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An anionic defensin from Plutella xylostella with potential activity against Bacillus thuringiensis

  • X.-X. Xu (a1), Y.-Q. Zhang (a1), S. Freed (a2), J. Yu (a1), Y.-F. Gao (a1), S. Wang (a1), L.-N. Ouyang (a1), W.-Y. Ju (a1) and F.-L. Jin (a1)...


Insect defensins, are cationic peptides that play an important role in immunity against microbial infection. In the present study, an anionic defensin from Plutella xylostella, (designated as PxDef) was first cloned and characterized. Amino acid sequence analysis showed that the mature peptide owned characteristic six-cysteine motifs with predicted isoelectric point of 5.57, indicating an anionic defensin. Quantitative real-time polymerase chain reaction analysis showed that PxDef was significantly induced in epidermis, fat body, midgut and hemocytes after injection of heat-inactivated Bacillus thuringiensis, while such an induction was delayed by the injection of live B. thuringiensis in the 4th instar larvae of P. xylostella. Knocking down the expression of nuclear transcription factor Dorsal in P. xylostella by RNA interference significantly decreased the mRNA level of PxDef, and increased the sensitivity of P. xylostella larvae to the infection by live B. thuringiensis. The purified recombinant mature peptide (PxDef) showed higher activity against Gram-positive bacteria, with the minimum inhibition concentrations of 1.6 and 2.6 µM against B. thuringiensis and Bacillus subtilis, respectively. To our knowledge, this is the first report about an anionic PxDef, which may play an important role in the immune system of P. xylostella against B. thuringiensis.


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Ando, K., Okada, M. & Natori, S. (1987) Purification of sarcotoxin II, antibacterial proteins of Sarcophaga peregrina (flesh fly) larvae. Biochemistry 26, 226230.
Badapanda, C. & Surendra, K.C. (2016) Lepidopteran antimicrobial peptides (AMPs): overview, regulation, modes of action, and therapeutic potentials of insect-derived AMPs. pp. 141163 in Chandrasekar, R., Goldsmith, M.R. & Agunbiade, T.A. (Eds) Short Views of Insect Genomics and Proteomics. Switzerland, Springer International Publishing.
Bao, Y.Y., Chandrasekar, R. & Chuan, X.Z. (2014) The innate immune network in a hemimetabolous insect, the brown plant hopper, Nilaparvata lugens . pp. 233252 in Chandrasekar, R., Tyagi, B.K., Gui, Z.Z. & Reeck, G. ( Eds ) Short Views on Insect Biochemistry and Molecular Biology, vol. 1. Manhattan, USA, International Book Mission, Academic Publisher.
Bates, J.M., Akerlund, J., Mittge, E. & Guillemin, K. (2007) Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota. Cell Host & Microbes 2, 371382.
Bhusan, K.K., Polamarasetty, A. & Pallu, R. (2010) EasyModeller: a graphical interface to modeller. BMC Research Notes 3, 226.
Bjellqvist, B., Hughes, G.J., Pasquali, C., Paquet, N., Ravier, F., Sancez, J.C., Frutiger, S. & Hochstrasser, D. (1993) The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 14, 10231031.
Boman, H. (1995) Peptide antibiotics and their role in innate immunity. Annual Review of Immunology 13, 6192.
Boulanger, N., Lowenberger, C., Volf, P., Ursic, R., Sigutova, L., Sabatier, L., Svobodova, M., Beverley, S.M., Späth, G., Brun, R., Pesson, B. & Bulet, P. (2004) Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major . Infection and Immunity 72, 71407146.
Bulet, P. & Stöcklin, R. (2005) Insect antimicrobial peptides: structures, properties and gene regulation. Protein & Peptide Letters 1, 311.
Bulet, P., Cociancich, S., Dimarcq, J.L., Lambert, J., Reichhart, J.M., Hoffmann, D., Hetru, C. & Hoffmann, J.A. (1991) Insect immunity. Isolation from a coleopteran insect of a novel inducible antibacterial peptide and of new members of the insect defensin family. Journal of Biological Chemistry 266, 2452024525.
Cecilia, V., Peter, J.W., Débora, P.M., Daniele, P.C., Cícero, B.M., Norman, A.R., Garcia, E.S. & Azambuja, P. (2014) Humoral responses in Rhodnius prolixus: bacterial feeding induces differential patterns of antibacterial activity and enhances mRNA levels of antimicrobial peptides in the midgut. Parasites & Vectors 7, 232.
Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T.J., Higgins, D.G. & Thompson, J.D. (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Research 31, 34973500.
Chernysh, S., Cociancich, S., Briand, J., Hetru, C. & Bulet, P. (1996) The inducible antibacterial peptides of the Hemipteran insect Palomena prasina: identification of a unique family of prolinerich peptides and of a novel insect defensin. Journal of Insect Physiology 42, 8189.
Dang, X.L., Wang, Y.S., Huang, Y.D., Yu, X.Q. & Zhang, W.Q. (2010) Purification and characterization of an antimicrobial peptide, insect defensin, from immunized house fly (Diptera, Muscidae). Journal of Medical Entomology 47, 11411145.
Dassanayake, R.S., Silva Gunawardene, Y.I.N. & Tobe, S.S. (2006) Evolutionary selective trends of insect/mosquito antimicrobial defensin peptides containing cysteine-stabilized α/β motifs. Peptides 28, 6275.
Dhume, A., Lu, S. & Horowits, R. (2006) Targeted disruption of N-RAP gene function by RNA interference: a role for N-RAP in myofibril organization. Cell Motility and the Cytoskeleton 63, 493511.
Dimarcq, J., Hoffmann, D., Meister, M., Bulet, P., Lanot, R., Reichhart, J. & Hoffmann, J.A. (1994) Characterization and transcriptional profiles of a Drosophila gene encoding an insect defensin: a study in insect immunity. European Journal of Biochemistry 221, 201209.
Dixit, R., Sharma, A., Patole, M.S. & Shouche, Y.S. (2008) Molecular and phylogenetic analysis of a novel salivary defensin cDNA from malaria vector Anopheles stephensi . Acta Tropica 106, 7579.
Dubovskiy, I.M., Martemyanov, V., Vorontsova, Y., Rantala, M., Gryzanova, E. & Glupov, V.V. (2008 a) Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera, Pyralidae). Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology 148, 15.
Dubovskiy, I.M., Krukova, N.A. & Glupov, V.V. (2008 b) Phagocytic activity and encapsulation rate of Galleria mellonella larval haemocytes during bacterial infection by Bacillus thuringiensis . Journal of Invertebrate Pathology 98, 360362.
El Shazely, B., Veverka, V., Fučík, V., Voburka, Z., Žďárek, J. & Čeřovský, V. (2013) Lucifensin II, a defensin of medicinal maggots of the blowfly Lucilia cuprina (Diptera, Calliphoridae). Journal of Medical Entomology 50, 571578.
Ericsson, J.D., Janmaat, A.F., Lowenberger, C. & Myers, J.H. (2009) Is decreased generalized immunity a cost of Bt resistance in cabbage loopers Trichoplusia ni? Journal of Invertebrate Pathology 100, 6167.
Ferrandon, D., Imler, J., Hetru, C. & Hoffmann, J.A. (2007) The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. Nature Reviews Immunology 7, 862874.
Gambichler, T. & Skrygan, M. (2015) Expression of human β-defensin-2 in psoriatic epidermis models treated with balneophototherapy. Journal of the European Academy of Dermatology and Venereology 29, 169173.
Gao, B. & Zhu, S.Y. (2013) An insect defensin-derived β-hairpin peptide with enhanced antibacterial activity. ACS Chemical Biology 9, 405413.
Gobert, V. (2003) Dual activation of the drosophila toll pathway by two pattern recognition receptors. Science 302, 21262130.
Hoffmann, J.A. & Reichhart, J. (2002) Drosophila innate immunity: an evolutionary perspective. Nature Immunology 3, 121126.
Huang, W., Xu, X., Freed, S., Zheng, Z., Wang, S., Ren, S.X. & Jin, F.L. (2015) Molecular cloning and characterization of a beta-1,3-glucan recognition protein from Plutella xylostella (L.). New Biotechnology 32, 290299.
Hultmark, D. (2003) Drosophila immunity: paths and patterns. Current Opinion in Immunology 15, 1219.
Jin, F., Sun, Q., Xu, X., Li, L., Gao, G., Xu, Y., Yu, X. & Ren, S. (2012) cDNA cloning and characterization of the antibacterial peptide cecropin 1 from the diamondback moth, Plutella xylostella L. Protein Expression and Purification 85, 230238.
