187:699–703

    Article  Google Scholar 

  • Habel MDA, Biglang-awa IM, Dulce A, Luu DD, Garcia P, Weers PMM, Haas-Stapleton EJ (2012) Inactivation of the budded virus of Autographa californica M nucleopolyhedrovirus by gloverin. J Invertebr Pathol 110:92–101

    Article  Google Scholar 

  • Haine ER, Moret Y, Siva-Jothy MT, Rolff J (2008) Antimicrobial defense and persistent infection in insects. Science 322:1257–1259

    Article  CAS  Google Scholar 

  • Han PF, Fan JQ, Liu Y, Cuthbertson AGS, Yan SQ, Qiu BL, Ren SX (2014) RNAi-mediated knockdown of serine protease inhibitor genes increases the mortality of Plutella xylostella challenged by destruxin A. PLoS ONE 9(5):e97863. doi:10.1371/journal.pone.0097863

    Article  Google Scholar 

  • Hoffmann JA (1995) Innate immunity of insects. Curr Opin Immun 7:4–10

    Article  CAS  Google Scholar 

  • Hoffmann JA (2003) The immune response of Drosophila. Nat 426:33–38

    Article  CAS  Google Scholar 

  • Hultmark D (2003) Drosophila immunity: paths and patterns. Curr Opin Immun 15:12–19

    Article  CAS  Google Scholar 

  • Hultmark D, Steiner H, Rasmuson T, Boman HG (1980) Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur J Biochem 106:7–16

    Article  CAS  Google Scholar 

  • Hwang J, Kim Y (2011) RNA interference of an antimicrobial peptide, gloverin, of the beet armyworm, Spodoptera exigua, enhances susceptibility to Bacillus thuringiensis. J Invertebr Pathol 108:194–200

    Article  CAS  Google Scholar 

  • Imler JL, Hoffmann JA (2000) Signaling mechanisms in the antimicrobial host defense of Drosophila. Curr Opin Microbiol 3:16–22

    Article  CAS  Google Scholar 

  • Iwanaga S, Lee BL (2005) Recent advances in the innate immunity of invertebrate animals. J Biochem Mol Biol 38:128

    Article  CAS  Google Scholar 

  • Jin FL, Xu XX, Yu X, Ren SX (2009a) Expression and characterization of antimicrobial peptide Cecropin AD in the methylotrophic yeast Pichia pastoris. Proc Biochem 44:11–16

    Article  CAS  Google Scholar 

  • Jin FL, Dong X, Xu X, Ren SX (2009b) cDNA cloning and recombinant expression of the general odorant binding protein II from Spodoptera litura. Sci China C Life Sci 52(1):80–87

    Article  CAS  Google Scholar 

  • Kawaoka S, Katsuma S, Daimon T, Isono R, Omuro N, Mita K, Shimada T (2007) Functional analysis of four Gloverin-like genes in the silkworm, Bombyx mori. Arch Insect Biochem Physiol 67:87–96

    Article  Google Scholar 

  • Korner P, Schmid HP (2004) In vivo dynamics of an immune response in the bumble bee Bombus terrestris. J Invertebr Pathol 87:59–66

    Article  CAS  Google Scholar 

  • Kwon Y, Kim H, Kim Y, Kang Y, Lee I, Jin B, Han Y, Cheon H, Ha N, Seo S (2008) Comparative analysis of two attacin genes from Hyphantria cunea. Comp Biochem Physiol Part B Biochem Mol Biol 151:213–220

    Article  CAS  Google Scholar 

  • Kylsten P, Samakovlis C, Hultmark D (1990) The cecropin locus in Drosophila; a compact gene cluster involved in the response to infection. EMBO J 9:217

    CAS  Google Scholar 

  • Lavine M, Chen G, Strand M (2005) Immune challenge differentially affects transcript abundance of three antimicrobial peptides in hemocytes from the moth Pseudoplusia includens. Insect Biochem Mol Biol 35:1335–1346

    Article  CAS  Google Scholar 

  • Lemaitre B, Hoffmann J (2007) The host defense of Drosophila melanogaster. Annu Rev Immunol 25:697–743

    Article  CAS  Google Scholar 

  • Lemaitre B, Reichhart JM, Hoffmann JA (1997) Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. PNAS 94:14614–14619

    Article  CAS  Google Scholar 

  • Lundström A, Liu G, Kang D, Berzins K, Steiner H (2002) Trichoplusia ni gloverin, an inducible immune gene encoding an antibacterial insect protein. Insect Biochem Mol Biol 32:795–801

    Article  Google Scholar 

  • Mackintosh JA, Gooley AA, Karuso PH, Beattie AJ, Jardine DR, Veal DA (1998) A gloverin-like antibacterial protein is synthesized in Helicoverpa armigera following bacterial challenge. Develop Comp Immunol 22:387–399

    Article  CAS  Google Scholar 

  • Molloy S, Bresnahan P, Leppla SH, Klimpel K, Thomas G (1992) Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg–XX–Arg and efficiently cleaves anthrax toxin protective antigen. J Biol Chem 267:16396–16402

    CAS  Google Scholar 

  • Mrinal N, Nagaraju J (2008) Intron loss is associated with gain of function in the evolution of the gloverin family of antibacterial genes in Bombyx mori. J Biol Chem 283:23376–23387

