scholarly journals In vivo nanoscale analysis of the dynamic synergistic interaction of Bacillus thuringiensis Cry11Aa and Cyt1Aa toxins in Aedes aegypti

2021 ◽  
Vol 17 (1) ◽  
pp. e1009199
Author(s):  
Samira López-Molina ◽  
Nathaly Alexandre do Nascimento ◽  
Maria Helena Neves Lobo Silva-Filha ◽  
Adán Guerrero ◽  
Jorge Sánchez ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Lower Layer ◽  
Larval Mortality ◽  
Membrane Insertion ◽  
Cell Cytoplasm ◽  
Mutant Proteins ◽  
Posterior Midgut ◽  
High Larval Mortality

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered “net like” structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.

scholarly journals Investigation of the pore-forming mechanism of a cytolytic δ-endotoxin from Bacillus thuringiensis

2003 ◽  
Vol 374 (1) ◽  
pp. 255-259 ◽  
Author(s):  
Boonhiang PROMDONKOY ◽  
David J. ELLAR
Keyword(s):  
Bacillus Thuringiensis ◽  
Blood Cells ◽  
Pore Formation ◽  
Response Curve ◽  
Effect Of Temperature ◽  
Membrane Insertion ◽  
Free Solution ◽  
Forming Mechanism ◽  
Close Proximity ◽  
Membrane Bound

Cyt2Aa1 is a cytolytic protein produced by Bacillus thuringiensis subsp. kyushuensis. Penetration of the toxin into membranes has been studied to learn more about membrane-insertion mechanisms and transmembrane-pore formation. The haemolysis assay of Cyt2Aa1 showed a steep and sigmoidal dose–response curve, indicating that toxin aggregation or oligomerization is required for pore formation. Studies of the effect of temperature on pore formation and fluorimetric studies of acrylodan-labelled toxin suggest that toxin inserts into the membrane before oligomerizing to form a pore. Low temperature neither inhibited membrane binding nor closed pores that have been formed, but markedly inhibited oligomerization of the toxin molecules. When toxin-treated red blood cells at 4 °C were transferred to a toxin-free solution at 37 °C, no significant increase in haemolysis was observed. This result suggests that membrane-bound toxin could not diffuse laterally and interact with other molecules to form a pore. From these results, we propose that Cyt2Aa1 binds and inserts into the membrane as a monomer. Oligomerization occurs when toxin molecules have bound in close proximity to each other and pores are formed from large oligomers.

scholarly journals Production of Chymotrypsin-Resistant Bacillus thuringiensis Cry2Aa1 δ-Endotoxin by Protein Engineering

1999 ◽  
Vol 65 (10) ◽  
pp. 4601-4605 ◽  
Author(s):  
Mongkon Audtho ◽  
Algimantas P. Valaitis ◽  
Oscar Alzate ◽  
Donald H. Dean
Keyword(s):  
Bacillus Thuringiensis ◽  
Major Protein ◽  
Cleavage Sites ◽  
Mutant Proteins ◽  
Protein Fragments ◽  
Protease Cleavage ◽  
Protease Cleavage Sites ◽  
Domain I ◽  
Active Fragment

ABSTRACT Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) fromBacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when125I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.

scholarly journals Bacillus thuringiensis Cry1Ac Toxin-Binding and Pore-Forming Activity in Brush Border Membrane Vesicles Prepared from Anterior and Posterior Midgut Regions of Lepidopteran Larvae

2008 ◽  
Vol 74 (6) ◽  
pp. 1710-1716 ◽  
Author(s):  
Ana Rodrigo-Simón ◽  
Silvia Caccia ◽  
Juan Ferré
Keyword(s):  
Bacillus Thuringiensis ◽  
Brush Border ◽  
Mode Of Action ◽  
Pore Formation ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Insect Species ◽  
Binding Parameters ◽  
Larval Midgut ◽  
Posterior Midgut

ABSTRACT It is generally accepted that Bacillus thuringiensis Cry toxins insert into the apical membrane of the larval midgut after binding to specific receptors, and there is evidence that the distribution of binding molecules along the midgut is not uniform. By use of the voltage-sensitive dye DiSC3(5) and 125I-labeled Cry1Ac, we have measured the effect of Cry1Ac in terms of permeabilization capacity and of binding parameters on brush border membrane vesicles (BBMV) prepared from the anterior and the posterior regions of the larval midgut from two insect species, Manduca sexta and Helicoverpa armigera. The permeabilizing activity was significantly higher with BBMV from the posterior region than with the one observed in the anterior region in both insect species. Instead, 125I-Cry1Ac bound specifically to BBMV from the two midgut regions, with no significant differences in the binding parameters between the anterior and posterior regions within an insect species. N-acetylgalactosamine inhibition patterns on pore formation and binding differed between anterior and posterior midgut regions and between species, providing evidence of a multifaceted involvement of the sugar in the Cry1Ac mode of action. The analysis of binding and pore formation in different midgut regions could be an effective method to study differences in the mode of action of Cry1Ac toxin in different species.