Kaneko, Y., Tanaka, H., Ishibashi, J., Iwasaki, T. & Yamakawa, M. (2008) Gene expression of a novel defensin antimicrobial peptide in the silkworm, Bombyx mori . Bioscience Biotechnology Biochemistry 72, 23532361.
Kennerdell, J.R. & Carthew, R.W. (1998) Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95, 10171026.
Lai, R., Lomas, L.O., Jonczy, J., Turner, P.C. & Rees, H.H. (2004) Two novel non-cationic defensin-like antimicrobial peptides from haemolymph of the female tick, Amblyomma hebraeum . Biochemistry Journal 379, 681685.
Lambert, J., Keppi, E., Dimarcq, J.L., Wicker, C., Reichhart, J.M., Dunbar, B., Lepage, P., Van Dorsselaer, A., Hoffmann, J. & Fothergill, J. (1989) Insect immunity: isolation from immune blood of the dipteran Phormia terranovae of two insect antibacterial peptides with sequence homology to rabbit lung macrophage bactericidal peptides. Proceedings of National Academy of Sciences of the United States of America 86, 262266.
Lauth, X., Nesin, A., Briand, J., Roussel, J. & Hetru, C. (1998) Isolation, characterization and chemical synthesis of a new insect defensin from Chironomus plumosus (Diptera). Insect Biochemistry and Molecular Biology 28, 10591066.
Lee, E., Kim, J., Shin, S., Jeong, K., Shin, A., Lee, J., Lee, D.G., Hwang, J. & Kim, Y. (2013) Insight into the antimicrobial activities of coprisin isolated from the dung beetle, Copris tripartitus, revealed by structure–activity relationships. Biochimica Biophysica Acta (BBA) – Biomembranes 1828, 271283.
Lee, Y.S., Yun, E.K., Jang, W.S., Kim, I., Lee, J.H., Park, S.Y., Ryu, K.S., Seo, S.J., Kim, C.H. & Lee, I.H. (2004) Purification, cDNA cloning and expression of an insect defensin from the great wax moth, Galleria mellonella . Insect Molecular Biology 13, 6572.
Lemaitre, B. & Hoffmann, J. (2007) The host defense of Drosophila melanogaster . Annual Review of Immunology 25, 697743.
Lemaitre, B., Reichhart, J.M. & Hoffmann, J.A. (1997) Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Proceedings of National Academy of Sciences of the United States of America 94, 1461414619.
Lin, Q., Jin, F., Hu, Z., Chen, H., Yin, F., Li, Z., Dong, X., Zhang, D., Ren, S. & Feng, X. (2013) Transcriptome analysis of chlorantraniliprole resistance development in the diamondback moth Plutella xylostella . PLoS ONE 208, e72314.
Maillet, F., Bischoff, V., Vignal, C., Hoffmann, J. & Royet, J. (2008) The Drosophila peptidoglycan recognition protein PGRP-LF blocks PGRP-LC and IMD/JNK pathway activation. Cell Host & Microbes 3, 293303.
Matsuyama, K. & Natori, S. (1988) Purification of three antibacterial proteins from the culture medium of NIH Sape-4, an embryonic cell line of Sarcophaga peregrina . Journal of Biological Chemistry 263, 17, 112–116.
Meister, M., Lemaitre, B. & Hoffmann, J.A. (1997) Antimicrobial peptide defense in Drosophila . Bioassays 19, 10191026.
Nakamura, M., Iwasaki, T., Tokino, S., Asaoka, A., Yamakawa, M. & Ishibashi, J. (2011) Development of a bioactive fiber with immobilized synthetic peptides designed from the active site of a beetle defensin. Biomacromolecules 12, 15401545.
Nenci, A., Becker, C., Wullaert, A., Gareus, R., van Loo, G., Danese, S., Huth, M., Nikolaev, A., Neufert, C., Madison, B., Gumucio, D., Neurath, M.F. & Pasparakis, M. (2007) Epithelial NEMO links innate immunity to chronic intestinal inflammation. Nature 446, 557561.
Ouyang, L.N., Xu, X.X., Freed, S., Gao, Y.F., Yu, J., Wang, S., Ju, W.Y., Zhang, Y.Q. & Jin, F.L. (2015) Cecropins from Plutella xylostella and their interaction with Metarhizium anisopliae . PloS ONE 10, e0142451.
