APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2011, p. 24–31 Vol. 77, No. 10099-2240/11/$12.00 doi:10.1128/AEM.01852-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Cadherin, Alkaline Phosphatase, and Aminopeptidase N as Receptors ofCry11Ba Toxin from Bacillus thuringiensis subsp.

jegathesan in Aedes aegypti�

Supaporn Likitvivatanavong,1 Jianwu Chen,1 Alejandra Bravo,2 Mario Soberon,2 and Sarjeet S. Gill1*Department of Cell Biology and Neuroscience, University of California, Riverside, California 92521,1 and Instituto de Biotecnología,

Universidad Nacional Autonoma de Mexico. Apdo. Postal 510-3, Cuernavaca 62250, Morelos, Mexico2

Received 3 August 2010/Accepted 22 October 2010

Cry11Ba is one of the most toxic proteins to mosquito larvae produced by Bacillus thuringiensis. It binds Aedesaegypti brush border membrane vesicles (BBMV) with high affinity, showing an apparent dissociation constant(Kd) of 8.2 nM. We previously reported that an anticadherin antibody competes with Cry11Ba binding toBBMV, suggesting a possible role of cadherin as a toxin receptor. Here we provide evidence of specific cadherinrepeat regions involved in this interaction. Using cadherin fragments as competitors, a C-terminal fragmentwhich contains cadherin repeat 7 (CR7) to CR11 competed with Cry11Ba binding to BBMV. This binding wasalso efficiently competed by the CR9, CR10, and CR11 peptide fragments. Moreover, we show CR11 to be animportant region of interaction with Cry11Ba toxin. An alkaline phosphatase (AaeALP1) and an aminopep-tidase-N (AaeAPN1) also competed with Cry11Ba binding to Ae. aegypti BBMV. Finally, we found that Cry11Baand Cry4Ba share binding sites. Synthetic peptides corresponding to loops �8, �2-�3 (loop 1), �8-�9, and�10-�11 (loop 3) of Cry4Ba compete with Cry11Ba binding to BBMV, suggesting Cry11Ba and Cry4Ba havecommon sites involved in binding Ae. aegypti BBMV. The data suggest that three different Ae. aegypti midgutproteins, i.e., cadherin, AaeALP1, and AaeAPN1, are involved in Cry11Ba binding to Ae. aegypti midgut brushborder membranes.

Microbiological control strategies involving Bacillus thurin-giensis subsp. israelensis or Bacillus sphaericus are increasinglyused worldwide for the control of insect vectors. B. thuringien-sis subsp. israelensis produces four major insecticidal Cry pro-teins (Cry4Aa, Cry4Ba, Cry10Aa, and Cry11Aa) and threecytolytic proteins (Cyt1Aa, Cyt2Ba, and Cyt1Ca) (6). Amongthem, Cry11Aa is the most active toxin against Aedes aegypti(13). However, B. thuringiensis strains producing other mos-quitocidal Cry toxins have been identified, including B. thurin-giensis subsp. jegathesan (27). Parasporal crystals of this speciescontain seven major proteins, one of which is a protein of 80kDa designated Cry11Ba. Cry11Ba exhibits 58% identity withCry11Aa at the amino acid level (15), and it is, to date, thetoxin with the highest activity against mosquitoes, having about6 to 40 times more activity (depending on the species of mos-quito tested) than Cry11Aa of B. thuringiensis subsp. israelensis(15). Consequently, the Cry11Ba toxin is an alternative tothose used in current control programs, since the use of thistoxin may directly address the risk of development of resis-tance to B. thuringiensis subsp. israelensis or B. sphaericus toxinsin mosquitoes.