scholarly journals Helix 4 Mutants of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa Display Altered Pore-Forming Abilities

2004 ◽  
Vol 70 (10) ◽  
pp. 6123-6130 ◽  
Author(s):  
Vincent Vachon ◽  
Gabrielle Préfontaine ◽  
Cécile Rang ◽  
Florence Coux ◽  
Marc Juteau ◽  
...  
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Membrane Vesicles ◽  
Amino Acid Position ◽  
Site Directed Mutagenesis ◽  
Insecticidal Toxin ◽  
Mutant Proteins ◽  
Charged Residues ◽  
Α Helix

ABSTRACT The role played by α-helix 4 of the Bacillus thuringiensis toxin Cry1Aa in pore formation was investigated by individually replacing each of its charged residues with either a neutral or an oppositely charged amino acid by using site-directed mutagenesis. The majority of the resulting mutant proteins were considerably less toxic to Manduca sexta larvae than Cry1Aa. Most mutants also had a considerably reduced ability to form pores in midgut brush border membrane vesicles isolated from this insect, with the notable exception of those with alterations at amino acid position 127 (R127N and R127E), located near the N-terminal end of the helix. Introducing a negatively charged amino acid near the C-terminal end of the helix (T142D and T143D), a region normally devoid of charged residues, completely abolished pore formation. For each mutant that retained detectable pore-forming activity, reduced membrane permeability to KCl was accompanied by an approximately equivalent reduction in permeability to N-methyl-d-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose and by a reduced rate of pore formation. These results indicate that the main effect of the mutations was to decrease the toxin's ability to form pores. They provide further evidence that α-helix 4 plays a crucial role in the mechanism of pore formation.

scholarly journals Evaluation of indigenous the nucleopolyhedrovirus (NPV) of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) in combination with chlorantraniliprole against Spodoptera species

2021 ◽  
Vol 31 (1) ◽  
Author(s):  
Ghulam Sarwar ◽  
Naeem Arshad Maan ◽  
Muhammad Ahsin Ayub ◽  
Muhammad Rafiq Shahid ◽  
Mubasher Ahmad Malik ◽  
...  
Keyword(s):  
Larval Mortality ◽  
Adult Emergence ◽  
Lethal Concentration ◽  
Pest Species ◽  
Vegetable Crops ◽  
Chemical Insecticide ◽  
Larval Instars ◽  
Novel Method ◽  
High Larval Mortality ◽  
Lepidoptera Noctuidae

Abstract Background The armyworms, Spodoptera exigua (Hübner), and S. litura (Fabricius) (Lepidoptera: Noctuidae) are polyphagous pests of many cash crops. Heavy crop losses have been reported for the fruit and vegetable crops each year owing to the diverse impact on global economies. The present study was aimed to sort out a novel method of pest control using the insect’s own nucleopolyhedrosis virus (NPV) alone and in combination with a new chemistry insecticide chlorantraniliprole. Results In the study, the effect of indigenous isolated nucleopolyhedrovirus (NPV) and the chemical insecticide (chlorantraniliprole) formulations against the 2nd and 4th larval instars of S. litura and S. exigua, collected from the different geographical region of Punjab (Pakistan) province, was evaluated. Three concentrations of the NPV isolate, sub-lethal (1 × 104, 6 × 104 POB ml−1), lethal (3 × 105 POB ml−1), and chlorantraniliprole 0.01 μl l−1, were applied alone and in combination against the 2nd and 4th larval instars of both pest species. The lethal concentration of NPV + chlorantraniliprole exhibited synergistic interaction and caused high larval mortality against both instars, while in all other combinations, additive effect was observed. Moreover, NPV + chlorantraniliprole at lethal concentration exhibited decreased pupation, adult emergence, and egg eclosion. Conclusion The implications of using NPV alone and in combination with an insecticide are discussed briefly in this study.

LONG TERM STUDY OF THE EFFECTIVENESS OF AERIAL APPLICATION OF BACILLUS THURINGIENSIS – ACEPHATE COMBINATIONS AGAINST THE SPRUCE BUDWORM, CHORISTONEURA FUMIFERANA (LEPIDOPTERA: TORTRICIDAE)

1977 ◽  
Vol 109 (9) ◽  
pp. 1239-1248 ◽  
Author(s):  
O. N. Morris
Keyword(s):  
Bacillus Thuringiensis ◽  
Choristoneura Fumiferana ◽  
Larval Mortality ◽  
Abies Balsamea ◽  
Spruce Budworm ◽  
White Spruce ◽  
Balsam Fir ◽  
Population Densities ◽  
Long Term Study

