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Article

Baculovirus Expression and Functional Analysis of Vpa2 Proteins from Bacillus thuringiensis

1
Institute for Multidisciplinary Research in Applied Biology, Universidad Pública de Navarra, 31006 Pamplona, Navarra, Spain
2
Instituto Académico Pedagógico de Ciencias Básicas y Aplicadas, Centro de Investigaciones y Transferencia de Villa María (CITVM-CONICET), Universidad Nacional De Villa María, Villa María, Córdoba 5900, Argentina
3
Departamento de Investigación y Desarrollo, Bioinsectis SL, Polígono Industrial Mocholi Plaza Cein 5, Nave A14, 31110 Noain, Navarra, Spain
4
Instituto de Ecología AC, Xalapa, Veracruz 91073, Mexico
*
Author to whom correspondence should be addressed.
Both authors contributed equally to this manuscript.
Toxins 2020, 12(9), 543; https://doi.org/10.3390/toxins12090543
Received: 24 July 2020 / Revised: 19 August 2020 / Accepted: 20 August 2020 / Published: 22 August 2020
(This article belongs to the Section Bacterial Toxins)
The mode of action underlying the insecticidal activity of the Bacillus thuringiensis (Bt) binary pesticidal protein Vpa1/Vpa2 is uncertain. In this study, three recombinant baculoviruses were constructed using Bac-to-Bac technology to express Vpa2Ac1 and two novel Vpa2-like genes, Vpa2-like1 and Vpa2-like2, under the baculovirus p10 promoter in transfected Sf9 cells. Pairwise amino acid analyses revealed a higher percentage of identity and a lower number of gaps between Vpa2Ac1 and Vpa2-like2 than to Vpa2-like1. Moreover, Vpa2-like1 lacked the conserved Ser-Thr-Ser motif, involved in NAD binding, and the (F/Y)xx(Q/E)xE consensus sequence, characteristic of the ARTT toxin family involved in actin polymerization. Vpa2Ac1, Vpa2-like1 and Vpa2-like2 transcripts and proteins were detected in Sf9 culture cells, but the signals of Vpa2Ac1 and Vpa2-like2 were weak and decreased over time. Sf9 cells infected by a recombinant bacmid expressing Vpa2-like1 showed typical circular morphology and produced viral occlusion bodies (OBs) at the same level as the control virus. However, expression of Vpa2Ac1 and Vpa2-like2 induced cell polarization, similar to that produced by the microfilament-destabilizing agent cytochalasin D and OBs were not produced. The presence of filament disrupting agents, such as nicotinamide and nocodazole, during transfection prevented cell polarization and OB production was observed. We conclude that Vpa2Ac1 and Vpa2-like2 proteins likely possess ADP-ribosyltransferase activity that modulated actin polarization, whereas Vpa2-like1 is not a typical Vpa2 protein. Vpa2-like2 has now been designated Vpa2Ca1 (accession number AAO86513) by the Bacillus thuringiensis delta-endotoxin nomenclature committee. View Full-Text
Keywords: vegetative pesticidal proteins; Vpa; entomopathogen; ADP-ribosyltransferase; recombinant baculovirus; lepidopteran cells; biopesticides; broad spectrum vegetative pesticidal proteins; Vpa; entomopathogen; ADP-ribosyltransferase; recombinant baculovirus; lepidopteran cells; biopesticides; broad spectrum
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MDPI and ACS Style

Simón, O.; Palma, L.; Fernández, A.B.; Williams, T.; Caballero, P. Baculovirus Expression and Functional Analysis of Vpa2 Proteins from Bacillus thuringiensis. Toxins 2020, 12, 543. https://doi.org/10.3390/toxins12090543

AMA Style

Simón O, Palma L, Fernández AB, Williams T, Caballero P. Baculovirus Expression and Functional Analysis of Vpa2 Proteins from Bacillus thuringiensis. Toxins. 2020; 12(9):543. https://doi.org/10.3390/toxins12090543

Chicago/Turabian Style

Simón, Oihane, Leopoldo Palma, Ana Beatriz Fernández, Trevor Williams, and Primitivo Caballero. 2020. "Baculovirus Expression and Functional Analysis of Vpa2 Proteins from Bacillus thuringiensis" Toxins 12, no. 9: 543. https://doi.org/10.3390/toxins12090543

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