The highly conserved structure of Cry toxins suggests thatthey may share a mode of action in which domains II and III,composed mainly of � sheets, are responsible for binding mem-brane receptors. A number of proteins have been identified asCry toxin receptors, such as cadherin, alkaline phosphatases

(ALPs), and aminopeptidase (APN), and these were first iden-tified in different lepidopteran insects as Cry1A toxin bindingproteins (4, 22, 23, 26, 29, 32, 36, 37, 42, 44). In mosquitolarvae, similar proteins have been described. Cadherin-likeproteins that bind Cry4Ba in Anopheles gambiae (24) andCry11Aa in Ae. aegypti (11) were described. Moreover, glyco-sylphosphatidylinositol (GPI)-anchored proteins, such asAPNs from Anopheles quadrimaculatus, An. gambiae, and Ae.aegypti (1, 12, 45, 46) and ALPs from Ae. aegypti and An.gambiae (18, 25), were also found to bind Cry11Aa andCry11Ba toxins and were proposed as potential toxin recep-tors. After binding receptors, Cry toxins are believed to createpores that are permeable to small ions and solutes by use ofmembrane-embedded oligomeric Cry structures, thereforecausing osmotic lysis of midgut cells in susceptible insects (16,39, 40). However, little is known about Ae. aegypti midgutproteins that bind the Cry11Ba toxin. Here we identify andcharacterize these proteins.

MATERIALS AND METHODS

Cry11Ba preparation and toxin biotinylation. A B. thuringiensis strain express-ing only the Cry11Ba (15) was grown in nutrient broth sporulation medium witherythromycin (25 �g/ml) at 30°C (30). Following autolysis, spores and inclusionswere harvested and washed three times with 1 M NaCl–10 mM EDTA, pH 8.0.The final pellet was resuspended in the same buffer (30 ml) and purified by useof NaBr gradients as previously described (10). Purified Cry11Ba inclusions weresolubilized in 50 mM Na2CO3 (pH 10.0) and activated with trypsin (1:20, wt/wt).Ion exchange chromatography (MonoQ fast protein liquid chromatography[FPLC]) (AKTA; Amersham Biosciences) was used to further purify the acti-vated-Cry11Ba toxin. The solubilized and activated Cry11Ba toxin was biotinyl-ated using a protein biotinylation module kit (40 �l of reagent with 1 mg toxin;Amersham Biosciences), and a Sephadex G25 column was used to removeuncoupled biotin.

* Corresponding author. Mailing address: Department of Cell Biol-ogy and Neuroscience, University of California, Riverside, CA 92521.Phone: (951) 827-4621/3547. Fax: (951) 827-3087. E-mail: sarjeet.gill@ucr.edu.

� Published ahead of print on 29 October 2010.

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Preparation of BBMV. Midguts were dissected from early fourth-instar Ae.aegypti larvae and kept at �80°C until use. Brush border membrane vesicles(BBMV) were prepared by the differential magnesium precipitation method(33). BBMV were resuspended in ice-cold buffer A (0.3 M mannitol, 0.5 MEGTA, 20 mM Tris-Cl, pH 8), and the concentration of total protein wasmeasured with a bicinchoninic acid (BCA) kit (Pierce). Alkaline phosphataseand leucine aminopeptidase activities were determined as previously described(33). Freshly prepared BBMV were kept on ice and used the same day.

Western blotting and toxin overlay assay. Purified cadherin repeats CR7 toCR11 (10 �g) were separated by 10% SDS-PAGE and electrotransferred, andthe membranes were incubated first in blocking buffer (phosphate-buffered sa-line [PBS] and 0.1% Tween 20 [PBST] with 5% skim milk) for 1 h and then with20 nM Cry11Ba toxins for 2 h. Unbound toxins were removed by washing themembrane four times with washing buffer (PBST) for 15 min each. Membraneswere then incubated with rabbit anti-Cry11Ba polyclonal antibody (1:1,500 dilu-tion) followed by a secondary goat anti-rabbit antibody conjugated with horse-radish peroxidase (HRP) (1:5,000 dilution), and bound toxin revealed usingluminol (ECL; Amersham Biosciences).

