scholarly journals Differential Role of Manduca sexta Aminopeptidase-N and Alkaline Phosphatase in the Mode of Action of Cry1Aa, Cry1Ab, and Cry1Ac Toxins from Bacillus thuringiensis

2013 ◽  
Vol 79 (15) ◽  
pp. 4543-4550 ◽  
Author(s):  
Biviana Flores-Escobar ◽  
Hector Rodríguez-Magadan ◽  
Alejandra Bravo ◽  
Mario Soberón ◽  
Isabel Gómez
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Thuringiensis ◽  
Larval Development ◽  
Manduca Sexta ◽  
Mode Of Action ◽  
Membrane Vesicles ◽  
Aminopeptidase N ◽  
Content Type ◽  
Insecticidal Effect ◽  
Protein Levels

ABSTRACTAminopeptidase-N (APN1) and alkaline phosphatase (ALP) proteins located in the midgut epithelium ofManduca sextahave been implicated as receptors for Cry1Aa, Cry1Ab, and Cry1Ac insecticidal proteins produced byBacillus thuringiensissubsp.kurstaki. In this study, we analyzed the roles of ALP and APN1 in the toxicity of these three Cry1A proteins. Ligand blot analysis using brush border membrane vesicles ofM. sextashowed that Cry1Aa and Cry1Ab bind preferentially to ALP during early instars while binding to APN was observed after the third instar of larval development. Cry1Ac binds to APN throughout all larval development, with no apparent binding to ALP. ALP was cloned fromM. sextamidgut RNA and expressed inEscherichia coli. Surface plasmon resonance binding analysis showed that recombinant ALP binds to Cry1Ac with 16-fold lower affinity than to Cry1Aa or Cry1Ab. Downregulation of APN1 and ALP expression by RNA interference (RNAi) using specific double-stranded RNA correlated with a reduction of transcript and protein levels. Toxicity analysis of the three Cry1A proteins in ALP- or APN1-silenced larvae showed that Cry1Aa relies similarly on both receptor molecules for toxicity. In contrast, RNAi experiments showed that ALP is more important than APN for Cry1Ab toxicity, while Cry1Ac relied principally on APN1. These results indicated that ALP and APN1 have a differential role in the mode of action of Cry1A toxins, suggesting thatB. thuringiensissubsp.kurstakiproduces different Cry1A toxins that in conjunction target diverse midgut proteins to exert their insecticidal effect.

The Bacillus thuringiensis Cry1Ac toxin-induced permeability change in Manduca sexta midgut brush border membrane vesicles proceeds by more than one mechanism

1997 ◽  
Vol 110 (24) ◽  
pp. 3099-3104
Author(s):  
J. Carroll ◽  
M.G. Wolfersberger ◽  
D.J. Ellar
Keyword(s):  
Bacillus Thuringiensis ◽  
Manduca Sexta ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Aminopeptidase N ◽  
Insecticidal Toxin ◽  
Cry1ac Toxin ◽  
Posterior Midgut

Aminopeptidase N purified from whole Manduca sexta midgut binds the Cry1Ac insecticidal toxin from Bacillus thuringiensis and this binding is inhibited by N-acetylgalactosamine (GalNAc). We have examined the membrane permeabilising activity of the Cry1Ac toxin using brush border membrane vesicles (BBMV) prepared from the anterior (A-BBMV) and posterior (P-BBMV) subregions of the M. sexta midgut. A toxin mixing assay demonstrated a faster rate of toxin activity on P-BBMV than on A-BBMV. In the presence of GalNAc this rapid activity on P-BBMV was reduced to the rate seen with A-BBMV. GalNAc had no effect on the rate of A-BBMV permeabilisation by Cry1Ac. Aminopeptidase N assays of A- and P-BBMV demonstrated that this Cry1Ac binding protein is concentrated in the posterior midgut region of M. sexta. It therefore appears that there are two mechanisms by which Cry1Ac permeabilises the M. sexta midgut membrane: a GalNAc-sensitive mechanism restricted to the posterior midgut region, probably involving aminopeptidase N binding, and a previously undetected mechanism found in both the posterior and anterior regions.

scholarly journals Denaturation of Either Manduca sexta Aminopeptidase N or Bacillus thuringiensis Cry1A Toxins Exposes Binding Epitopes Hidden under Nondenaturing Conditions

2002 ◽  
Vol 68 (5) ◽  
pp. 2106-2112 ◽  
Author(s):  
Anu Daniel ◽  
Sreedhara Sangadala ◽  
Donald H. Dean ◽  
Michael J. Adang
Keyword(s):  
Bacillus Thuringiensis ◽  
Manduca Sexta ◽  
Brush Border ◽  
Binding Proteins ◽  
Membrane Vesicles ◽  
Aminopeptidase N ◽  
Toxin Binding ◽  
Cry Toxin ◽  
Domain I ◽  
Ligand Blot

