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Protein & Peptide Letters

Author(s): Patcharaporn Boonyos, Chutchanun Trakulnalueamsai, Amporn Rungrod, Sukumal Chongthammakun and Boonhiang Promdonkoy*

DOI: 10.2174/0929866527666200625205846

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Antagonistic Effect of Truncated Fragments of Bacillus thuringiensis Vip3Aa on the Larvicidal Activity of its Full-length Protein

Page: [131 - 139] Pages: 9

  • * (Excluding Mailing and Handling)

Abstract

Background: Vip3Aa is a vegetative insecticidal protein produced by Bacillus thuringiensis. The protein is produced as an 88-kDa protoxin that could be processed by insect gut proteases into a 22-kDa N-terminal and a 66-kDa C-terminal fragments. The C-terminal part could bind to a specific receptor while the N-terminal part is required for toxicity and structural stability.

Objective: To demonstrate the antagonistic effect of truncated fragments on the insecticidal activity of the full-length Vip3Aa.

Methods: The full-length protein (Vip3Aa), a 66-kDa C-terminal fragment (Vip3Aa-D199) and a predicted carbohydrate binding module (CBM) were produced in Escherichia coli. Purified proteins were mixed at different ratios and fed to Spodoptera litura and Spodoptera exigua larvae. Mortality was recorded and compared between larvae fed with individual toxin and mixtures of the full-length and truncated toxins.

Results: Production level of the Vip3Aa-D199 was significantly decreased comparing to that of the full-length protein. Vip3Aa-D199 and CBM fragment were not toxic to insect larvae whereas Vip3Aa showed high toxicity with LC50 about 200 ng/cm2. Feeding the larvae with mixtures of the Vip3Aa and Vip3Aa-D199 at different ratios revealed antagonistic effect of the Vip3Aa-D199 on the toxicity of Vip3Aa. Results showed that the lethal time (LT 50 and LT 95) of larvae fed the mixture toxins was longer than those fed the Vip3Aa alone. In addition, a CBM fragment could inhibit toxicity of the full-length Vip3Aa.

Conclusion: Our results demonstrated that the Vip3Aa-D199 and a CBM fragment could complete for the membrane binding thus rendering activity of the full-length Vip3Aa.

Keywords: Antagonistic effect, Bacillus thuringiensis, larvicidal activity, Spodoptera litura, truncated protein, Vip3Aa.

