Single-reversal charge in the β10-β11 receptor-binding loop of Bacillus thuringiensis Cry4Aa and Cry4Ba toxins reflects their different toxicity against Culex spp. larvae

Visitsattapongse, Sarinporn; Sakdee, Somsri; Leetacheewa, Somphob; Angsuthanasombat, Chanan

doi:10.1016/j.bbrc.2014.06.090

Cited by 4 publications

(5 citation statements)

References 30 publications

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“…As at this stage, we still could not obtain a purified functional Cq -mALP1264 isoform to be used for binding studies, in silico attempts were therefore made instead to calculate ΔΔG bind for determining affinity binding changes in Cry4Aa- Cq -mALP1264 interactions upon such single substitutions. The computed results revealed that the K514D mutation showed the largest ΔΔG bind value or the greatest decrease in binding affinity as compared to three other Ala-substitutions (i.e., T512A, Y513A, K514A), signifying an impact of a negative charge at this position on Cq -mALP1264-binding; this perception seems to be consistent with our previous findings for an negatively charged impact ofCry4Ba-Asp 454, which can be superimposed with Cry4Aa-Lys 514 , seeing that a large increase in the Cry4Batoxicity against Culex larvae was achieved toward an opposite-charge conversion of Asp 454 to Lys or Arg [ 25 ]. In this context, when the Cry4Ba-D454K modeled structure was docked with Cq -mALP1264, the mutant toxin appeared to change its binding orientation, being able to utilize DII instead of DIII particularly through the mutated residue—Lys 454 along with other residues in β2-β3 (Tyr 332 and Asp 334 ) and β10-β11 (Tyr 455 ) loops (see Supplementary Figure S2 ).…”

Section: Discussionsupporting

confidence: 88%

“…For a functional role in receptor recognition of DII, although most other studies are restricted to only three β-hairpin loops, i.e.,β2-β3, β6-β7 and β10-β11loops (originally assigned as loops 1, 2 and 3, respectively [ 9 ]), we have demonstrated that two other Cry4Ba-loops, i.e., β4-β5 and β8-β9loops, also play a critical role in receptor binding, and hence larval toxicity [ 23 , 24 ]. Moreover, we have shown that charge-reversal mutations at Asp 454 located within the β10-β11loop ofCry4Ba-DII noticeably enhance the toxin activity against less susceptible Culex larvae, suggesting a role of the charged side-chain in determining target specificity [ 25 ]. We have also disclosed that the structural stability of two receptor-binding hairpins ( i.e .…”

Section: Introductionmentioning

confidence: 99%

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Cry4Aa and Cry4Ba Mosquito-Active Toxins Utilize Different Domains in Binding to a Particular Culex ALP Isoform: A Functional Toxin Receptor Implicating Differential Actions on Target Larvae

Dechkla

Charoenjotivadhanakul

Imtong³

et al. 2022

Toxins

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The three-domain Cry4Aa toxin produced from Bacillus thuringiensis subsp. israelensis was previously shown to be much more toxic to Culexmosquito larvae than its closely related toxin—Cry4Ba. The interaction of these two individual toxins with target receptors on susceptible larval midgut cells is likely to be the critical determinant in their differential toxicity. Here, two full-length membrane-bound alkaline phosphatase (mALP) isoforms from Culex quinquefasciatus larvae, Cq-mALP1263and Cq-mALP1264, predicted to be GPI-linked was cloned and functionally expressed inSpodoptera frugiperda(Sf9) cells as 57- and 61-kDa membrane-bound proteins, respectively. Bioinformatics analysis disclosed that bothCq-mALP isoforms share significant sequence similarity to Aedes aegypti-mALP—a Cry4Ba toxin receptor.In cytotoxicity assays, Sf9 cells expressing Cq-mALP1264, but not Cq-mALP1263, showed remarkably greater susceptibility to Cry4Aa than Cry4Ba, while immunolocalization studies revealed that both toxins were capable of binding to each Cq-mALPexpressed on the cell membrane surface. Molecular docking of the Cq-mALP1264-modeled structure with individual Cry4 toxins revealed that Cry4Aa could bind to Cq-mALP1264 primarily through particular residues on three surface-exposed loops in the receptor-binding domain—DII, including Thr512, Tyr513 and Lys514 in the b10-b11loop. Dissimilarly, Cry4Ba appeared to utilize only certain residues in its C-terminal domain—DIII to interact with such a Culex counterpart receptor. Ala-substitutions of selected b10-b11loop residues (T512A, Y513A and K514A) revealed that only the K514A mutant displayed a drastic decrease in biotoxicity against C. quinquefasciatuslarvae. Further substitution of Lys514 with Asp (K514D) revealed a further decrease in larval toxicity. Furthermore, in silico calculation of the binding affinity change (ΔΔGbind) in Cry4Aa-Cq-mALP1264 interactions upon these single-substitutions revealed that the K514D mutation displayed the largest ΔΔGbind value as compared to three other mutations, signifying an adverse impact of a negative charge at this critical receptor-binding position.Altogether, our present study has disclosed that these two related-Cry4 mosquito-active toxins conceivably exploited different domains in functional binding to the same Culex membrane-bound ALP isoform—Cq-mALP1264 for mediating differential toxicity against Culex target larvae.

