Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Aedes aegypti Galectin Competes with Cry11Aa for Binding to ALP1 To Modulate Cry Toxicity Ling-Ling Zhang,†,‡ Xiao-Hua Hu,† Song-Qing Wu,† Khadija Batool,† Munmun Chowdhury,‡ Yi Lin,∥ Jie Zhang,‡ Sarjeet S. Gill,⊥ Xiong Guan,*,† and Xiao-Qiang Yu*,‡,§
J. Agric. Food Chem. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 12/18/18. For personal use only.
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State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops and School of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China ‡ Division of Cell Biology and Biophysics, University of Missouri − Kansas City, Kansas City, Missouri 64110, United States § Guangzhou Key Laboratory of Insect Development Regulation and Application Research, Institute of Insect Science and Technology, and School of Life Sciences, South China Normal University, Guangzhou 510631, China ∥ Department of Bioengineering & Biotechnology, College of Chemical Engineering, Huaqiao University, Xiamen 361021, China ⊥ Department of Molecular, Cell and Systems Biology, University of California, Riverside, California 92521, United States ABSTRACT: The key step for the toxicity of Bacillus thuringiensis subsp. israelensis (Bti) is the interaction between toxins and putative receptors; thus, many studies focus on identification of new toxin receptors and engineering of toxins with higher affinity/specificity for receptors. In the larvae of Aedes aegypti, galectin-14 was one of the genes upregulated by Bti treatment. RNAi knockdown expression of galectin-14 and feeding recombinant galectin-14-thioredoxin fusion protein significantly affected survival of Ae. aegypti larvae treated with Bti toxins. Recombinant galectin-14 protein bound to brush border membrane vesicles (BBMVs) of Ae. aegypti larvae, ALP1 and APN2, and galectin-14 and Cry11Aa bound to BBMVs with a similarly high affinity. Competitive binding results showed that galectin-14 competed with Cry11Aa for binding to BBMVs and ALP1 to prevent effective binding of toxin to receptors. These novel findings demonstrated that midgut proteins other than receptors play an important role in modulating the toxicity of Cry toxins. KEYWORDS: Alkaline phosphatase, Bacillus thuringiensis, Cry11A, Galectin, Toxicity
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INTRODUCTION
cadherin, alkaline phosphatase (ALP), aminopeptidase-N (APN), and ATP-binding cassette (ABC) transporters.14−17 Although the interaction of Cry toxins with putative receptors is relatively well understood, less is known about other midgut proteins that may interact with Cry toxins or toxin receptors, and thus interfere with the interaction between Cry toxins and receptors, resulting in alteration of the toxicity. Since toxin binding is, in part, dependent on receptor glycosylation,18 proteins that contain carbohydrate-recognition domains (CRDs) may affect toxicity. Galectins are a large family of β-galactoside-binding lectins with diverse biological functions.19−24 Intracellular mammalian galectins participate in signaling pathways and alter biological responses, including apoptosis, cell differentiation, and cell motility,23,25 whereas extracellular galectins can exhibit bivalent or multivalent interaction with cell-surface glycans to exert different effects, such as production of cytokines, cell adhesion, apoptosis, and chemoattraction.25 In contrast, insect galectins have not been well studied, and they may play a role in immunity and development.26 A midgut sand fly galectin with two CRDs is used by Leishmania major to increase parasite survival.27
Aedes mosquitoes transmit several deadly diseases in humans and are the primary vectors of yellow fever, dengue, and Chikungunya viruses, as well as the recent outbreak of Zika virus.1,2 Since vaccines and antiviral drugs are not available for arboviruses, vector control is still one of the major strategies for controlling mosquito-borne diseases.1,3 Among these strategies is the use of biological agents, such as Bacillus thuringiensis (Bt), which has been widely used in pest management in agriculture, forestry, and public health.4−7 Among Bt strains, subsp. israelensis (Bti) is highly toxic to mosquitoes and has been used for mosquito control.8−11 In Bt Cry toxins with a three-domain architecture, domain II is the most variable of the domains and is important for toxin specificity and binding to putative receptors. Domain II is a βprism structure that is structurally similar to the plant lectin jacalin and Maclura pomifera agglutinin.12 Domain III adopts a β-sandwich fold and may be involved in determining insect specificity.13 The mode of action of Cry toxins includes solubilization of protoxins in the midgut, proteolytic activation of protoxins by midgut proteases, interaction of the active toxins with putative receptors, and toxin oligomerization followed by insertion of these oligomers into the midgut epithelial cells, finally leading to formation of pores in the midgut cell plasma membrane and resulting in cell death.6,14 The key step in killing of insects by Cry toxins is the interaction between Cry toxins and putative receptors, such as © XXXX American Chemical Society
Received: August 30, 2018 Revised: December 3, 2018 Accepted: December 7, 2018
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DOI: 10.1021/acs.jafc.8b04665 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry Interestingly, the CRD of mammalian galectins adopts a βsandwich fold composed of 5-stranded (F1−F5) and 6stranded (S1−S6) β-sheets,28,29 structurally similar to the domain III of Cry toxins. Thus, galectin CRDs may compete with domain III of Cry toxins for binding to toxin receptors or directly bind to glycans on the receptors to modulate the toxicity of Cry toxins. To test our hypothesis, we selected galectin-14 (Genbank accession number: AAEL009850) in this study since it was one of the upregulated genes in the transcriptome analysis of Ae. aegypti larvae treated with Bti LLP29 total toxins (unpublished data). Indeed, we showed that Ae. aegypti galectin-14 bound to larval brush border membrane vesicles (BBMVs), ALP1 and APN2, but not to cadherin. Furthermore, RNAi knockdown of galectin-14 significantly decreased larvae survival after exposure to Bti total toxins, whereas feeding recombinant galectin-14-thioredoxin fusion protein to Ae. aegypti larvae significantly increased survival of larvae treated with Bti toxins. Competitive binding results showed that galectin-14 competed with Cry11Aa for binding to ALP1 to prevent effective binding of toxin to ALP1, thus altering the toxicity of Cry11Aa.