ABSTRACT The effect of polypeptide denaturation of Bacillus thuringiensis Cry1A toxins or purified Manduca sexta 120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with 125I-labeled Cry1Ac, Cry1Ac mutant 509QNR-AAA511 (QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the N-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both 125I-Cry1Ac and 125I-QNR-AAA bound to 210- and 120-kDa proteins from M. sexta brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots 125I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, 125I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. 125I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured 125I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) 125I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.

scholarly journals Pore-forming properties of the Bacillus thuringiensis toxin Cry9Ca in Manduca sexta brush border membrane vesicles

2010 ◽  
Vol 1798 (6) ◽  
pp. 1111-1118 ◽  
Author(s):  
Jean-Frédéric Brunet ◽  
Vincent Vachon ◽  
Marc Juteau ◽  
Jeroen Van Rie ◽  
Geneviève Larouche ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Manduca Sexta ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Bacillus Thuringiensis Toxin

scholarly journals Aedes aegypti Membrane-Bound Alkaline Phosphatase Expressed in Escherichia coli Retains High-Affinity Binding for Bacillus thuringiensis Cry4Ba Toxin

2011 ◽  
Vol 77 (19) ◽  
pp. 6836-6840 ◽  
Author(s):  
Anon Thammasittirong ◽  
Manasave Dechklar ◽  
Somphob Leetachewa ◽  
Kusol Pootanakit ◽  
Chanan Angsuthanasombat
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Nitrilotriacetic Acid ◽  
Affinity Column ◽  
Dot Blot ◽  
Content Type ◽  
Binding Characteristics ◽  
High Affinity

ABSTRACTGlycosylphosphatidylinositol-linked alkaline phosphatase (GPI-ALP) from the epithelial membrane of the larval midgut ofAedes aegyptiwas previously identified as a functional receptor of theBacillus thuringiensisCry4Ba toxin. Here, heterologous expression inEscherichia coliof the cloned ALP, lacking the secretion signal and GPI attachment sequences, and assessment of its binding characteristics were further investigated. The 54-kDa His tag-fused ALP overexpressed as an inclusion body was soluble when phosphate buffer (pH 7.5) was supplemented with 8 M urea. After renaturation in a nickel-nitrilotriacetic acid (Ni-NTA) affinity column, the refolded ALP protein was able to retain its phosphatase activity. This refolded ALP also showed binding to the 65-kDa activated Cry4Ba toxin under nondenaturing (dot blot) conditions. Quantitative binding analysis using a quartz crystal microbalance revealed that the purified ALP immobilized on a gold electrode was bound by the Cry4Ba toxin in a stoichiometry of approximately 1:2 and with high affinity (dissociation constant [Kd] of ∼14 nM) which is comparable to that calculated from kinetic parameters (dissociation rate constant [koff]/binding constant [kon]). Altogether, the data presented here of theE. coli-expressed ALP fromA. aegyptiretaining high-affinity toxin binding support our notion that glycosylation of this receptor is not required for binding to its counterpart toxin, Cry4Ba.

scholarly journals A Binding Site for Bacillus thuringiensis Cry1Ab Toxin Is Lost during Larval Development in Two Forest Pests

2000 ◽  
Vol 66 (4) ◽  
pp. 1553-1558 ◽  
Author(s):  
Carolina Rausell ◽  
Amparo Consuelo Martínez-Ramírez ◽  
Inmaculada García-Robles ◽  
María Dolores Real
Keyword(s):  
Bacillus Thuringiensis ◽  
Larval Development ◽  
Binding Sites ◽  
Specific Binding ◽  
Membrane Vesicles ◽  
Thaumetopoea Pityocampa ◽  
Binding Assays ◽  
Forest Pests ◽  
First Instar ◽  
Nun Moth

ABSTRACT The insecticidal activity and receptor binding properties ofBacillus thuringiensis Cry1A toxins towards the forest pests Thaumetopoea pityocampa (processionary moth) andLymantria monacha (nun moth) were investigated. Cry1Aa, Cry1Ab, and Cry1Ac were highly toxic (corresponding 50% lethal concentration values: 956, 895, and 379 pg/μl, respectively) to first-instar T. pityocampa larvae. During larval development, Cry1Ab and Cry1Ac toxicity decreased with increasing age, although the loss of activity was more pronounced for Cry1Ab. Binding assays with 125I-labelled Cry1Ab and brush border membrane vesicles from T. pityocampa first- and last-instar larvae detected a remarkable decrease in the overall Cry1Ab binding affinity in last-instar larvae, although saturable Cry1Ab binding to both instars was observed. Homologous competition experiments demonstrated the loss of one of the two Cry1Ab high-affinity binding sites detected in first-instar larvae. Growth inhibition assays with sublethal doses of Cry1Aa, Cry1Ab, and Cry1Ac in L. monacha showed that all three toxins were able to delay molting from second instar to third instar. Specific saturable binding of Cry1Ab was detected only in first- and second-instar larvae. Cry1Ab binding was not detected in last-instar larvae, although specific binding of Cry1Aa and Cry1Ac was observed. These results demonstrate a loss of Cry1Ab binding sites during development on the midgut epithelium of T. pityocampa and L. monacha, correlating in T. pityocampa with a decrease in Cry1Ab toxicity with increasing age.