Toxin binding assays. The kinetics of Cry11Ba binding to BBMV were mea-sured using a modified microplate assay. Briefly, 96-well plates coated with 4 �gBBMV were incubated overnight at 4°C and then in PBST for 1 h at roomtemperature. Increasing concentrations of biotinylated Cry11Ba toxin (0.01 to200 nM) in 100 �l binding buffer (0.1% bovine serum albumin [BSA], 0.1%Tween 20, 1� PBS, pH 7.4) were then transferred to the BBMV-coated plates.Parallel plates were run under identical conditions except in the presence of 10�M unlabeled Cry11Ba. After 2 h, the plates were washed with 100 �l PBSTthree times. Bound biotinylated Cry11Ba protein was detected by incubationwith streptavidin-horseradish peroxidase (HRP) conjugate (1:1,500) for 1 h.After washing three times with PBST, HRP activity was revealed with a freshlyprepared luminol substrate (Supersignal enzyme-linked immunosorbent assay[ELISA] pico; Thermo Scientific). An X-ray film was place over the microplatein a darkroom for 1 to 5 min, and the data were quantified with NIH Image Jsoftware and analyzed using Origin (Origin Lab). Specific binding was calculatedfrom total minus nonspecific binding. The concentration corresponding to halfthe saturation response of specific binding was considered the dissociation con-stant (Kd) and was obtained from two independent experiments using differentBBMV preparations.

For competition assays, biotinylated Cry11Ba toxin (10 nM) was equilibratedwith increasing amounts of unlabeled Cry11Ba or Cry4Ba protein (0.01 to 1,500nM) in PBST (100 �l) for 1 h at room temperature. The mixtures were thentransferred to plates previously coated with BBMV for 2 h. The plates werewashed with PBST (100 �l). Bound biotinylated Cry11Ba protein was detected asdescribed above. The concentration corresponding to half the maximal responsewas considered the 50% inhibitory concentration (IC50) and was obtained fromtwo independent experiments using different BBMV preparations.

Competition binding assays with cadherin, ALP, and APN. Assays of toxinbinding to BBMV were done in binding buffer (100 �l, 0.1% BSA, 0.1% Tween20, 1� PBS, pH 7.4). BBMV (10 �g protein) were incubated with biotinylatedCry11Ba toxin (10 nM) in the presence or absence of unlabeled Cry11Ba toxin,cadherin fragments, cadherin repeats (CR), Ae. aegypti ALP1 (AaeALP1) to-3, AaeAPN1, and Cry4Ba loop peptides for 1 h at room temperature. The mixtureswere centrifuged at 10,000 � g for 10 min to remove unbound toxins, and thepellet then was washed three times with binding buffer. BBMV were resuspendedin 20 �l of PBS and 4 �l of 6� Laemmli sample loading buffer (60% glycerol, 300mM Tris-Cl [pH 6.8], 12 mM EDTA, 12% SDS, 864 mM 2-mercaptoethnol,0.05% bromophenol blue). The samples were then boiled for 5 min, separated bySDS-PAGE, and electrotransferred to polyvinylidene difluoride (PVDF) mem-branes (Immobilon; Amersham Biosciences). The membranes were incubatedwith streptavidin-peroxidase conjugate (1:1,500 dilution; Amersham Bio-sciences) for 1 h and then visualized using luminal (ECL; Amersham Bio-sciences).

Expression of different proteins in E. coli. Constructs in pQE30 encoding theCry11Ba wild-type and the mutant toxins (�8-V256A/G257A/E258A, L1-R303A/E304A/N305, L3-N454A/K455A/L456A, and L1-H307A) (28) as well as Esche-richia coli clones of cadherin fragments G10, G7, and C13 (11), AaeALP1 to -3(10), and AaeAPN1 (12) were transformed into E. coli M15 competent cells forprotein expression. Bacterial clones harboring the different plasmids were grownat 37°C in Luria-Bertani medium containing ampicillin (l00 �g/ml) and kanamy-cin (50 �g/ml) until the optical density at 600 nm (OD600) of the culture reached0.3 to 0.5. Protein expression was induced with isopropyl-�-D-thiogalactopyrano-side (IPTG) at a final concentration of 0.1 mM for 4 h. Cells containing cyto-plasmic inclusions were harvested by centrifugation and resuspended in B-PERsolution (Amersham). Cells were then disrupted by sonication for 10 min (Soni-

fier 450). After centrifugation at 10,000 � g and 4°C for 10 min, the pellets werewashed three times in 1% Triton X-l00 and suspended by sonication. Proteinconcentrations of the partially purified inclusions were determined by using aBCA kit (Pierce). Inclusions (1 to 2 mg/ml) were solubilized by incubation at37°C for 2 to 3 h in 50 mM NaOH and then dialyzed in 50 mM Na2CO3 (pH 10.0)at 4°C overnight. The quality of these proteins was analyzed by 10% SDS-PAGE.

