Insecticidal spectrum and mode of action of the Bacillus thuringiensis Vip3Ca insecticidal protein

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Vip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its structure. The Vip3Aa protoxin (of 89 kDa) was treated with trypsin at concentrations from 1:100 to 120:100 (trypsin:Vip3A, w:w). If the action of trypsin was 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. However, when the proteolytic reaction was efficiently stopped, it was revealed that the protoxin was only cleaved at a primary cleavage site, regardless of 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. The trypsin treatment of the protoxin in the presence of SDS revealed the presence of secondary cleavage sites at S-509, and presumably at T-466 and V-372, rendering C-terminal fragments of approximately 29, 32, and 42 kDa, respectively. The fact that the predicted secondary structure of the Vip3Aa protein shows a cluster of beta sheets in the C-terminal region of the protein might be the reason behind the higher stability to proteases compared to the rest of the protein, which is mainly composed of alpha helices.

Section: Introductionmentioning

confidence: 99%

Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis

Bel

Banyuls

Chakroun

et al. 2017

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“…The current understanding of the mode of action of Vip3 proteins remains limited, although a number of steps towards intoxication are known [19]. Proteolysis of the ~88 kDa full-length Vip3A proteins to ~65 kDa by trypsin or the gut juices of both susceptible and non-susceptible insects has been reported [12,13,20,21].…”

Section: Introductionmentioning

confidence: 99%

The Vip3Ag4 Insecticidal Protoxin from Bacillus thuringiensis Adopts A Tetrameric Configuration That Is Maintained on Proteolysis

Palma

Scott

Harris

et al. 2017

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The Vip3 proteins produced during vegetative growth by strains of the bacterium Bacillus thuringiensis show insecticidal activity against lepidopteran insects with a mechanism of action that may involve pore formation and apoptosis. These proteins are promising supplements to our arsenal of insecticidal proteins, but the molecular details of their activity are not understood. As a first step in the structural characterisation of these proteins, we have analysed their secondary structure and resolved the surface topology of a tetrameric complex of the Vip3Ag4 protein by transmission electron microscopy. Sites sensitive to proteolysis by trypsin are identified and the trypsin-cleaved protein appears to retain a similar structure as an octomeric complex comprising four copies each of the ~65 kDa and ~21 kDa products of proteolysis. This processed form of the toxin may represent the active toxin. The quality and monodispersity of the protein produced in this study make Vip3Ag4 a candidate for more detailed structural analysis using cryo-electron microscopy.

“…They are produced by certain B. thuringiensis strains and bind to receptors that are different from those of Cry proteins [33,34], and thus, they have a spectrum of activity complementary to that of the Cry proteins. Therefore, a combination of Cry and Vip proteins could broaden the spectrum of insecticidal activity [35,36,37,38] and prevent the evolution of resistance of insects to Cry proteins [39,40,41]. …”

Section: Introductionmentioning

confidence: 99%

Assessment of the Antimicrobial Activity and the Entomocidal Potential of Bacillus thuringiensis Isolates from Algeria

Djenane

Natèche

Amziane

et al. 2017

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This work represents the first initiative to analyze the distribution of B. thuringiensis in Algeria and to evaluate the biological potential of the isolates. A total of 157 isolates were recovered, with at least one isolate in 94.4% of the samples. The highest Bt index was found in samples from rhizospheric soil (0.48) and from the Mediterranean area (0.44). Most isolates showed antifungal activity (98.5%), in contrast to the few that had antibacterial activity (29.9%). A high genetic diversity was made evident by the finding of many different crystal shapes and various combinations of shapes within a single isolate (in 58.4% of the isolates). Also, over 50% of the isolates harbored cry1, cry2, or cry9 genes, and 69.3% contained a vip3 gene. A good correlation between the presence of chitinase genes and antifungal activity was observed. More than half of the isolates with a broad spectrum of antifungal activity harbored both endochitinase and exochitinase genes. Interestingly, 15 isolates contained the two chitinase genes and all of the above cry family genes, with some of them harboring a vip3 gene as well. The combination of this large number of genes coding for entomopathogenic proteins suggests a putative wide range of entomotoxic activity.