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the gel slice containing recombinant galectin-14-Trx was used as an antigen to produce rabbit polyclonal antiserum at Cocalico Biologicals, Inc. (Reamstown, PA, USA). Recombinant thioredoxin (Trx) was also purified by Ni-NTA resins as a control protein. Expression vectors for recombinant Ae. aegypti ALP1, APN2, and cadherin as well as rabbit polyclonal antibodies to these putative receptors were made available by the Gill Laboratory at the University of California, Riverside, and recombinant ALP1, APN2, cadherin, Cry11Aa, and LLP29 toxins were all purified as described previously.32−37 Bioassays. Newly hatched Ae. aegypti larvae (25 per group) were transferred into a 100-mL glass beaker containing 50 mL of deionized water. Gel slices containing 16 μg of dsRNA in nanoparticles were added into each beaker once a day to feed mosquito larvae for 4 consecutive days. Transcription and protein levels of galectin-14 were determined by RT-PCR (and/or real-time PCR) and Western blot analysis, respectively, and the susceptibility of these mosquito larvae to LLP29 toxins (bioassays) were assessed after feeding total toxins. For bioassays with recombinant galectin-14-Trx fusion protein, 25 of the fourth instar Ae. aegypti larvae were fed LLP29 toxins (7.65 μg) or LLP29 toxins (7.65 μg) mixed with recombinant galectin-14-Trx or thioredoxin (control) protein (0, 6.12, 24.48, and 36.72 μg) in 30 mL of dechlorinated water, and survival of mosquito larvae was recorded from 12 to 48 h after protein feeding. Bioassay experiments were repeated at least three times with three replicates for each treatment. Preparation of Ae. aegypti BBMVs. Over 1000 of the fourth instar Ae. aegypti larvae were dissected and midguts were collected. BBMVs were prepared from these midguts as described previously.32,33 Protein concentration of BBMVs was determined by the Bradford method.38 Western Blot and Far-Western Blot Analyses. For Western blot analysis, purified recombinant Ae. aegypti galectin-14-Trx, ALP1, APN2, and cadherin were separated on 10% or 12% SDS-PAGE and transferred to nitrocellulose membranes. The membrane was blocked with 5% dry skim milk in Tris-buffer saline (TBS) containing 0.05% Tween-20 (TBS-T) and incubated with primary rabbit polyclonal antibody (1:2000) to each protein. Antibody binding was visualized by a color reaction catalyzed by alkaline phosphatase conjugated to goat antirabbit antibody (1:3000) (Bio-Rad) as described previously.34,39 Far-Western blot analysis was performed as described previously.39 Target proteins, including purified recombinant Ae. aegypti ALP1, APN2, cadherin, and galectin-14-Trx, were separated on 10% SDSPAGE and transferred to nitrocellulose membrane. The membrane was washed with TBS-T several times, blocked with 5% dry skim milk in TBS-T, and then probed with purified recombinant galectin-14-Trx, ALP1, APN2, or cadherin in TBS-T (pH 9) containing 0.1% bovine serum albumin overnight at 4 °C with gentle rocking. After washing, the membrane was then incubated with the primary rabbit polyclonal antibody (1:2000) to each probe protein, and antibody binding was visualized by a color reaction catalyzed by alkaline phosphatase conjugated to goat antirabbit antibody (1:3000) (Bio-Rad). Plate ELISA Assays. For plate enzyme-linked immunosorbent assay (ELISA) and Forte-Bio ORS experiment (see below), the purified recombinant galectin-14-Trx, Cry11Aa, and thioredoxin were biotinylated with EZ-Link-NHS-Biotin following the manufacturer’s instructions (ThermoFisher Scientific, Waltham, MA, USA). Binding of galectin-14-Trx fusion protein to BBMVs and ALP1, as well as Cry11Aa to ALP1, was performed by plate ELISA assays as described previously.40 Briefly, 96-well microtiter plates were coated with BBMVs or recombinant ALP1 (4 μg/well) overnight at 4 °C and the plates were washed with TBS three times. Then increasing concentrations of biotinylated galectin-14-Trx, Cry11Aa, or thioredoxin (1, 2, 5, 10, 40, and 80 nM) in 100 μL of TBS-T (pH 7.4) were added to each well and incubated at room temperature for 2 h. The plates were washed with TBS-T three times, and the bound biotinylated protein was detected by incubating the plates with streptavidin horseradish peroxidase (HRP) conjugate antibody (1:3000) (Beyotime, Shanghai, China) according to the manufacturer’s instruction, at 450 nm on the Multiskan GO Microplate Spectrophotometer (Thermo 180 Scientific).