Competitive enzyme-linked immunosorbent assay-based binding assays. Inbrief, 10 nM G10 was equilibrated with increasing concentrations (0.01 to 1,000nM) of mutants of Cry11Ba (�8-V256A/G257A/E258A, L1-R303A/E304A/N305A, and L3-N454A/K455A/L456A) in PBST (100 �l) for 1 h at room tem-perature. The mixtures were then transferred to ELISA plate wells previouslycoated with wild-type Cry11Ba toxin and treated with blocking buffer (PBS, 0.1%Tween 20, 0.5% gelatin). After washing, the bound G10 protein was detected bypolyclonal anticadherin antibody (1:2,500 dilution), followed by incubation witha goat anti-rabbit alkaline phosphatase (ALP) conjugate antibody (1:1,500 dilu-tion). The ALP enzymatic activity was revealed with a freshly prepared substrate(3 mM nitrophenyl phosphate), and the absorbance was read at 405 nm (Mo-lecular Devices, Sunnyvale, CA).

Mass spectrometry. Protein bands of activated Cry11Ba toxin were excisedfrom the SDS-polyacrylamide gel, digested with trypsin, and analyzed by nano-ultra-performance liquid chromatography/tandem mass spectrometry (nano-UPLC/MS/MS) for de novo sequencing of both peptides (Institute of IntegrativeGenome Biology [IIGB], University of California, Riverside). The sequenceswere compared to the full-length Cry11Ba sequence to identify putative cleavagesites.

Larval bioassays. Larvicidal assays were performed using fourth-instar larvae.The assays were done at room temperature in water (200 ml), each with 25larvae. The 50% lethal concentration (LC50) and the 95% fiducial limits wereobtained by using a probit analysis program, version 1.5 (U.S. EnvironmentalProtection Agency). Any overlap of the fiducial limits indicates a lack of statis-tical difference at the 95% level of confidence.

RESULTS

Dissociation constant and competitive assays of Cry11Batoxin binding to Ae. aegypti BBMV. A number of studiesshowed the interaction of Cry11Aa or Cry4Ba toxins with mos-quito BBMV (5, 17, 20). Toxin affinity to BBMV has beenmeasured with Cry11Aa, Cry4Aa and Cry4Ba, Cry1C, and thebinary toxin from B. sphaericus (2, 3, 14, 33, 34). Here wedetermined the binding affinity of Cry11Ba toxin to Ae. aegyptiBBMV using biotinylated Cry11Ba. We first demonstrated thatbiotin-labeled Cry11Ba remains toxic to mosquito larvae. Thecalculated 50% lethal concentration (LC50) of soluble Cry11Bawas 8,050 ng/ml, whereas the LC50 of biotin-labeled Cry11Bawas in the same range (10,000 ng/ml). The trypsin-activatedforms of Cry11Ba or biotinylated Cry11Ba showed lower tox-icity, with LC50s of 40,000 and 49,000 ng/ml, respectively. Over-all, no significant difference in toxicity was observed afterbiotinylation of the toxin (Table 1).

The affinity of Cry11Ba binding to BBMV was then deter-mined using increasing concentrations of biotinylatedCry11Ba. Binding of Cry11Ba toxin to proteins on BBMVisolated from Ae. aegypti was specific and dose dependentshowing a dissociation constant (Kd) of 8.2 nM (Fig. 1A). In analternative assay, binding of biotinylated Cry11Ba to BBMVwas measured in the presence of increasing concentrations ofunlabeled Cry11Ba toxin (0.001 to 1,000 nM). A half-maximalinhibitory concentration (IC50) of 3.6 nM was obtained (Fig.1B. Furthermore, it is important to note that freshly preparedBBMV were required, presumably due to rapid degradation ofBBMV proteins that bound Cry11Ba.