Graphical Abstract

[1]
Chakroun M, Banyuls N, Bel Y, Escriche B, Ferré J. Bacterial vegetative insecticidal proteins (Vip) from entomopathogenic bacteria. Microbiol Mol Biol Rev 2016; 80(2): 329-50.
[http://dx.doi.org/10.1128/MMBR.00060-15] [PMID: 26935135]
[2]
Estruch JJ, Warren GW, Mullins MA, Nye GJ, Craig JA, Koziel MG. Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proc Natl Acad Sci USA 1996; 93(11): 5389-94.
[http://dx.doi.org/10.1073/pnas.93.11.5389] [PMID: 8643585]
[3]
Li C, Xu N, Huang X, et al. Bacillus thuringiensis Vip3 mutant proteins: insecticidal activity and trypsin sensitivity. Biocontrol Sci Technol 2007; 17: 699-708.
[http://dx.doi.org/10.1080/09583150701527177]
[4]
Milne R, Liu Y, Gauthier D, van Frankenhuyzen K. Purification of Vip3Aa from Bacillus thuringiensis HD-1 and its contribution to toxicity of HD-1 to spruce budworm (Choristoneura fumiferana) and gypsy moth (Lymantria dispar) (Lepidoptera). J Invertebr Pathol 2008; 99(2): 166-72.
[http://dx.doi.org/10.1016/j.jip.2008.05.002] [PMID: 18585733]
[5]
Yu CG, Mullins MA, Warren GW, Koziel MG, Estruch JJ. The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut epithelium cells of susceptible insects. Appl Environ Microbiol 1997; 63(2): 532-6.
[http://dx.doi.org/10.1128/AEM.63.2.532-536.1997] [PMID: 9023933]
[6]
Abdelkefi-Mesrati L, Boukedi H, Dammak-Karray M, Sellami-Boudawara T, Jaoua S, Tounsi S. Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis. J Invertebr Pathol 2011; 106(2): 250-4.
[http://dx.doi.org/10.1016/j.jip.2010.10.002] [PMID: 20965198]
[7]
Liu JG, Yang AZ, Shen XH, Hua BG, Shi GL. Specific binding of activated Vip3Aa10 to Helicoverpa armigera brush border membrane vesicles results in pore formation. J Invertebr Pathol 2011; 108(2): 92-7.
[http://dx.doi.org/10.1016/j.jip.2011.07.007] [PMID: 21824478]
[8]
Boukedi H, Tounsi S, Abdelkefi-Mesrati L. Insecticidal activity, putative binding proteins and histopathological effects of Bacillus thuringiensis Vip3(459) toxin on the lepidopteran pest Ectomyelois ceratoniae. Acta Trop 2018; 182: 60-3.
[http://dx.doi.org/10.1016/j.actatropica.2018.02.006] [PMID: 29448008]
[9]
Hernández-Martínez P, Gomis-Cebolla J, Ferré J, Escriche B. Changes in gene expression and apoptotic response in Spodoptera exigua larvae exposed to sublethal concentrations of Vip3 insecticidal proteins. Sci Rep 2017; 7(1): 16245.
[http://dx.doi.org/10.1038/s41598-017-16406-1] [PMID: 29176692]
[10]
Nimsanor S, Srisaisup M, Jammor P, Promdonkoy B, Boonserm P. Intracellular localization and cytotoxicity of Bacillus thuringiensis Vip3Aa against Spodoptera frugiperda (Sf9) cells. J Invertebr Pathol 2020; 171: 107340.
[http://dx.doi.org/10.1016/j.jip.2020.107340] [PMID: 32044359]
[11]
Zheng M, Evdokimov AG, Moshiri F, Lowder C, Haas J. Crystal structure of a Vip3B family insecticidal protein reveals a new fold and a unique tetrameric assembly. Protein Sci 2020; 29(4): 824-9.
[http://dx.doi.org/10.1002/pro.3803] [PMID: 31840313]
[12]
Kunthic T, Surya W, Promdonkoy B, Torres J, Boonserm P. Conditions for homogeneous preparation of stable monomeric and oligomeric forms of activated Vip3A toxin from Bacillus thuringiensis. Eur Biophys J 2017; 46(3): 257-64.
[http://dx.doi.org/10.1007/s00249-016-1162-x] [PMID: 27473845]
[13]
Selvapandiyan A, Arora N, Rajagopal R, et al. Toxicity analysis of N- and C-terminus-deleted vegetative insecticidal protein from Bacillus thuringiensis. Appl Environ Microbiol 2001; 67(12): 5855-8.
[http://dx.doi.org/10.1128/AEM.67.12.5855-5858.2001] [PMID: 11722946]
[14]
Chen J, Sun F, Tang L, et al. Expression of Bacillus thuringiensis full-length and N-terminally truncated vip184 gene in an acrystalliferous strain of subspecies kurstaki. World J Microbiol Biotechnol 2003; 19: 883-9.
[http://dx.doi.org/10.1023/B:WIBI.0000007288.31371.7d]
[15]
Chen J, Yu J, Tang L, Tang M, Shi Y, Pang Y. Comparison of the expression of Bacillus thuringiensis full-length and N-terminally truncated vip3A gene in Escherichia coli. J Appl Microbiol 2003; 95(2): 310-6.