MATERIALS AND METHODS
Mosquitoes and Bacterial Strains. Ae. aegypti were reared in an environmentally controlled room at 28 °C and 85% relative humidity with a photoperiod of 14 h of light and 10 h of dark. Bti strain LLP29 was isolated and stored in our laboratory, and it is highly toxic to mosquitoes.30,31 Escherichia coli strain JM109 was used for recombinant DNA cloning and BL21 for Cry toxin expression. Plasmid pMD18-T (TaKaRa, Dalian, China) was used for DNA cloning and sequencing, and pGEX-4T-1 (Amersham Pharmacia Biotech, Japan) was used for expression of Cry11Aa protein. Bacteria were cultured in Luria−Bertani (LB) medium at 30 °C (37 °C for E. coli) with constant rotary shaking at 230 rpm. RNA Interference (RNAi) Knockdown of galectin-14 in Ae. aegypti Larvae. Nanoparticle-mediated RNAi was performed to knock down expression of galectin-14.11 Double-stranded RNA (dsRNA) corresponding to a fragment of galectin-14 (372 bp, nucleotides 24−395) or green fluorescent protein (GFP) (500 bp, nucleotides 151−650) was synthesized with a MEGAscript RNAi Kit (Ambion, Austin, TX, USA), purified, and mixed with chitosan to form nanoparticles. Then nanoparticles with entrapped dsRNA (32 μg) were mixed with 6 mg of ground mosquito larval food (ground brewer’s yeast, lactalbumin, and rat food in a 1:1:1 ratio). The mixture of food and nanoparticles was then coated by thoroughly mixing it with 40 μL of 2% premelted low-melting agarose gel solution at 55 °C. After solidification, the gel containing both the food and nanoparticles was cut into pieces to feed newly hatched Ae. aegypti larvae for 4 consecutive days before feeding LLP29 toxins. The survival of mosquito larvae was recorded at 24 h after feeding LLP29 toxins. Expression and Purification of Recombinant galectin-14 and Production of Antibody. The cDNA of Ae. aegypti galectin-14 (Genbank accession number: AAEL009850) was amplified by polymerase chain reaction (PCR) with gene-specific forward (5′CAT GCC ATG GAT GAA CGA CCG TTT CAA-3′) and reverse (5′-CCC AAG CTT GAT TTA GGT AAA ATC ACC GC-3′) primers, and the DNA fragment was cloned into pMD-18T vector (TaKaRa, Dalian, China) for selection of positive clones and sequencing. Then the DNA fragment was cloned into pET-32a expression vector (YouBia, China). The recombinant galectin-14thioredoxin (Trx) fusion protein was expressed in E. coli BL21 in LB medium after induction with 0.3 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 12 h at 16 °C, and purified by Ni-NTA resins (TransGen Biotech, China) following the manufacturer’s instructions. The purified galectin-14-Trx was further separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and B
DOI: 10.1021/acs.jafc.8b04665 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 1. Ae. aegypti galectin-14 alters the toxicity of Bti toxins to mosquito larvae. (A) RNAi knockdown expression of galectin-14 in Ae. aegypti larvae detected by real-time PCR. (B) Knockdown of galectin-14 expression in mosquito larvae significantly decreased the survival of larvae treated with Bti LLP29 total toxins. (C and D) Feeding recombinant galectin-14-Trx fusion protein significantly increased the survival of Ae. aegypti larvae treated with LLP29 total toxins at 12 h (C) and 24 h (D) after feeding. The bars (A and B) and points (C and D) represent the mean ± SEM (n ≥ 3). Significance of difference was calculated by one way ANOVA followed by Tukey’s multiple comparison tests using GraphPad Prism (A and B), and identical letters are not significantly different (p > 0.05) while different letters indicate significant difference (p < 0.05). Significance of difference was also determined by an unpaired t test with the GraphPad InStat software (C and D): *p < 0.05, **p < 0.01, and ***p < 0.001.
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For competitive binding assays, a constant concentration (10 nM) of recombinant biotin-labeled galectin-14-Trx with excess amounts of unlabeled Cry11Aa or thioredoxin (0−500 nM), or a constant concentration (10 nM) of biotin-labeled Cry11Aa with excess amounts of unlabeled galectin-14-Trx or thioredoxin (0−500 nM), were added to BBMVs-coated or ALP1-coated plates, and the binding assays were performed in the same manner as described above. Forte-Bio ORS Biolayer Interferometry. To determine the binding affinity and kinetics of galectin-14-Trx and Cry11Aa to BBMVs, binding of galectin-14-Trx or Cry11Aa to BBMVs was also performed by Forte-Bio ORS on the Octet Systems (Pall ForteBio, New York). Biotinylated Cry11Aa or galectin-14-Trx was immobilized to Streptavidin biosensors, and increasing concentrations (62.5, 125, 250, 500, and 1000 nM) of BBMVs were run through the proteincoated biosensors. The dissociation constant (Kd) for the binding of Cry11Aa or galectin-14-Trx to BBMVs was analyzed by the Octet System Data Analysis software. Protein Modeling and Molecular Docking. Three-dimensional structures of Cry11Aa, ALP1, and galectin were predicted using Bt δendotoxin (PDB: 1DLC), shrimp alkaline phosphatase (PDB: 1K7H), and human galectin-8 (PDB: 4FQZ) as templates, respectively, by PHYRE2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page). Protein− protein docking was predicted using online analysis software GRAMM-X Protein−Protein Docking Web Server v.1.2.0 (http:// vakser.compbio.ku.edu/resources/gramm/grammx). These results were analyzed and displayed by the Discovery Studio2.5 by adjusting the atom, form of filling, color, and lighting. Amino acids involved in binding were separately analyzed by the Discovery Studio2.5.41
