Abstract
Baculoviruses have a long history of safe use as specific, environmentally friendly insecticides that provide alternatives to chemical pesticides for controlling insect pests. However, their use has been limited by several factors, particularly their slow pathogenicity. In this study, we constructed a recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) and an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) that expressed an insect-specific cyto-insectotoxin (Cit1a) from the venom of the central Asian spider Lachesana tarabaevi. Cit1a is a comparatively long linear cytolytic molecule that contains a predicted α-helix structure composed of two short membrane-acting antimicrobial peptides (MAMPs) that are joined together in a “head-to-tail” shape. Cit1a fused to polyhedrin gene (polh) (polh-cit1a) was expressed in the nuclei as polyhedra in silkworm larvae, Bm5 and Sf9 cells. An early death of Bm5 and Sf9 cells by recombinant BmNPV/Polh-Cit1a and AcMNPV/Polh-Cit1a was observed compared with control viruses that lacked the toxin gene. The infected cells showed a loss of cytoplasm, membrane integrity, and structural changes, suggesting that recombinant baculovirus-infected cells were killed by the necrosis caused by Cit1a. In addition, the BmNPV/Polh-Cit1a showed a significant reduction in the median lethal time (LT50) against silkworm larvae compared with those of control BmNPV that lacked the cit1a gene.
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References
Ali MP, Yoshimatsu K, Suzuki T, Kato T, Park EY (2014) Expression and purification of cyto-insectotoxin (Cit1a) using silkworm larvae targeting for an antimicrobial therapeutic agent. Appl Microbiol Biotechnol 98:6973–6982
Ardisson-Araújo DMP, Morgado FDS, Schwartz EF, Corzo G, Ribeiro BM (2013) A new theraphosid spider toxin causes early insect cell death by necrosis when expressed in vitro during recombinant baculovirus infection. PLoS One 8, e84404
Chang JH, Choi JY, Jin BR, Roh JY, Olszewski JA, Seo SJ, O’Reilly DR, Je YH (2003) An improved baculovirus insecticide producing occlusion bodies that contain Bacillus thuringiensis insect toxin. J Invertebr Pathol 84:30–37
Choi JY, Wang Y, Kim YS, Kang JN, Roh JY, Woo SD, Jin BR, Je YH (2008) Insecticidal activities of recombinant Autographa californica nucleopolyhedrovirus containing a scorpion neurotoxin gene using promoters from Cotesia plutellae bracovirus. J Asia-Pac Entomol 11:155–159
Douris V, Swevers L, Labropoulou V, Andronopoulou E, Georgoussi Z, Iatrou K (2006) Stably transformed insect cell lines: Tools for expression of secreted and membrane-anchored proteins and high-throughput screening platforms for drug and insecticide discovery. Adv Virus Res 68:113–156
El-Menofy W, Osman G, Assaeedi A, Salama M (2014) A novel recombinant baculovirus overexpressing a Bacillus thuringiensis Cry1Ab toxin enhances insecticidal activity. Biol Proced Online 16:7. doi:10.1186/1480-9222-16-7
Gramkow AW, Perecmanis S, Sousa RLB, Noronha EF, Felix CR, Nagata T, Ribeiro BM (2010) Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses. Virol J 7:143
Harrison RL, Bonning BC (2010) Proteases as Insecticidal Agents. Toxins 2:935–953
Hiyoshi M, Kageshima A, Kato T, Park EY (2007) Construction of a cysteine protease deficient Bombyx mori multiple nucleopolyhedrovirus bacmid and its application to improve expression of a fusion protein. J Virol Methods 144:91–97
Hong HK, Woo SD, Choi JY, Lee HK, Kim MH, Je YH, Kang SK (2000) Characterization of four isolates of Bombyx mori nucleopolyhedrovirus. Arch Virol 145:2351–2361
Inceoglu AB, Kamita S, Hinton AC, Hung Q, Severson TF, Kang KK, Hammock BD (2001) Recombinant baculoviruses for insect control. Pest Manag Sci 57:981–987