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Section: Discussionsupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Cry4Aa and Cry4Ba Mosquito-Active Toxins Utilize Different Domains in Binding to a Particular Culex ALP Isoform: A Functional Toxin Receptor Implicating Differential Actions on Target Larvae

Dechkla

Charoenjotivadhanakul

Imtong³

et al. 2022

Toxins

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“…Further studies, to better understand more critical insights into Cry4Ba toxin-receptor interactions, charged reversed mutagenesis on these putative interaction sites would be of great interest. Likewise, point mutations to positively charged sidechains on receptor-binding loop regions of the Cry4Ba toxin itself might perhaps improve the toxin activity and specificity against Aedes larvae, consistent with previous findings that Cry4Bacharged-reversal mutants (D454R and D454K) exhibit increased binding affinity and larval toxicity to Culex larvae [6].…”

Section: Common Structural Features Of Aaeapn Isoforms and Implicatiosupporting

confidence: 89%

“…DI and DII have been revealed as membrane-pore formation and receptor recognition, respectively [5]. Recently, we have demonstrated that charge-reversal mutations at Asp 454 (D454R and D454K) located within a receptor-binding loop of Cry4Ba-DII markedly increases the toxin activity against lesssusceptible Culex larvae, suggesting a role of the charged sidechain in determining target specificity [6].…”

Section: Introductionmentioning

confidence: 99%

Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity

Aroonkesorn

Pootanakit

Katzenmeier

et al. 2015

Biochemical and Biophysical Research Communications

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The interaction between Bacillus thuringiensis Cry toxins and their receptors on midgut cells of susceptible insect larvae is the critical determinant in toxin specificity. Besides GPI-linked alkaline phosphatase in Aedes aegypti mosquito-larval midguts, membrane-bound aminopeptidase N (AaeAPN) is widely thought to serve as a Cry4Ba receptor. Here, two full-length AaeAPN isoforms, AaeAPN2778 and AaeAPN2783, predicted to be GPI-linked were cloned and successfully expressed in Spodoptera frugiperda (Sf9) cells as 112- and 107-kDa membrane-bound proteins, respectively. In the cytotoxicity assay, Sf9 cells expressing each of the two AaeAPN isoforms showed increased sensitivity to the Cry4Ba mosquito-active toxin. Double immunolocalization revealed specific binding of Cry4Ba to each individual AaeAPN expressed on the cell membrane surface. Sequence analysis and homology-based modeling placed these two AaeAPNs to the M1 aminopeptidase family as they showed similar four-domain structures, with the most conserved domain II being the catalytic component. Additionally, the most variable domain IV containing negatively charged surface patches observed only in dipteran APNs could be involved in insect specificity. Overall results demonstrated that these two membrane-bound APN isoforms were responsible for mediating Cry4Ba toxicity against AaeAPN-expressed Sf9 cells, suggesting their important role as functional receptors for the toxin counterpart in A. aegypti mosquito larvae.

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“…Previous substitution of amino acid Asp 454 on β10- β11 loops of Cry4Ba domain II with Lys or Arg (D454K and D454R) was found to increase Cry4Ba toxicity toward Cx. quinquefasciatus larvae [ 45 ]. In this study, treatment of Cx.…”

Section: Discussionmentioning

confidence: 99%

Enhancement of insect susceptibility and larvicidal efficacy of Cry4Ba toxin by calcofluor

et al. 2018

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BackgroundMosquitoes transmit many vector-borne infectious diseases including malaria, dengue, chikungunya, yellow fever, filariasis, and Japanese encephalitis. The insecticidal δ-endotoxins Cry4, Cry11, and Cyt produced from Bacillus thuringiensis have been used for bio-control of mosquito larvae. Cry δ-endotoxins are synthesised as inactive protoxins in the form of crystalline inclusions in which they are processed to active toxins in larval midgut lumen. Previously, we demonstrated that the activated Cry4Ba toxin has to alter the permeability of the peritrophic membrane (PM), allowing toxin passage across PM to reach specific receptors on microvilli of larval midgut epithelial cells, where the toxin undergoes conformational changes, followed by membrane insertion and pore formation, resulting in larval death. A peritrophic membrane (PM)-binding calcofluor has been proposed to inhibit chitin formation and enhance baculovirus infection of lepidopteran Trichoplusia ni.MethodsIn this study, Aedes aegypti larvae were fed with the calcofluor and Cry4Ba toxin to investigate the effect of this agent on the toxicity of the Cry4Ba toxin.ResultsCalcofluor displayed an enhancing effect when co-fed with the Cry4Ba wild-type toxin. The agent could restore the killing activity of the partially active Cry4Ba mutant E417A/Y455A toward Ae. aegypti larvae. PM destruction was observed after larval challenge with calcofluor together with the toxin. Interestingly, calcofluor increased Cry4Ba toxin susceptibility toward semi-susceptible Culex quinquefasciatus larvae. However, calcofluor alone or in combination with the toxin showed no mortality effect on non-susceptible fresh-water fleas, Moina macrocopa.ConclusionsOur results suggest that PM may contribute to the resistance of the mosquito larvae to Cry4Ba toxin. The PM-permeability alternating calcofluor might be a promising candidate for enhancing insect susceptibility, which will consequently improve Cry4Ba efficacy in field settings in the future.

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Single-reversal charge in the β10-β11 receptor-binding loop of Bacillus thuringiensis Cry4Aa and Cry4Ba toxins reflects their different toxicity against Culex spp. larvae

Cited by 4 publications

References 30 publications

Cry4Aa and Cry4Ba Mosquito-Active Toxins Utilize Different Domains in Binding to a Particular Culex ALP Isoform: A Functional Toxin Receptor Implicating Differential Actions on Target Larvae

Cry4Aa and Cry4Ba Mosquito-Active Toxins Utilize Different Domains in Binding to a Particular Culex ALP Isoform: A Functional Toxin Receptor Implicating Differential Actions on Target Larvae

Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity

Enhancement of insect susceptibility and larvicidal efficacy of Cry4Ba toxin by calcofluor

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