RESULTS AND DISCUSSION galectin-14 Inhibits Larvicidal Activity of Cry Toxins. To investigate whether galectin-14 plays a role in the toxicity of Bti toxins, RNAi knockdown was performed. Nanoparticlemediated RNAi experiment showed that the expression of galectin-14 transcript in Ae. aegypti larvae was significantly reduced compared to the controls (Figure 1A), indicating that RNAi was effective in mosquito larvae. When these mosquito larvae were fed LLP29 total toxins, survival of larvae treated with dsRNA to galectin-14 was significantly lower than that of the controls (dsRNA to GFP and no dsRNA treatment) (Figure 1B), suggesting that galectin-14 may affect the activity of Cry toxins. To further confirm that galectin-14 indeed can alter the activity of Cry toxins, protein feeding experiments were performed. Purified recombinant galectin-14-Trx fusion protein and thioredoxin control protein were used for feeding mosquito larvae. Bioassay results showed that survival of mosquito larvae fed on LLP29 total toxins mixed with galectin14-Trx fusion protein was significantly increased compared to larvae fed on LLP29 total toxins mixed with the thioredoxin control protein at 12 h (Figure 1C) and 24 h (Figure 1D) postfeeding. Together, these results suggest that galectin-14 can inhibit larvicidal activity of Cry toxins. galectin-14 Binds to BBMVs, ALP1, and APN2. The key step in toxicity of Cry toxins is the interaction between the toxins and putative receptors. Thus, inhibition of larvicidal activity of Cry toxins by galectin-14 may be due to interaction
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DOI: 10.1021/acs.jafc.8b04665 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 2. galectin-14 interacts with Ae. aegypti ALP1 and APN2. (A) Western blot analysis of recombinant galectin-14-Trx fusion protein, ALP1, APN2, and cadherin. Purified recombinant galectin-14-Trx, ALP1, APN2, and cadherin were detected by rabbit polyclonal antibodies specific to galectin-14 (a), ALP1 (b), APN2 (c), and cadherin (d), respectively. (B) galectin-14 interacts with ALP1 and APN2, but not with cadherin. galectin14-Trx band was detected by antibody specific to ALP1 and APN2 after the membrane was probed with ALP1 (e) and APN2 (f); ALP1 and APN2 bands were detected by antibody specific to galectin-14 after the membrane was probed with galectin-14-Trx (g and h). galectin-14-Trx band was not detected by antibody specific to cadherin after the membrane was probed with cadherin (i).
Figure 3. galectin-14 and Cry11Aa compete with each other for binding to Ae. aegypti BBMVs and ALP1. Binding of galectin-14-Trx to immobilized BBMVs (A) and ALP1 (B), Cry11Aa to immobilized ALP1 (C), biotinylated galectin-14-Trx to immobilized BBMVs (D) and ALP1 (E) in the presence of excess Cry11Aa or thioredoxin, and biotinylated Cry11Aa to immobilized BBMVs in the presence of excess galectin-14-Trx (F). All the plate ELISA binding assays were performed as described in Materials and Methods. The data showed total binding.
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DOI: 10.1021/acs.jafc.8b04665 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 4. Cry11Aa and galectin-14 bind to BBMVs with a similar high affinity. Biotinylated Cry11Aa or galectin-14-Trx was immobilized to a Streptavidin biosensor, and increasing concentrations of BBMVs from Ae. aegypti larvae were run through the coated biosensor. Dissociation constant (Kd) of Cry11Aa or galectin-14-Trx for BBMVs was calculated according to the values of kon and koff using the Octet System Data Analysis software. (A) Cry11Aa binding to BBMVs; (B) galectin-14-Trx binding to BBMVs.
of galectin-14 with Cry toxins or with putative receptors such as cadherin, ALP, APN, and/or ABC transporter.15,31−34 To determine binding of galectin-14-Trx to individual toxin receptors, Far-Western blot analysis was performed with purified recombinant proteins. Western blot analysis with purified recombinant Ae. aegypti galectin-14-Trx, ALP1, APN2, and cadherin showed that protein bands corresponding to galectin-14-Trx fusion protein (∼38 kDa) (Figure 2A-a), ALP1 (∼66 kDa) (Figure 2A-b), APN2 (∼73 kDa) (Figure 2A-c), and cadherin (∼88 kDa) (Figure 2A-d) were recognized by polyclonal antibodies specific to galectin-14, ALP1, APN2, and cadherin, respectively. When galectin-14-Trx in the membrane was probed with ALP1 or APN2 and detected with antibody specific to ALP1 or APN2, the 38 kDa galectin-14-Trx fusion protein was observed (Figure 2A-e,f). Similarly, the 66 kDa ALP1 and 73 kDa APN2 in the membrane was detected by antibody specific to galectin-14 after probing with galectin-14Trx (Figure 2A-g,h). However, when galectin-14-Trx in the membrane was probed with cadherin, the 38 kDa protein band was not observed after detection with polyclonal antibody specific to cadherin (Figure 2A-i). These results indicate that
galectin-14 can interact with ALP1 and APN2, but not with cadherin. We then tested the binding of galectin-14 to midgut BBMVs of Ae. aegypti larvae. Plate ELISA assay showed that increasing amounts of recombinant galectin-14-Trx bound to immobilized BBMVs when higher concentrations of the fusion protein were applied, and the binding was saturated at 10 nM galectin-14Trx, whereas negligible amounts of the control thioredoxin protein bound to BBMVs even at 40 nM thioredoxin (Figure 3A). To confirm binding of galectin-14 to ALP1, plate ELISA assay was performed. With increasing concentrations of galectin-14-Trx, more galectin-14-Trx bound to immobilized ALP1, and the binding was not saturated at 80 nM galectin-14Trx (Figure 3B). As a comparison, binding of Cry11Aa to ALP1 was also performed. More Cry11Aa bound to immobilized ALP1 with increasing concentrations of Cry11Aa and the binding was saturated at 40 nM Cry11Aa (Figure 3C). To test whether galectin-14 and Cry11Aa compete with each other for binding to BBMVs and ALP1, competitive plate ELISA assays were performed. The results showed that 25-fold E
DOI: 10.1021/acs.jafc.8b04665 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 5. Modeling structures of ALP1, Cry11Aa, galectin-14, and domains II and III of Cry11Aa. Three-dimensional structures of Ae. aegypti ALP1 (A), LLP29 Cry11Aa (B), Ae. aegypti galectin-14 (C), and domain II (D) and domain III (E) of Cry11Aa were modeled using PHYRE 2 engine. Residues in domains II and III of Cry11Aa (B) that are predicted to participate in interaction with ALP1 were colored in yellow, green, and red.