Pardo-Lopez, L., Soberon, M. & Bravo, A. (2013) Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection. FEMS Microbiology Reviews 37, 322.
Porto, W.F., Fensterseifer, G.M. & Franco, O.L. (2014) In silico identification, structural characterization, and phylogenetic analysis of MdesDEF-2: a novel defensin from the Hessian fly, Mayetiola destructor . Journal of Molecular Modeling 20, 18.
Pütsep, K., Brändén, C., Boman, H.G. & Normark, S. (1999) Antibacterial peptide from Helicobacter pylori. Nature 398, 671672.
Richman, A.M., Bulet, P., Hetru, C., Barillas, M.C., Hoffmann, J.A. & Kafatos, F.C. (1996) Inducible immune factors of the vector mosquito Anopheles gambiae: biochemical purification of a defensin antibacterial peptide and molecular cloning of preprodefensin cDNA. Insect Molecular Biology 5, 203210.
Seufi, A.M., Hafez, E.E. & Galal, F.H. (2011) Identification, phylogenetic analysis and expression profile of an anionic insect defensin gene, with antibacterial activity, from bacterial-challenged cotton leafworm, Spodoptera littoralis . BMC Molecular Biology 12, 47.
Song, K.J., Park, B.R., Kim, S.Y. & Park, K.S. (2010) Molecular characterization of anionic defensin-like peptide inimmune response of silkworm, Bombyx mori L. (Lepidoptera). Genes & Genomics 32, 447453.
Tabashnik, B. (1994) Evolution of resistance to Bacillus thurinigienis . Annual Review of Entomology 39, 4779.
Tabashnik, B.E. & Cushing, N.L. (1987) Leaf residue vs topical bioassays for assessing insecticide resistance in the diamondback moth, Plutella xyillstella L. Food and Agriculture Organization of the United Nations Plant Protection Bulletin 35, 1114.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28, 27312739.
Telleria, E.L., Sant'Anna, M.R., Alkurbi, M.O., Pitaluga, A.N., Dillon, R.J. & Traub-Csekö, Y.M. (2013) Bacterial feeding, Leishmania infection and distinct infection routes induce differential defensin expression in Lutzomyia longipalpis . Parasite and Vectors 6, 12.
Vallet, G.I., Lemaitre, B. & Boccard, F. (2008) Bacterial strategies to overcome insect defences. Nature Review of Microbiology 6, 302313.
Wang, Y.P., Zhang, Z.Y., Chen, L., Guang, H., Li, Z., Yang, H.L., Li, J.X., You, D.W., Yu, H.N. & Lai, R. (2012) Cathelicidin-BF, a snake cathelicidin-derived antimicrobial peptide, could be an excellent therapeutic agent for acne vulgaris. PLoS ONE 6, e22120.
Wen, H.X., Lan, X.Q., Cheng, T.C., He, N.J., Shiomi, K., Kajiura, Z., Zhou, Z.Y., Xia, Q.Y., Xiang, Z.H. & Nakagaki, M. (2009) Sequence structure and expression pattern of an anionic defensin-like gene from silkworm (Bombyx mori). Molecular Biology Reports 36, 711716.
Xu, W. & Faisal, M. (2010) Defensin of the zebra mussel (Dreissena polymorpha): molecular structure, in vitro expression, antimicrobial activity, and potential functions. Molecular Immunology 47, 21382147.
Xu, X., Jin, F., Yu, X., Ji, S., Wang, J., Cheng, H., Wang, C. & Zhang, W. (2007) Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli . Protein Expression and Purification 53, 293301.
Yang, H.L., Wang, X., Liu, X.H., Wu, J., Liu, C.B., Gong, W., Zhao, Z., Hong, J., Lin, D., Wang, Y. & Lai, R. (2009) Antioxidant peptidomics reveals novel skin antioxidant system. Molecular & Cell Proteomics 8, 571583.
Yi, H., Chowdhury, M., Huang, Y. & Yu, X. (2014) Insect antimicrobial peptides and their applications. Applied Microbiology and Biotechnology 98, 58075822.
You, M., Yue, Z., He, W., Yang, X., Yang, G., Xie, M., Zhan, D., Baxter, S.W., Vasseur, L., Gurr, G.M., Douglas, C.J., 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, J.W., Smith, S.M., 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. Nature Genetics 45, 220225.


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