Cry11Ba inclusions were purified from a recombinant B.thuringiensis strain by use of density gradients. Upon activa-tion, the 80-kDa Cry11Ba protein generated 33- and 36-kDa

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fragments. Both fragments were analyzed using tandem massspectrometry, which showed that proteolytic cleavage occurs atthe N and C termini, resulting in 24Leu and 651Arg as thecorresponding termini. Intramolecular processing occurs be-tween 343Lys and 354Gly (Fig. 2), but these two fragments arekept together by salt bridges. The N-terminal 36-kDa fragmentconsists of all of domain I and loops �8 and 1 of domain II,while the 33-kDa fragment represents the C-terminal end con-taining the rest of domain II, including loops 2 and 3, and thecomplete domain III of Cry11Ba toxin (Fig. 2).

The activated 33- and 36-kDa toxin fragments were labeledwith biotin, and their binding to Ae. aegypti BBMV was ana-lyzed. Homologous competition assays show that at a molarratio of 1:1 of labeled to unlabeled Cry11Ba toxin, the unla-beled toxin competed with binding of the 36-kDa fragment,followed by competition with the 33-kDa fragment (Fig. 1C).

At ratios of 1:50 and higher, binding to both fragments of thebiotinylated Cry11Ba was nearly totally competed (Fig. 1C).

Aedes cadherin fragments compete with Cry11Ba binding toBBMV. The Ae. aegypti cadherin fragments G7 (includes cad-herin repeats 1 to 5), C13 (containing CR3 to -7), and G10(containing CR7 to -11) were examined for their ability tocompete Cry11Ba binding to Ae. aegypti BBMV. As shown inFig. 3 and Table 2, the G10 fragment (Fig. 3, lane 2) signifi-cantly competed with the binding of Cry11Ba to BBMV. TheG7 and C13 fragments showed lower competition, particularlyto the 33-kDa fragment. In contrast, the negative-control pep-tide NHE8, derived from Ae. aegypti NHE ion exchanger pro-tein, does not compete (Table 2; Fig. 3). Since the G10 frag-ment showed the highest competition, we further analyzed thebinding competition of individual CR regions that are presentin G10 (CR7 to CR11). The cadherin repeats were expressed

TABLE 1. Toxicity of Cry11Ba toxins with or without biotinylation against Ae. aegypti mosquito larvae

Cry11Ba toxinLC50, ng/ml (95% fiducial limit)

Inclusion body Soluble toxin Trypsin-activated toxin

Unlabeled 15.81 (10.2–29.37) 8,053 (5,260–10,546) 39,770 (31,193–50,637)Biotin labeled NDa 9,997 (5,487–10,906) 49,064 (35,796–57,412)

a ND, not determined.

FIG. 1. Binding of Cry11Ba toxin to Ae. aegypti BBMV. (A) Specific binding of biotinylated Cry11Ba toxin to BBMV was determined in thepresence of increasing concentrations of labeled Cry11Ba toxin. Specific binding was obtained from total binding minus nonspecific binding. Thedissociation constant (Kd) (8.2 nM) for toxin binding affinity was determined by Origin plot analysis from two different BBMV preparations.The curve shown is from a single experiment. (B) Binding of biotinylated Cry11Ba toxin to BBMV was determined in the presence of increasingconcentrations of unlabeled Cry11Ba toxin. The IC50 (3.6 nM) for toxin binding affinity was determined by Origin plot analysis from three differentBBMV preparations. The curve shown is from a single experiment. (C) Homologous competition in the binding of biotinylated Cry11Ba toxin toBBMV was also analyzed in solution; bound toxins to the BBMV were recovered by centrifugation, subjected to SDS-PAGE, and transferred toPVDF membranes. Biotinylated toxins were visualized with streptavidin-HRP. Different ratios of labeled to unlabeled toxins were used. At a ratioof 1:50 or 1:75, binding of biotinylated Cry11Ba was competed.