[http://dx.doi.org/10.1046/j.1365-2672.2003.01977.x] [PMID: 12859763]
[16]
Bhalla R, Dalal M, Panguluri SK, et al. Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis. FEMS Microbiol Lett 2005; 243(2): 467-72.
[http://dx.doi.org/10.1016/j.femsle.2005.01.011] [PMID: 15686851]
[17]
Fang J, Xu X, Wang P, et al. Characterization of chimeric Bacillus thuringiensis Vip3 toxins. Appl Environ Microbiol 2007; 73(3): 956-61.
[http://dx.doi.org/10.1128/AEM.02079-06] [PMID: 17122403]
[18]
Chi B, Li H, Zhang J, Wei P, Gao J, Liu R. In silico structure-based identification and validation of keyresidues of Vip3Aa involving in Lepidopteran brush border receptor binding. Appl Biochem Biotechnol 2019; 187(4): 1448-59.
[http://dx.doi.org/10.1007/s12010-018-2880-6] [PMID: 30251230]
[19]
Lee MK, Walters FS, Hart H, Palekar N, Chen JS. The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab delta-endotoxin. Appl Environ Microbiol 2003; 69(8): 4648-57.
[http://dx.doi.org/10.1128/AEM.69.8.4648-4657.2003] [PMID: 12902253]
[20]
Soares Figueiredo C, Nunes Lemes AR, Sebastião I, Desidério JA. Synergism of the Bacillus thuringiensis Cry1, Cry2, and Vip3 proteins in Spodoptera frugiperda control. Appl Biochem Biotechnol 2019; 188(3): 798-809.
[http://dx.doi.org/10.1007/s12010-019-02952-z] [PMID: 30706415]
[21]
Wang Z, Fang L, Zhou Z, et al. Specific binding between Bacillus thuringiensis Cry9Aa and Vip3Aa toxins synergizes their toxicity against Asiatic rice borer (Chilo suppressalis). J Biol Chem 2018; 293(29): 11447-58.
[http://dx.doi.org/10.1074/jbc.RA118.003490] [PMID: 29858245]
[22]
Yu X, Liu T, Sun Z, et al. Co-expression and synergism analysis of Vip3Aa29 and Cyt2Aa3 insecticidal proteins from Bacillus thuringiensis. Curr Microbiol 2012; 64(4): 326-31.
[http://dx.doi.org/10.1007/s00284-011-0070-7] [PMID: 22218570]
[23]
Abdelkefi-Mesrati L, Boukedi H, Chakroun M, et al. Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin. J Invertebr Pathol 2011; 107(3): 198-201.
[http://dx.doi.org/10.1016/j.jip.2011.05.014] [PMID: 21600212]
[24]
Boukedi H, Ben Khedher S, Abdelkefi-Mesrati L, Van Rie J, Tounsi S. Comparative analysis of the susceptibility/tolerance of Spodoptera littoralis to Vip3Aa, Vip3Ae, Vip3Ad and Vip3Af toxins of Bacillus thuringiensis. J Invertebr Pathol 2018; 152: 30-4.
[http://dx.doi.org/10.1016/j.jip.2018.01.006] [PMID: 29378203]
[25]
Jiang K, Hou X, Han L, Tan T, Cao Z, Cai J. Fibroblast growth factor receptor, a novel receptor for vegetative insecticidal protein Vip3Aa. Toxins (Basel) 2018; 10(12): 546.
[http://dx.doi.org/10.3390/toxins10120546] [PMID: 30567360]
[26]
Jiang K, Hou XY, Tan TT, et al. Scavenger receptor-C acts as a receptor for Bacillus thuringiensis vegetative insecticidal protein Vip3Aa and mediates the internalization of Vip3Aa via endocytosis. PLoS Pathog 2018; 14(10): e1007347.
[http://dx.doi.org/10.1371/journal.ppat.1007347] [PMID: 30286203]
[27]
de Maagd RA, Bravo A, Berry C, Crickmore N, Schnepf HE. Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annu Rev Genet 2003; 37: 409-33.
[http://dx.doi.org/10.1146/annurev.genet.37.110801.143042] [PMID: 14616068]
[28]
Promdonkoy B, Ellar DJ. Investigation of the pore-forming mechanism of a cytolytic delta-endotoxin from Bacillus thuringiensis. Biochem J 2003; 374(Pt 1): 255-9.
[http://dx.doi.org/10.1042/bj20030437] [PMID: 12795638]
[29]
Sriwimol W, Aroonkesorn A, Sakdee S, et al. Potential prepore trimer formation by the Bacillus thuringiensis mosquito-specific toxin: molecular insights into a critical prerequisite of membrane-bound monomers. J Biol Chem 2015; 290(34): 20793-803.
[http://dx.doi.org/10.1074/jbc.M114.627554] [PMID: 26112409]
[30]
Kunthic T, Watanabe H, Kawano R, et al. pH regulates pore formation of a protease activated Vip3Aa from Bacillus thuringiensis. Biochim Biophys Acta Biomembr 2017; 1859(11): 2234-41.
[http://dx.doi.org/10.1016/j.bbamem.2017.08.018] [PMID: 28865796]
[31]