scholarly journals Enhancement ofBacillus thuringiensisCry1Ab and Cry1Fa Toxicity toSpodoptera frugiperdaby Domain III Mutations Indicates There Are Two Limiting Steps in Toxicity as Defined by Receptor Binding and Protein Stability

2018 ◽  
Vol 84 (20) ◽  
Author(s):  
Isabel Gómez ◽  
Josue Ocelotl ◽  
Jorge Sánchez ◽  
Christina Lima ◽  
Erica Martins ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Manduca Sexta ◽  
Spodoptera Frugiperda ◽  
Transgenic Maize ◽  
Invasive Pest ◽  
Effective Control ◽  
Alanine Scanning Mutagenesis ◽  
Content Type ◽  
Lepidopteran Pests ◽  
Domain Iii

ABSTRACTBacillus thuringiensisCry1Ab and Cry1Fa toxins are environmentally safe insecticides that control important insect pests.Spodoptera frugiperdais an important maize pest that shows low susceptibility to Cry1A toxins, in contrast to Cry1Fa, which is highly active against this pest and is used in transgenic maize forS. frugiperdacontrol. The β16 region from domain III of Cry1Ab has been shown to be involved in interactions with receptors such as alkaline phosphatase (ALP) or aminopeptidase (APN) in different lepidopteran insects. Alanine-scanning mutagenesis of amino acids of Cry1Ab β16 (509STLRVN514) revealed that certain β16 mutations, such as N514A, resulted in increased toxicity of Cry1Ab forS. frugiperdawithout affecting the toxicity for other lepidopteran larvae, such asManduca sextalarvae. Exhaustive mutagenesis of N514 was performed, showing that the Cry1Ab N514F, N514H, N514K, N514L, N514Q, and N514S mutations increased the toxicity towardS. frugiperda. A corresponding mutation was constructed in Cry1Fa (N507A). Toxicity assays of wild-type and mutant toxins (Cry1Ab, Cry1AbN514A, Cry1AbN514F, Cry1Fa, and Cry1FaN507A) against fourS. frugiperdapopulations from Mexico and one from Brazil revealed that Cry1AbN514A and Cry1FaN507A consistently showed 3- to 18-fold increased toxicity against four of fiveS. frugiperdapopulations. In contrast, Cry1AbN514F showed increased toxicity in only two of theS. frugiperdapopulations analyzed. The mutants Cry1AbN514A and Cry1AbN514F showed greater stability to midgut protease treatment. In addition, binding analysis of the Cry1Ab mutants showed that the increased toxicity correlated with increased binding to brush border membrane vesicles and increased binding affinity forS. frugiperdaALP, APN, and cadherin receptors.IMPORTANCESpodoptera frugiperdais the main maize pest in South and North America and also is an invasive pest in different African countries. However, it is poorly controlled byBacillus thuringiensisCry1A toxins expressed in transgenic crops, which effectively control other lepidopteran pests. In contrast, maize expressing Cry1Fa is effective in the control ofS. frugiperda, although its effectiveness is being lost due to resistance evolution. Some of the Cry1Ab domain III mutants characterized here show enhanced toxicity forS. frugiperdawithout loss of toxicity toManduca sexta. Thus, these Cry1Ab mutants could provide useful engineered toxins that, along with other Cry toxins, would be useful for developing transgenic maize expressing stacked proteins for the effective control ofS. frugiperdaand other lepidopteran pests in the field.

scholarly journals Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism

2015 ◽  
Vol 81 (15) ◽  
pp. 5184-5195 ◽  
Author(s):  
Xiaozhao Song ◽  
Wendy Kain ◽  
Douglas Cassidy ◽  
Ping Wang
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Thuringiensis ◽  
Trichoplusia Ni ◽  
Genetic Linkage ◽  
Genetic Mechanism ◽  
Cross Resistance ◽  
Genetic Linkage Analysis ◽  
Recessive Trait ◽  
Content Type ◽  
Significant Difference

ABSTRACTThe resistance to theBacillus thuringiensis(Bt) toxin Cry2Ab in a greenhouse-originatedTrichoplusia nistrain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in theT. nistrain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab inT. ni. From the dual-toxin-resistantT. nistrain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptibleT. nistrain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptibleT. nilarvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratoryT. nistrains. Genetic linkage analysis showed that the Cry2Ab resistance inT. niwas not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter geneABCC2. Therefore, the Cry2Ab resistance inT. niis conferred by a novel but unknown genetic mechanism.

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