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in E. coli individually and used in toxin overlay assays. Ourresults indicated that CR7 to CR11 bound Cry11Ba toxin.However, CR11 appeared to be show the highest binding toCry11Ba (data not shown). In addition, to demonstrate therole of these cadherin repeats in the binding interaction withCry11Ba toxin, heterologous competition assays using indi-vidual CR peptides were performed. Repeats CR9 to CR11were the most critical regions for interaction with Cry11Batoxin, whereas CR8 competed off only half of Cry11Ba bind-ing and CR7 did not compete at all (Fig. 3, lanes 8 to 12, andTable 2). A synthetic peptide of CR11 also showed highcompetition with Cry11Ba binding to BBMV (Fig. 3, lanes16 to 18).

Role of ALPs and APN in binding interaction with Cry11Batoxin. We also determined if AaeALPs (AaeALP1, AaeALP2,and AaeALP3) and AaeAPN1 are able to compete in Cry11Ba

binding to BBMV. The binding of biotinylated Cry11Ba on Ae.aegypti BBMV can be competed principally with AaeALP1,while AaeALP2 and AaeALP3 barely competed Cry11Babinding to BBMV (Fig. 4, lanes 3 to 5). For the 36-kDaCry11Ba fragment, 64% was competed off with AaeALP1,whereas competitors such as AaeALP2 and AaeALP3 com-peted only 29% and 24% of toxin binding, respectively. Simi-larly, the 33-kDa Cry11Ba fragment was competed withAaeALP1 at 50%, whereas AaeALP2 and AaeALP3 compete insmaller amounts, at 17% and 3%, respectively. Recently, GPI-anchored APNs from An. quadrimaculatus and An. gambiaewere determined to bind Cry11Ba and considered potentialtoxin receptors (1, 45). Here we also observed competitivebinding between biotinylated Cry11Ba and AaeAPN1 (Fig. 4,lanes 8 and 9), suggesting a possible role of APN as a toxinreceptor for Cry11Ba in Ae. aegypti mosquito larvae.

FIG. 2. Schematic analysis of Cry11Ba toxin proteolysis. The 80-kDa Cry11Ba protoxin was processed in vitro by trypsin. The Cry11Ba toxinis cleaved between 343Lys and 354Gly, generating a 36-kDa N-terminal fragment and a 33-kDa C-terminal fragment. Loop region sequences in theCry11Ba toxin are underlined in insets. Loop �8 and loop 1 were identified in the N-terminal region of Cry11Ba, whereas loop 2 and loop 3 werein the C-terminal fragment.

FIG. 3. Cadherin is involved in binding of Cry11Ba to Ae. aegypti midgut membranes. Homologous and heterologous competition assays ofbinding of biotinylated Cry11Ba to Ae. aegypti BBMV were performed in the presence of different competitors, including cadherin fragments,cadherin repeats, and a cadherin synthetic peptide. Cadherin fragments G7, G10, and C13 (lanes 1 to 3, respectively) and unlabeled Cry11Ba (lane5) were used to compete the binding of biotinylated Cry11Ba to BBMV at a 250-fold molar excess. Lane 6 shows uncompeted toxin binding.Cadherin repeats CR7 to CR11, also at 250-fold-higher concentrations, were used as competitors in lanes 8 to 12, respectively. Finally, a syntheticpeptide corresponding to CR11 sequence (lanes 16 to 18) was used at molar excesses of 1:50, 1:100, and 1:250, respectively (Cry11Ba biotinylatedtoxin to CR11 peptide). NHE8 (lanes 4, 13, and 15), an unrelated peptide, was used as a negative control in competition experiments.

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Analyses of binding of different Cry11Ba mutant toxins tothe G10 cadherin fragment. Four Cry11Ba mutants with mu-tations located in different loop regions of domain II wereexpressed in E. coli and used to assess binding to the G10cadherin fragment. Mutants with multiple mutations in threeloop regions (loop �8-V256A/G257A/E258A, loop 1-R303A/E304A/N305A, and loop 3-N454A/K455A/L456A), all ofwhich lost toxicity to mosquito larvae, and a single mutant(loop 1-H307A), which had slightly lower toxicity than thewild-type Cry11Ba toxin, were analyzed for their ability to bindthe G10 cadherin fragment. There are differences, though mi-nor, in the 50% effective concentrations (EC50s) of bindingbetween the wild-type and mutant Cry11Ba toxins (Fig. 5).Thus, binding to just the cadherin fragment does not appear tobe correlated with larval toxicity.