    Article  CAS  Google Scholar 

  • Rao XJ, Yu XQ (2010) Lipoteichoic acid and lipopolysaccharide can activate antimicrobial peptide expression in the tobacco hornworm Manduca sexta. Develop Comp Immunol 34:1119–1128

    Article  CAS  Google Scholar 

  • Rao XJ, Xu XX, Yu XQ (2011) Functional analysis of two lebocin-related proteins from Manduca sexta. Insect Biochem Mol Biol 34:1119–1128

    Google Scholar 

  • Rayaprolu S, Wang Y, Kanost MR, Hartson S, Jiang H (2010) Functional analysis of four processing products from multiple precursors encoded by a lebocin-related gene from Manduca sexta. Develop Comp Immunol 34:638–647

    Article  CAS  Google Scholar 

  • Schuler TH, Denholm I, Clark SJ, Stewart CN, Poppy GM (2004) Effects of Bt plants on the development and survival of the parasitoid Cotesia plutellae (Hymenoptera: Braconidae) in susceptible and Bt-resistant larvae of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). J Insect Physiol 50:435–443

    Article  CAS  Google Scholar 

  • Shiomi K, Ishida Y, Ikeda M, Sato Y, Saito H, Imai K, Isobe M, Yamashita O (1994) Induction of non-diapause eggs by injection of anti-diapause hormone rabbit serum into the diapause type of the silkworm, Bombyx mori. J Insect Physiol 40:693–699

    Article  CAS  Google Scholar 

  • Steiner H, Hultmark D, Engstrom A, Bennich H, Boman H (1981) Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 292:246–248

    Article  CAS  Google Scholar 

  • Sugiyama M, Kuniyoshi H, Kotani E, Taniai K, Kadono-Okuda K, Kato Y, Yamamoto M, Shimabukuro M, Chowdhury S, Xu J (1995) Characterization of a Bombyx mori cDNA encoding a novel member of the attacin family of insect antibacterial proteins. Insect Biochem Mol Biol 25:385–392

    Article  CAS  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  CAS  Google Scholar 

  • Tanaka H, Ishibashi J, Fujita K, Nakajima Y, Sagisaka A, Tomimoto K, Suzuki N, Yoshiyama M, Kaneko Y, Iwasaki T (2008) A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori. Insect Biochem Mol Biol 38:1087–1110

    Article  CAS  Google Scholar 

  • Tanji T, Ip YT (2005) Regulators of the Toll and Imd pathways in the Drosophila innate immune response. Trends Immunol 26:193

    Article  CAS  Google Scholar 

  • Tauszig S, Jouanguy E, Hoffmann JA, Imler JL (2000) Toll-related receptors and the control of antimicrobial peptide expression in Drosophila. PNAS 97:10520–10525

    Article  CAS  Google Scholar 

  • Xu XX, Zhong X, Yi HY, Yu XQ (2012) Manduca sexta gloverin binds microbial components and is active against bacteria and fungi. Dev Com Immunol 38:275–284

    Article  CAS  Google Scholar 

  • You M, Yue Z, He W, Yang X, Yang G, Xie M, Zhan D, Baxter SW, Vasseur L, Gurr GM, Douglas CJ, Bai J, Wang P, Cui K, Huang S, Li X, Zhou Q, Wu Z, Chen Q, Liu C, Wang B, Li X, Xu X, Lu C, Hu M, Davey JW, Smith SM, Chen M, Xia X, Tang W, Ke F, Zheng D, Hu Y, Song F, You Y, Ma X, Peng L, Zheng Y, Liang Y, Chen Y, Yu L, Zhang Y, Liu Y, Li G, Fang L, Li J, Zhou X, Luo Y, Gou C, Wang J, Wang J, Yang H, Wang J (2013) A heterozygous moth genome provides insights into herbivory and detoxification. Nat Genet 45:220–225

    Article  CAS  Google Scholar 

  • Zhong X, Xu XX, Yi HY, Lin C, Yu XQ (2012) A Toll-Spätzle pathway in the tobacco hornworm, Manduca sexta. Insect Biochem Mol Biol 42:514–524

    Article  CAS  Google Scholar 

  • Zhu Y, Johnson T, Myers A, Kanost M (2003) Identification by subtractive suppression hybridization of bacteria-induced genes expressed in Manduca sexta fat body. Insect Biochem Mol Biol 33:541–559

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by grant from the National Natural Science Foundation of China (31071734; 31371989; 30900943). We also thank editors, anonymous referees for their invaluable comments and suggestions.

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    Authors and Affiliations

    1. College of Natural Resources and Environment, Engineering Research Centre of Biological Control, Ministry of Education, South China Agricultural University Guangzhou, Guangzhou, 510642, China

      X. X. Xu, F. L. Jin, Q. B. Hu & S. X. Ren

    2. State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical College, Guangzhou, 510230, China

      Y. S. Wang

    3. Department of Entomology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, 60800, Pakistan

      Shoaib Freed

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    1. X. X. Xu
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    Correspondence to F. L. Jin.

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    X. X. Xu and F. L. Jin have contributed equally to this work.

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    Xu, X.X., Jin, F.L., Wang, Y.S. et al. Molecular cloning and characterization of gloverin from the diamondback moth, Plutella xylostella L. and its interaction with bacterial membrane. World J Microbiol Biotechnol 31, 1529–1541 (2015). https://doi.org/10.1007/s11274-015-1901-7

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