AbstractBacillus thuringiensis (Dipel® 36B) mixed with a sublethal concentration of acephate (Orthene®) (O, S-dimethyl acetylphosphoramidothioate), an organophosphorous insecticide, was applied at 2.35–14 l./ha to white spruce (Picea glauca) and balsam fir (Abies balsamea) trees infested with spruce budworm, Choristoneura fumiferana (Clem.). The treatment rate was 20 Billion International Units of B. thuringiensis (B.t.) activity with or without 42 g of active ingredient of acephate/ha.The ground deposit of the standard Dipel wettable powder formulation was 12% of emitted volume compared with 21–32% for the Dipel 36B flowable. The viability of B.t. spores was drastically reduced after 1 day of weathering but a high level of biological activity by the spore–crystal complex persisted for up to 20 days post-spray due probably to crystal activity.The addition of about 10% of the recommended operational rate of acephate to the B.t. suspension increased larval mortality by 34% when applied at 4.7 l./ha. Reductions in budworm populations were 97–99% in B.t. + acephate plots and 86–90% in B.t. alone plots.Plots with moderate budworm densities of up to 27 larvae/100 buds on white spruce and 36/100 on balsam fir were satisfactorily protected from excessive defoliation in the year of spray by B.t. with or without acephate. Plots with higher population densities were not satisfactorily protected based on the branch sample examination but aerial color photographs indicated good protection to the top third of the trees. Population declines were greater and defoliation and oviposition were lower in the treated plots than in the untreated checks 1 year later without further treatment. Two years later the larval population densities in all plots were low but the density was twice as high in the untreated check as in the treated plots, indicating long term suppression by the treatments. Defoliation was negligible in all plots.The treatments had no deleterious effect on spruce budworm parasitism. The data indicate that the integrated approach using Bacillus thuringiensis – chemical pesticide combinations is a viable alternative to the use of chemical pesticides alone in spruce budworm control. Large scale testing is now warranted.

scholarly journals Flagellar Morphogenesis: Protein Targeting and Assembly in the Paraflagellar Rod of Trypanosomes

1999 ◽  
Vol 19 (12) ◽  
pp. 8191-8200 ◽  
Author(s):  
Philippe Bastin ◽  
Thomas H. MacRae ◽  
Susan B. Francis ◽  
Keith R. Matthews ◽  
Keith Gull
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Brucei ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Reporter ◽  
Mutant Proteins ◽  
Green Fluorescent ◽  
A Minor ◽  
Fluorescent Protein Reporter

ABSTRACT The paraflagellar rod (PFR) of the African trypanosomeTrypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.

Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A

1989 ◽  
Vol 9 (6) ◽  
pp. 2453-2463
Author(s):  
P Yaciuk ◽  
J K Choi ◽  
D Shalloway
Keyword(s):  
Protein Kinase ◽  
3T3 Cells ◽  
Triple Mutant ◽  
Dependent Protein Kinase ◽  
Mutant Proteins ◽  
Tetradecanoyl Phorbol Acetate ◽  
Dependent Protein ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.

Effects of truncated neurofilament proteins on the endogenous intermediate filaments in transfected fibroblasts

1991 ◽  
Vol 99 (2) ◽  
pp. 335-350 ◽  
Author(s):  
S.S. Chin ◽  
P. Macioce ◽  
R.K. Liem
Keyword(s):  
Intermediate Filament ◽  
Dominant Negative ◽  
Deletion Mutants ◽  
Tail Domain ◽  
Cultured Fibroblasts ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Novel Approach ◽  
Carboxyl Terminal

The expression and assembly characteristics of carboxyl- and amino-terminal deletion mutants of rat neurofilament low Mr (NF-L) and neurofilament middle Mr (NF-M) proteins were examined by transient transfection of cultured fibroblasts. Deletion of the carboxyl-terminal tail domain of either protein indicated that this region was not absolutely essential for co-assembly into the endogenous vimentin cytoskeleton. However, deletion into the alpha-helical rod domain resulted in an inability of the mutant proteins to co-assemble with vimentin into filamentous structures. Instead, the mutant proteins appeared to be assembled into unusual tubular-vesicular structures. Additionally, these latter deletions appeared to act as dominant negative mutants which induced the collapse of the endogenous vimentin cytoskeleton as well as the constitutively expressed NF-H and NF-M cytoskeletons in stably transfected cell lines. Thus, an intact alpha-helical rod domain was essential for normal IF co-assembly whereas carboxyl-terminal deletions into this region resulted in dramatic alterations of the existing type III and IV intermediate filament cytoskeletons in vivo. Deletions from the amino-terminal end into the alpha-helical rod region gave different results. With these deletions, the transfected protein was not co-assembled into filaments and the endogenous vimentin IF network was not disrupted, indicating that these deletion mutants are recessive. The dominant negative mutants may provide a novel approach to studying intermediate filament function within living cells.

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