Figure 6. Molecular docking of ALP1 with Cry11Aa and galectin-14. Three-dimensional structures were modeled using PHYRE 2 engine and molecular docking was performed by the ZDock program of the Discovery Studio 2.5. Residues in yellow and green in Cry11Aa (A) and residues in yellow in galectin-14 (B) were predicted to participate in interaction with ALP1. (C) Residues in ALP1 that are involved in interaction with Cry11Aa (colored in green), with galectin-14 (colored in yellow), or with both Cry11Aa and galectin-14 (colored in red).
excess Cry11Aa completely blocked binding of galectin-14-Trx to immobilized BBMVs (Figure 3D) and ALP1 (Figure 3E); F
DOI: 10.1021/acs.jafc.8b04665 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 7. Expression of Ae. Aegypti galectin genes. Expression of Ae. aegypti galectin genes in L1-L3 larvae, L4 larvae, pupae, and a mixture of male and female adult mosquitoes (A) as well as in the midgut and carcass of L4 larvae (B) was detected by RT-PCR. ,: 100 bp DNA ladders; lanes 1−9: galectin-1, -2, -5, -6, -8, -11, -12, -13, and -14; lane 10, rsp40.
Figure 8. Residues in Cry11Aa and galectin-14 that are involved in binding to ALP1. Three-dimensional structures of LLP29 Cry11Aa (A) and Ae. aegypti galectin-14 (B) were modeled using PHYRE 2 engine. Residues in Cry11Aa (green, panel A) or in galectin-14 (yellow, panel B) were predicted to be involved in binding to Ae. aegypti ALP1.
involved in binding to both Cry11Aa and galectin-14 (Figure 6C, residues in red). These results together with the competitive binding results indicate that galectin-14 can compete with Cry11Aa for binding to ALP1, thus inhibiting the toxicity of Cry11Aa against mosquito larvae. Bti toxins have been used for mosquito control.10,29,30,42,43 However, primarily because of high costs, Bti has not been used as the main agent for control of larval mosquitoes in disease endemic countries. Furthermore, although there has been no evidence of resistance to Bti, resistance has been observed under laboratory conditions to individual Bti toxins.43 In this study, we showed that tolerance to individual Bti toxins in Ae. aegypti can be modulated by midgut proteins that may interfere with the interaction between Cry toxins and toxin receptors. One such protein is galectin-14, which was upregulated in larvae following LLP29 toxin treatment. Galectin CRDs adopt a β-sandwich fold that is similar to that of Cry toxin domain III. Our results demonstrated that change in galectin-14 mRNA level by RNAi or protein level by feeding recombinant galectin-14-Trx fusion protein indeed modulated the toxicity of Bti toxins against mosquito larvae, and galectin-14-Trx can bind to BBMVs, ALP1, and APN2.