Assays of binding competition between Cry11Ba andCry4Ba toxins. Analyses of Cry11Ba and Cry4Ba bindingcompetition with Ae. aegypti BBMV were conductedthrough heterologous competitions. Competitions were con-ducted with labeled Cry11Ba and unlabeled Cry11Ba or

Cry4Ba to determine if the two toxins share a binding sitepresent in BBMV. The binding of biotinylated Cry11Ba toBBMV was competed in the presence of increasing concen-trations of unlabeled Cry11Ba toxin as well as unlabeledCry4Ba toxin (Fig. 6). As expected, homologous competi-tion with Cry11Ba was more effective than heterologouscompetition with the Cry4Ba toxin.

To identify the critical loop regions of Cry4Ba involved inthis binding, we used heterologous binding competition assaysto determine common binding sites of the two toxins. Six syn-thetic peptides corresponding to the loop regions of domain IIof Cry4Ba (loop �8, loop �2-�3, loop �4-�5, loop �6-�7, loop�8-�9, and loop �10-�11) were synthesized and used to com-pete with binding of biotinylated Cry11Ba to larval midgutBBMV. Four peptides corresponding to loops �8, �2-�3, �8-�9, and �10-�11 significantly competed with Cry11Ba binding(Fig. 7). However, peptides corresponding to loops �4-�5 and�6-�7 showed negligible competition.

DISCUSSION

Of the Cry toxins, Cry11Ba has one of the highest mosqui-tocidal activities against Ae. aegypti larvae, but its mode ofaction in this species has been barely analyzed. Here we mea-sured the binding affinity between Cry11Ba and Ae. aegyptiBBMV and found that the toxin has a high binding affinity toBBMV. This interaction was saturable and dose dependent,with a dissociation constant (Kd) of 8.2 nM. The Cry11Babinding affinity to Ae. aegypti BBMV is higher than that ofCry11Aa to Culex pipiens BBMV, which is known to be around20 to 30 nM (S. M. Dai and S. S. Gill, unpublished data).Previous work by de Barros Moreira Beltrao et al. (14) usingwhole Ae. aegypti larval membranes showed that Cry11Aa hadan IC50 of 88 nM while Cry4Aa and Cry4Ba had IC50s of 99and 521 nM, respectively. Here we show Cry11Ba has an IC50

of 3.6 nM with midgut BBMV. According to previous LC50

bioassay data, Cry11Ba is more toxic to Ae. aegypti (18.8 ng/ml)(Table 1) than Cry11Aa toxin is to C. pipiens (372.4 ng/ml)(15). Thus, there appears to be a correlation between affinity ofbinding to BBMV and toxicity of Cry11Aa and Cry11Ba, atleast in these two mosquito species.

Upon activation, the 80-kDa Cry11Ba toxin generated 36-

TABLE 2. Cadherin fragments, cadherin repeats, AaeALPs, andAaeAPN1 compete with Cry11Ba binding to BBMV

Competitorb

% of binding of biotinylated-Cry11Ba toAe. aegypti BBMVa

Domains I-II,36-kDa

fragment

Domains II-III,33-kDa

fragment

Cry11Ba toxin 26.9 33.2Cadherin fragment G7 73.3 77.3Cadherin fragment G10 30.9 32.7Cadherin fragment C13 37.7 62.1Cadherin repeat 7 86.4 91.3Cadherin repeat 8 68.8 67.6Cadherin repeat 9 44.2 51.1Cadherin repeat 10 46.2 52.5Cadherin repeat 11 49.6 53.2AaeALP1 35.6 49.4AaeALP2 71.2 83.5AaeALP3 75.8 97.5AaeAPN1 45.7 49.0Peptide NHE8 92.9 100.8

a Experiments were repeated at least two times to obtain mean data.b Competitor peptides were used at a 1:250 molar ratio excess of Cry11Ba.