Kato T, Manoha SL, Tanaka S, Park EY (2009) High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV) displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization. BMC Biotechnol 9:55. doi:10.1186/1472-6750-9-55
Kato T, Thompson JR, Park EY (2013) Construction of new ligation-independent clonig vectors for the expression and purification of recombinant proteins in silkworms using BmNPV bacmid system. PLoS One 8, e64007. doi:10.1371/journal.pone.0064007
Lazarev VN, Polin NF, Shkarupeta MM, Kostrjukova ES, Vassilevski AA, Kozlov SA, Grishin EV, Govorun VM (2011) Spider venom peptides for gene therapy of Chlamydia infection. Antimicrob Agents Chemother 55:5367–5369
Lo HR, Chao YC (2004) Rapid titer determination of baculovirus by Quantitative Real-Time Polymerase Chain Reaction. Biotechnol Prog 20:354–360
Mintz IM (1994) Block of Ca channels in rat central neurons by the spider toxin omega-Aga-IIIA. J Neurosci 14:2844–2853
Mintz IM, Bean BP (1993) Block of calcium channels in rat neurons by synthetic omega-Aga-IVA. Neuropharmacol 32:1161–1169
Oerke EC (2006) Crop losses to pests. J Agric Sci 144:31–43
Osman G (2012) Detection, cloning and bioinformatics analysis of vip1/vip2 genes from local Bacillus thuringiensis. Afr J Biotechnol 15:11678–11685
Sanguinetti MC, Johnson JH, Hammerland LG, Kelbaugh PR, Volkmann RA, Saccomano NA, Muelle A (1997) Heteropodatoxins: peptides isolated from spider venom that block Kv4.2 potassium channels. Mol Pharmacol 51:491–498
Shim HJ, Cho JY, Wang Y, Tao XY, Li Q, Roh JY, Kim JS, Kim WJ, Woo SW, Ji BR, Je YH (2013) NeuroBactrus, a novel, highly effective, and environmentally friendly recombinant baculovirus insecticide. Appl Environ Microbiol 79:141–149
Slack J, Arif BM (2007) The baculoviruses occlusion-derived virus: virion structure and function. Adv Virus Res 69:99–165
Smith GE, Vlak JM, Summers MD (1983) Physical analysis of Autographa californica nuclear polyhedrosis virus transcripts for polyhedrin and 10,000-molecular-weight protein. J Virol 45:215–225
Stewart LMD, Hirst M, Ferber ML, Weather ATM, Cayley PJ, Possee RD (1991) Construction of an improved baculovirus insecticide containing an insect-specific toxin gene. Nature 352:85–88
Szewczyk B, Hoyos-Carvajal L, Paluszek M, Skrzecz I, Lobo de Souza M (2006) Baculoviruses – re-emerging biopesticides. Biotechnol Adv 24:143–160
Vassilevski AA, Kozlov SA, Samsonova OA, Egorova NS, Karpunin DV, Pluzhninkov KA, Feofanov AV, Grishin EV (2008) Cyto-insectotoxins, a novel class of cytolytic and insecticidal peptides from siper venom. Biochem J 411:687–696
Vassilevski AA, Kozlov SA, Grishin EV (2009) Molecular diversity of spider venom. Biochemistry-Moscow 74:1505–1534
Vorontsova OV, Egorova NS, Arseniev AS, Feofanov AV (2011) Haemolytic and cytotoxic action of latarcin Ltc2a. Biochimie 93:227–241
Wang R, Deng F, Hou D, Zhao Y, Guo L, Wang H, Hu Z (2010) Proteomics of the Autographa californica nucleopolyhedrovirus budded virions. J Virol 84:7233–7242
Windley MJ, Herzig V, Dziemborowicz SA, Hardy MC, King GF, Nicholson GM (2012) Spider-venom peptides as bioinsecticides. Toxins 4:191–227
Acknowledgments
MPA was supported by Shizuoka University Corporation Environmental Leaders Program (ELSU), Japan.
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The authors declare no commercial or financial conflict of interest.
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Ali, M.P., Kato, T. & Park, E.Y. Improved insecticidal activity of a recombinant baculovirus expressing spider venom cyto-insectotoxin. Appl Microbiol Biotechnol 99, 10261–10269 (2015). https://doi.org/10.1007/s00253-015-6846-0
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DOI: https://doi.org/10.1007/s00253-015-6846-0