similarly, 25-fold excess galectin-14-Trx also blocked binding of Cry11Aa to BBMVs (Figure 3F). These results suggest that galectin-14 can compete with Cry11Aa for binding to ALP1. galectin-14 and Cry11Aa Bind to BBMVs with a Similar Affinity. galectin-14-Trx and Cry11Aa can compete with each other for binding to BBMVs and ALP1 (Figure 3D− F). To determine the affinity of galectin-14-Trx and Cry11Aa for BBMVs, binding of BBMVs to immobilized galectin-14-Trx and Cry11Aa was performed by Forte-Bio ORS on the Octet System (Figure 4). galectin-14-Trx and Cry11Aa bound to Ae. Aegypti BBMVs with a similarly high affinity; both proteins had Kd = 1.7 × 10−7 M for BBMVs. Since carbohydrate-recognition domain (CRD) of galectins is structurally similar to domain III of Cry toxins with a βsandwich fold,28 it is possible that galectin-14 CRD competes with Cry11Aa for binding to ALP1 and BBMVs. We modeled the three-dimensional structures of ALP1, Cry11Aa, galectin14, and domain II and domain III of Cry11Aa (Figure 5) and performed molecular docking. The results showed that ALP1 can interact with both Cry11Aa and galectin-14 at two different interfaces (Figure 6A,B), but the two interfaces overlap and several residues in the overlapped interfaces of ALP1 are G
DOI: 10.1021/acs.jafc.8b04665 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Notes
The Ae. aegypti genome encodes 9 galectin genes (galectin-1, -2, -5, -6, -8, -11, -12, -13, and -14), and we found that transcripts of galectin-6, -8, -12, -13, and -14 were expressed in larvae, pupae, and adults, as well as in the midgut and carcass of adult mosquitoes (Figure 7). It must be noticed that RNAi knockdown expression of galectin-14 in Ae. aegypti larvae was efficient, but RNAi of galectin-14 on the survival of mosquito larvae was less effective than feeding recombinant galectin-14 fusion protein on the mosquito survival (compare Figure 1B− D). This may be because other galectin genes in the midgut have functions similar to galectin-14. Indeed, galectin-6 and other galectins in Ae. aegypti can also modulate the toxicity of Bti toxins (data unpublished). galectin-14 inhibited Cry toxicity by apparently competing with Cry11Aa for binding to ALP1 receptor. Galectin CRD and Cry toxin domain III adopt similar β-sandwich fold, and the CRD structure of galectins also shares similarity to the βprism structure of Cry toxin domain II (Figure 5). Molecular docking results showed that residues from both domains II and III of Cry11Aa might be involved in binding to ALP1 (Figure 5B and Figure 6A), and the predicted α-8 helix of domain II has been shown to bind ALP1.15,35 It must be noticed that modeling data did not show binding of residues 561−570 in domain III of Cry11Aa15,35 to ALP1 (Figure 6A), but predicted additional residues in both domains II and III of Cry11Aa that might be involved in interaction with ALP1 (Figure 5B and Figure 6A). Therefore, midgut proteins such as galectin-14 may interfere with the binding of both domains II and III of Cry11Aa to ALP1 (Figure 8), resulting in overall less effective binding of Cry11Aa to ALP1 and decrease of Cry11Aa toxicity to mosquito larvae. Our results are novel and open up a new research direction to study interaction between Cry toxins and toxin receptors. Because there are several putative receptors for Cry toxins, there may be different midgut proteins that can bind to and shield different receptors from toxin binding to alter the toxicity of Cry toxins. Future research is needed to identify other midgut proteins that can interfere with interaction between Cry toxins and different receptors, and to identify polysaccharides or chemicals that can neutralize the effect of galectins in the midgut to increase the toxicity of Cry toxins for mosquito control.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors particularly want to thank Professor Michael R. Kanost at Kansas State University, Manhattan, Kansas, for his critical comments on the manuscript.
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ABBREVIATIONS USED ABC transporters,ATP-binding cassette transporters; ALP,alkaline phosphatase; APN,aminopeptidase-N; BBMVs,brush border membrane vesicles; Bt,Bacillus thuringiensis; Bti,Bacillus thuringiensis subsp. israelensis; CRD,carbohydrate-recognition domain; dsRNA,double-stranded RNA; ELISA,enzyme-linked immunosorbent assay; GFP,green fluorescent protein; HRP,horseradish peroxidase; IPTG,isopropyl-β-D-1-thiogalactopyranoside; LB,Luria−Bertani; RNAi,RNA interference; PCR,polymerase chain reaction; RT-PCR,reverse-transcription PCR; SDS-PAGE,sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBS,Tris-buffer saline; TBS-T,Tris-buffer saline containing Tween-20; Trx,thioredoxin.
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REFERENCES
(1) Alto, B. W.; Lord, C. C. Transstadial Effects of Bti on Traits of Aedes aegypti and Infection with Dengue Virus. PLoS Neglected Trop. Dis. 2016, 10, e0004370. (2) Caminade, C.; Turner, J.; Metelmann, S.; Hesson, J. C.; Blagrove, M. S. C.; Solomon, T.; Morse, A. P.; Baylis, M. Global risk model for vector-borne transmission of Zika virus reveals the role of El Niño 2015. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 119−124. (3) Canton, P. E.; Cancino-Rodezno, A.; Gill, S. S.; Soberón, M.; Bravo, A. Transcriptional cellular responses in midgut tissue of Aedes aegypti larvae following intoxication with Cry11Aa toxin from Bacillus thuringiensis. BMC Genomics 2015, 16, 1042. (4) Wang, P.; Zhang, C.; Guo, M.; Guo, S.; Zhu, Y.; Zheng, J.; Zhu, L.; Ruan, L.; Peng, D.; Sun, M. Complete genome sequence of Bacillus thuringiensis YBT-1518, a typical strain with high toxicity to nematodes. J. Biotechnol. 2014, 171, 1−2. (5) Wu, S.; Lan, Y.; Huang, D.; Peng, Y.; Huang, Z.; Xu, L.; Gelbic, I.; Carballar-Lejarazu, R.; Guan, X.; Zhang, L.; Zou, S. Use of spent mushroom substrate for production of Bacillus thuringiensis by solidstate fermentation. J. Econ. Entomol. 