Characterisation of the structure-function relationship of the bacillus thuringiensis vip3a insecticidal proteins
- Autores: Núria Banyuls i Ferrando
- Directores de la Tesis: Juan Ferré Manzanero (dir. tes.)
- Lectura: En la Universitat de València ( España ) en 2017
- Idioma: español
- Tribunal Calificador de la Tesis: Primitivo Caballero (presid.), Patricia Hernández-Martínez (secret.), Silvia Caccia (voc.)
- Programa de doctorado: Programa de Doctorado en Biomedicina y Biotecnología por la Universitat de València (Estudi General)
- Materias:
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- Resumen
Modern agriculture demands for more sustainable agrochemicals to reduce the environmental and health impact. Some entomopathogenic bacteria produce insecticidal proteins which accumulate in inclusion bodies or parasporal crystals (such as the Cry and Cyt proteins), as well as insecticidal proteins which are secreted to the culture media. Among the latter, there are the Vip proteins, which are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some Coleoptera and Hemiptera. For the most recently reported Vip4 family, no target insects have been found as yet. Vip3 have no sequence similarity to any other proteins families known and are toxic to a wide variety of Lepidoptera. Its mode of action is yet not completely elucidated but a general mode of action similar to that of the Cry proteins (proteolytic activation, binding to the brush border membrane of the midgut epithelium, and pore formation) is assumed. Vip3A proteins do not share binding sites with Cry proteins, which makes them good candidates to be combined with Cry proteins in transgenic plants (Bt-crops) to prevent or delay insect resistance, and to broaden the insecticidal spectrum. Vip3A are an important tool for crop protection against caterpillar pests in integrated pest management (IPM) strategies.
This thesis aimed to deeper characterise the function and structure of the Vip3A proteins. In chapter 1, the toxicity of 5 different Vip3A proteins against 8 different caterpillar pests was first assessed: Vip3Aa, Vip3Ab, Vip3Ad, Vip3Ae and Vip3Af and their corresponding trypsin-activated toxins were tested for their toxicity against Agrotis ipsilon, Helicoverpa armigera, Mamestra brassicae, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis, Ostrinia nubilalis and Lobesia botrana. No major differences were found when comparing protoxins vs. trypsin-activated toxins. Vip3Aa, Vip3Ae and Vip3Af displayed overall good insecticidal activity against all insect species with the exception of O. nubilalis, which was found to be tolerant to all Vip3A tested and only marginal mortality was caused by the Vip3Af. Vip3Ad protein was not toxic to any of the tested species whereas Vip3Af showed the broadest range of activity.
The·3D-structure of the Vip3A proteins are not known, therefore, consecutives steps were performed to achieve a better insight on the Vip3Aprotein folding and structure. Vip3 proteins are expressed as a precursor that is to be activated by the insect gut proteases. In the 2nd chapter, stability of the Vip3Aa against proteases was investigated in the presence of SDS. Vip3Aa16 protoxin (of 89 kDa) was treated at high concentrations of trypsin and Agrotis ipsilon midgut juice. When the reactions were not properly neutralized, the results of SDS-PAGE analysis (as well as those with Agrotis ipsilon midgut juice) equivocally indicated that the protoxin could be completely processed, although it retained full toxicity against A. ipsilon. However, when the proteolytic reaction was efficiently stopped, there was revealed that the protoxin was only cleaved at a primary cleavage site, regardless the amount of trypsin used. The 66 kDa and the 19 kDa peptides generated by the proteases co-eluted after gel filtration chromatography, indicating that they remain together after cleavage. The 66 kDa fragment was found to be extremely resistant to proteases and that the misleading degradation observed in the SDS-PAGE was a consequence of the inefficient neutralisation of the enzymatic reaction and the interaction of the SDS molecules with the Vip3A protein. These results were reproduced in the 3d chapter with the different Vip3Af protein and the different pest species S. frugiperda. The misleading degradation previously reported for the closely related Vip3Aa16 was also observed in the Vip3Af at the highest concentration of peptidases used. The apparently degraded protein was active against S. frugiperda neonates. When the trypsin-activated toxin was further treated with trypsin, the misleading over-processing of the 62 kDa core was no longer observed in the SDS-PAGE. The proteolytic activation of the Vip3A is proposed to be stepwise fashion, which first step involves the formation of a toxin core of 62-66 kDa fragment that undergoes a subtle folding change likely involved in the insecticidal mechanism.
In the 4th chapter, the alanine scanning technique was performed on 558 out of the total of 788 amino acids of the Vip3Af1 protein. Alanine scanning is a successful technique for mapping crucial positions or epitopes in a protein and allows a greater insight into protein structure-function relationships. From the 558 residue substitutions, 19 impaired protein expression and 11 compromised the insecticidal activity against S. frugiperda. Substitutions that reduced insecticidal activity mainly clustered in two regions of the protein sequence (amino acids 167-272 and amino acids 689-741). Most of the substitutions that impaired the activity to S. frugiperda behave likewise to Agrotis segetum, with few exceptions. The characterisation of the sensitivity to proteases of these 11 mutant proteins displaying decreased insecticidal activity revealed 6 different band patterns as evaluated by SDS-PAGE. The study of the intrinsic fluorescence of all selected mutants revealed only slight shifts in the emission peak, likely indicating only minor changes in the tertiary structure. An in silico modelled 3D structure of Vip3Af1 is proposed for the first time.
Finally, in the 5th chapter, 12 different mutants were generated by site-directed mutagenesis all along the Vip3Af1 protein sequence. Site-directed mutagenesis is a common approach to function improvement as well as to deepen in the protein knowledge, especially when the protein structure is unknown. Ten of these mutants were successfully expressed and were functionally active, highlighting the high resilience of the Vip3Af1 sequence. None of the Vip3Af mutations caused an improvement of the insecticidal potency against S. frugiperda. Mutations in position 689 (G689S, G689E and N682K-G689S), as well as mutations E483H and W552H gave proteolytic patterns different to the native profile of the wild type. The intrinsic emission fluorescence spectra did not show a significant folding change. Implications on the intramolecular interactions and on the conformation of the protein are further discussed.
The results obtained in this Thesis give to a better understanding of the protein structure and function of Vip3A proteins, which will be helpful for the decision making when and how using B. thuringiensis or its insecticidal proteins as a phytosanitary resource in pest management programs and resistance management strategies.