Jiang K, Mei SQ, Wang TT, Pan JH, Chen YH, Cai J. Vip3Aa induces apoptosis in cultured Spodoptera frugiperda (Sf9) cells. Toxicon 2016; 120: 49-56.
[http://dx.doi.org/10.1016/j.toxicon.2016.07.019] [PMID: 27476462]
[32]
Bae B, Ohene-Adjei S, Kocherginskaya S, et al. Molecular basis for the selectivity and specificity of ligand recognition by the family 16 carbohydrate-binding modules from Thermoanaerobacterium polysaccharolyticum ManA. J Biol Chem 2008; 283(18): 12415-25.
[http://dx.doi.org/10.1074/jbc.M706513200] [PMID: 18025086]
[33]
Coutinho PM, Henrissat B. Recent Advances in Carbohydrate Bioengineering. In: Carbohydrate-active enzymes: an integrated database approach 1999; 3-12.
[34]
Finney D. Probit Analysis 1971.
[35]
Soonsanga S, Rungrod A, Audtho M, Promdonkoy B. Tyrosine-776 of Vip3Aa64 from Bacillus thuringiensis is important for retained larvicidal activity during high-temperature storage. Curr Microbiol 2019; 76(1): 15-21.
[http://dx.doi.org/10.1007/s00284-018-1578-x] [PMID: 30302555]
[36]
Liu M, Liu R, Luo G, Li H, Gao J. Effects of site-mutations within the 22 kDa no-core fragment of the Vip3Aa11 insecticidal toxin of Bacillus thuringiensis. Curr Microbiol 2017; 74(5): 655-9.
[http://dx.doi.org/10.1007/s00284-017-1233-y] [PMID: 28321527]
[37]
Shao E, Zhang A, Yan Y, et al. Oligomer formation and insecticidal activity of Bacillus thuringiensis Vip3Aa toxin. Toxins (Basel) 2020; 12(4): 274.
[http://dx.doi.org/10.3390/toxins12040274] [PMID: 32340293]
[38]
Palma L, Scott DJ, Harris G, et al. The Vip3Ag4 insecticidal protoxin from Bacillus thuringiensis adopts a tetrameric configuration that is maintained on proteolysis. Toxins (Basel) 2017; 9(5): 165.
[http://dx.doi.org/10.3390/toxins9050165] [PMID: 28505109]
[39]
Brun E, Johnson PE, Creagh AL, et al. Structure and binding specificity of the second N-terminal cellulose-binding domain from Cellulomonas fimi endoglucanase C. Biochemistry 2000; 39(10): 2445-58.
[http://dx.doi.org/10.1021/bi992079u] [PMID: 10704194]
[40]
Sainz-Polo MA, González B, Menéndez M, Pastor FI, Sanz-Aparicio J. Exploring multimodularity in plant cell wall deconstruction: Structural and functional analysis of Xyn10C containing the CBM22-1-CBM22-2 tandem. J Biol Chem 2015; 290(28): 17116-30.
[http://dx.doi.org/10.1074/jbc.M115.659300] [PMID: 26001782]
[41]
Gomis-Cebolla J, Ferreira Dos Santos R, Wang Y, et al. Domain shuffling between Vip3Aa and Vip3Ca: chimera stability and insecticidal activity against European, American, African, and Asian pests. Toxins (Basel) 2020; 12(2): 99.
[http://dx.doi.org/10.3390/toxins12020099] [PMID: 32033215]
[42]
Banyuls N, Hernández-Rodríguez CS, Van Rie J, Ferré J. Critical amino acids for the insecticidal activity of Vip3Af from Bacillus thuringiensis: Inference on structural aspects. Sci Rep 2018; 8(1): 7539.
[http://dx.doi.org/10.1038/s41598-018-25346-3] [PMID: 29765057]
[43]
Duchesne L, Tissot B, Rudd TR, Dell A, Fernig DG. N-glycosylation of fibroblast growth factor receptor 1 regulates ligand and heparan sulfate co-receptor binding. J Biol Chem 2006; 281(37): 27178-89.
[http://dx.doi.org/10.1074/jbc.M601248200] [PMID: 16829530]
[44]
Fernandez LE, Aimanova KG, Gill SS, Bravo A, Soberón M. A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae. Biochem J 2006; 394(Pt 1): 77-84.
[http://dx.doi.org/10.1042/BJ20051517] [PMID: 16255715]
[45]
Knight PJ, Carroll J, Ellar DJ. Analysis of glycan structures on the 120 kDa aminopeptidase N of Manduca sexta and their interactions with Bacillus thuringiensis Cry1Ac toxin. Insect Biochem Mol Biol 2004; 34(1): 101-12.
[http://dx.doi.org/10.1016/j.ibmb.2003.09.007] [PMID: 14976987]
[46]
Schnepf E, Crickmore N, Van Rie J, et al. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 1998; 62(3): 775-806.
[http://dx.doi.org/10.1128/MMBR.62.3.775-806.1998] [PMID: 9729609]
[47]
Valaitis AP, Jenkins JL, Lee MK, Dean DH, Garner KJ. Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity. Arch Insect Biochem Physiol 2001; 46(4): 186-200.
[http://dx.doi.org/10.1002/arch.1028] [PMID: 11304752]