2014, 107, 137−143. (6) Bravo, A.; Likitvivatanavong, S.; Gill, S. S.; Soberon, M. Bacillus thuringiensis: A story of a successful bioinsecticide. Insect Biochem. Mol. Biol. 2011, 41, 423−431. (7) Palma, L.; Munoz, D.; Berry, C.; Murillo, J.; Caballero, P. Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins 2014, 6, 3296−3325. (8) Boyce, R.; Lenhart, A.; Kroeger, A.; Velayudhan, R.; Roberts, B.; Horstick, O. Bacillus thuringiensis israelensis (Bti) for the control of dengue vectors: systematic literature review. Trop. Med. Int. Health 2013, 18, 564−577. (9) Ben-Dov, E. Bacillus thuringiensis subsp. israelensis and its dipteran-specific toxins. Toxins 2014, 6, 1222−1243. (10) Pruszynski, C. A.; Hribar, L. J.; Mickle, R.; Leal, A. L. A large scale biorational approach using Bacillus thuringiensis israeliensis (Strain AM65−52) for managing Aedes aegypti populations to prevent dengue, chikungunya and zika transmission. PLoS One 2017, 12, e0170079. (11) Zhang, X.; Zhang, J.; Zhu, K. Y. Chitosan/double-stranded RNA nanoparticle-mediated RNA interference to silence chitin synthase genes through larval feeding in the African malaria mosquito (Anopheles gambiae). Insect Mol. Biol. 2010, 19, 683−693. (12) Boonserm, P.; Mo, M.; Angsuthanasombat, C.; Lescar, J. Structure of the functional form of the mosquito larvicidal Cry4Aa
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Corresponding Authors
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[email protected] (X.G.). *Tel.: 816-235-6379. E-mail:
[email protected] (X.-Q.Y.). ORCID
Xiong Guan: 0000-0003-3505-7464 Funding
This work was funded by the National Institute of Health (Grant No. AI117808), National Natural Science Foundation of China (NSFC Grant Nos. 31472049, 31301724, and 31772227), Science and Technology Project of Fuzhou (2018-G-70), Foreign Cooperative Project of Fujian Province (Grant No. 2018I0023), Open Project Funds of the State Key Laboratory of Pathogen and Biosecurity (SKLPBS1838), the Special Fund for Scientific and Technological Innovation of Fujian Agriculture and Forestry University (CXZX2017136, CXZX2017306, CXZX2017214, and KF2015064-65). H
DOI: 10.1021/acs.jafc.8b04665 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry toxin from Bacillus thuringiensis at a 2.8-angstrom resolution. J. Bacteriol. 2006, 188, 3391−3401. (13) Boonserm, P.; Davis, P.; Ellar, D. J.; Li, J. Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications. J. Mol. Biol. 2005, 348, 363−382. (14) Bravo, A.; Gill, S. S.; Soberon, M. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 2007, 49, 423−435. (15) Fernandez, L. E.; Aimanova, K. G.; Gill, S. S.; Bravo, A.; Soberon, M. A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae. Biochem. J. 2006, 394, 77−84. (16) Park, Y.; Gonzalez-Martinez, R. M.; Navarro-Cerrillo, G.; Chakroun, M.; Kim, Y.; Ziarsolo, P.; Blanca, J.; Canizares, J.; Ferre, J.; Herrero, S. ABCC transporters mediate insect resistance to multiple Bt toxins revealed by bulk segregant analysis. BMC Biol. 2014, 12, 46. (17) Guo, Z.; Kang, S.; Chen, D.; Wu, Q.; Wang, S.; Xie, W.; Zhu, X.; Baxter, S. W.; Zhou, X.; Jurat-Fuentes, J. L.; Zhang, Y. MAPK signaling pathway alters expression of midgut ALP and ABCC genes and causes resistance to Bacillus thuringiensis Cry1Ac toxin in diamondback moth. PLoS Genet. 2015, 11, e1005124. (18) Oltean, D. I.; Pullikuth, A. K.; Lee, H. K.; Gill, S. S. Partial purification and characterization of Bacillus thuringiensis Cry1A toxin receptor A from Heliothis virescens and cloning of the corresponding cDNA. Appl. Environ. Microbiol. 1999, 65, 4760−4766. (19) Hernandez, J. D.; Baum, L. G. Ah, sweet mystery of death! Galectins and control of cell fate. Glycobiology 2002, 12, 127R−136R. (20) Ideo, H.; Fukushima, K.; Gengyo-Ando, K.; Mitani, S.; Dejima, K.; Nomura, K.; Yamashita, K. A Caenorhabditis elegans glycolipidbinding galectin functions in host defense against bacterial infection. J. Biol. Chem. 2009, 284, 26493−26501. (21) Takeuchi, T.; Tamura, M.; Nishiyama, K.; Iwaki, J.; Hirabayashi, J.; Takahashi, H.; Natsugari, H.; Arata, Y.; Kasai, K. Mammalian galectins bind galactosebeta1−4fucose disaccharide, a unique structural component of protostomial N-type glycoproteins. Biochem. Biophys. Res. Commun. 2013, 436, 509−513. (22) Viguier, M.; Advedissian, T.; Delacour, D.; Poirier, F.; Deshayes, F. Galectins in epithelial functions. Tissue barriers 2014, 2, e29103. (23) Chen, W. S.; Cao, Z.; Sugaya, S.; Lopez, M. J.; Sendra, V. G.; Laver, N.; Leffler, H.; Nilsson, U. J.; Fu, J.; Song, J.; Xia, L.; Hamrah, P.; Panjwani, N. Erratum: Pathological lymphangiogenesis is modulated by galectin-8-dependent crosstalk between podoplanin and integrin-associated VEGFR-3. Nat. Commun. 2016, 7, 12063. (24) Rao, S. P.; Ge, X. N.; Sriramarao, P. Regulation of Eosinophil Recruitment and Activation by Galectins in Allergic Asthma. Front. Med. 2017, 4, 68. (25) Liu, F. T.; Yang, R. Y.; Hsu, D. K. Galectins in acute and chronic inflammation. Ann. N. Y. Acad. Sci. 2012, 1253, 80−91. (26) Pace, K. E.; Lebestky, T.; Hummel, T.; Arnoux, P.; Kwan, K.; Baum, L. G. Characterization of a novel Drosophila melanogaster galectin. Expression in developing immune, neural, and muscle tissues. J. Biol. Chem. 2002, 277, 13091−13098. (27) Kamhawi, S.; Ramalho-Ortigao, M.; Pham, V. M.; Kumar, S.; Lawyer, P. G.; Turco, S. J.; Barillas-Mury, C.; Sacks, D. L.; Valenzuela, J. G. A role for insect galectins in parasite survival. Cell 2004, 119, 329−341. (28) Seetharaman, J.; Kanigsberg, A.; Slaaby, R.; Leffler, H.; Barondes, S. H.; Rini, J. M. X-ray crystal structure of the human galectin-3 carbohydrate recognition domain at 2.1-A resolution. J. Biol. Chem. 1998, 273, 13047−13052. (29) Rapoport, E. M.; Kurmyshkina, O. V.; Bovin, N. V. Mammalian galectins: structure, carbohydrate specificity, and functions. Biochemistry 2008, 73, 393−405. (30) Zhang, L.; Huang, E.; Lin, J.; Gelbic, I.; Zhang, Q.; Guan, Y.; Huang, T.; Guan, X. A novel mosquitocidal Bacillus thuringiensis strain LLP29 isolated from the phylloplane of Magnolia denudata. Microbiol. Res. 2010, 165, 133−141.
(31) Zhang, L.; Wu, S.; Peng, Y.; Li, M.; Sun, L.; Huang, E.; Guan, X.; Gelbic, I. The potential of the novel mosquitocidal Bacillus thuringiensis strain LLP29 for use in practice. J. Vector Ecol.: J. Soc. Vector Ecol. 2011, 36, 458−460. (32) Chen, J.; Aimanova, K. G.; Fernandez, L. E.; Bravo, A.; Soberon, M.; Gill, S. S. Aedes aegypti cadherin serves as a putative receptor of the Cry11Aa toxin from Bacillus thuringiensis subsp. israelensis. Biochem. J. 2009, 424, 191−200. (33) Chen, J.; Aimanova, K. G.; Pan, S.; Gill, S. S. Identification and characterization of Aedes aegypti aminopeptidase N as a putative receptor of Bacillus thuringiensis Cry11A toxin. Insect Biochem. Mol. Biol. 2009, 39, 688−696. (34) Chen, J.; Likitvivatanavong, S.; Aimanova, K. G.; Gill, S. S. A 104 kDa Aedes aegypti aminopeptidase N is a putative receptor for the Cry11Aa toxin from Bacillus thuringiensis subsp. israelensis. Insect Biochem. Mol. Biol. 2013, 43, 1201−1208. (35) Fernandez, L. E.; Aimanova, K. G.; Gill, S. S.; Bravo, A.; Soberon, M. A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae. Biochem. J. 2006, 394, 77−84. (36) Fernandez, L. E.; Martinez-Anaya, C.; Lira, E.; Chen, J.; Evans, A.; Hernandez-Martinez, S.; Lanz-Mendoza, H.; Bravo, A.; Gill, S. S.; Soberon, M. Cloning and epitope mapping of Cry11Aa-binding sites in the Cry11Aa-receptor alkaline phosphatase from Aedes aegypti. Biochemistry 2009, 48, 8899−8907. (37) Chen, J.; Aimanova, K.; Gill, S. S. Functional characterization of Aedes aegypti Alkaline phosphatase Alp1 involved in the toxicity of cry toxins from Bacillus thuringiensis subsp. israelensis and jegathesan. Peptides 2017, 98, 78−85. (38) Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein− dye binding. Anal. Biochem. 1976, 72, 248−254. (39) Zhang, L. L.; Zhao, G. H.; Hu, X. H.; Liu, J. N.; Li, M. W.; Batool, K.; Chen, M. F.; Wang, J. X.; Xu, J.; Huang, T. P.; Pan, X. H.; Xu, L.; Yu, X. Q.; Guan, X. Cry11Aa interacts with the ATP-Binding Protein from Culex quinquefasciatus to improve the toxicity. J. Agric. Food Chem. 2017, 65, 10884−10890. (40) El-Kersh, T. A.; Ahmed, A. M.; Al-Sheikh, Y. A.; Tripet, F.; Ibrahim, M. S.; Metwalli, A. A. Isolation and characterization of native Bacillus thuringiensis strains from Saudi Arabia with enhanced larvicidal toxicity against the mosquito vector Anopheles gambiae (s.l.). Parasites Vectors 2016, 9, 647. (41) Li, Y.; Shi, X.; Zhang, Q.; Hu, J.; Chen, J.; Wang, W. Computational evidence for the detoxifying mechanism of epsilon class glutathione transferase toward the insecticide DDT. Environ. Sci. Technol. 2014, 48, 5008−5016. (42) Kang, S.; Odom, O. W.; Thangamani, S.; Herrin, D. L. Toward mosquito control with a green alga: Expression of Cry toxins of Bacillus thuringiensis subsp. israelensis (Bti) in the chloroplast of Chlamydomonas. J. Appl. Phycol. 2017, 29, 1377−1389. (43) Georghiou, G. P.; Wirth, M. C. Influence of exposure to single versus multiple toxins of Bacillus thuringiensis subsp. israelensis on development of resistance in the mosquito Culex quinquefasciatus (Diptera: Culicidae). Appl. Environ. Microbiol. 1997, 63, 1095−1101.
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DOI: 10.1021/acs.jafc.8b04665 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