FIG. 4. APN and ALP compete with Cry11Ba binding to Ae. aegypti membranes. Heterologous competition assays of binding of biotinylatedCry11Ba to Ae. aegypti BBMV were performed in the presence of different competitors, including AaeAPN1 and AaeALP1 to -3. PurifiedAaeAPN1 and AaeALP1 to -3 at a 100- or 250-fold molar excess were incubated with biotinylated Cry11Ba overnight before binding to Ae. aegyptiBBMV. NHE8 (lane 6 and 12), an unrelated peptide, was used as a negative control.

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and 33-kDa fragments on SDS-PAGE. Cleavage occurs atprotease-sensitive sites between loop 1 and loop 2. We showthat at a molar ratio of 1:1 of labeled to unlabeled Cry11Batoxins, the unlabeled toxin competed principally the bindingof the 36-kDa fragment (32%), followed by competitionwith the 33-kDa fragment binding (12%). At higher ratios(1:50 or 1:100), binding to both fragments was nearly totallycompeted. The 36-kDa fragment was shown to be the N-terminal fragment, which consists of domain I and criticalloops of domain II of Cry11Ba toxin (31). Hence, it is notsurprising that the 36-kDa fragment was more readily com-peted off than the 33-kDa fragment with most of the com-petitors.

Since all binding experiments were conducted with bio-tinylated toxin, the toxicity upon labeling of Cry11Ba toxinwas tested in a mosquito larva bioassay. The biotinylatedtoxins retained similar toxicity, comparable to that of theunlabeled toxins. The toxicities of soluble and activatedCry11Ba toxins were 500 and 2,500 times lower than that ofinclusion bodies of Cry11Ba. Previous results showed solu-bilized crystals of B. thuringiensis subsp. israelensis were7,000 times less toxic to Ae. aegypti larvae than intact crystals(38). Presumably, this is because mosquito larvae are filterfeeders, which selectively concentrate particles while exclud-ing water and soluble molecules.

Based on information gathered from prior studies, cad-herin is a likely candidate binding site for Cry4Ba andCry11Aa toxins in different mosquito species (11, 24). Wepreviously showed that the anticadherin antibody inhibitsthe binding of Cry11Ba toxin to Ae. aegypti BBMV (31). Todetermine the specificity of cadherin interaction withCry11Ba toxin, the roles of three cadherin fragments (G7,C13, and G10) covering the cadherin repeats that have beenshown to be involved in Cry11Aa binding to Ae. aegyptiBBMV (11) were analyzed. Our results suggest that frag-ment G10 (which contains cadherin repeats CR7 to CR11)was able to significantly compete with Cry11Ba binding to

BBMV. Cadherin fragments G7 and C13 also competed,albeit at lower levels. We then analyzed the individual cad-herin repeats that are present on G10. Binding competitionassays using BBMV revealed that CR9 to CR11 are thebinding regions of this receptor that interact with theCry11Ba toxin. Moreover, a CR11 peptide can compete withCry11Ba binding to Ae. aegypti BBMV. These results indi-cate that CR11 of cadherin, which is the cadherin repeatmost proximal to the cell membrane, is critical for bindingand plays an important role in Cry11Ba binding. Previouswork performed with Cry11Aa toxin showed that CR11 fromAe. aegypti cadherin bound Cry11Aa toxin and that thisregion binds loop 3 of Cry11Aa toxin (11). A CR11 peptidefrom An. gambiae cadherin (CR11-MPED) was reported tosynergize the toxicity of Cry4Ba to mosquito larvae (35),suggesting that this peptide fragment also contains a Cry4Batoxin binding site and plays a fundamental role in toxininteraction. Importantly, Cry4Ba binds with high affinity (23nM) to the An. gambiae cadherin fragment, CR11-MPED(35), further suggesting that binding to cadherin is impor-tant for toxicity.

ALP and APN were identified as functional receptors inmany insects. A 65-kDa ALP (AaeALP1) was identified as aCry11Aa receptor in Ae. aegypti larvae (18–20). Isoforms ofALP were also shown to be Cry4Ba binding proteins in Ae.aegypti (18–20). Recently, AgALP1 was recognized as afunctional receptor for Cry11Ba in An. gambiae (25). How-ever, there are no data on whether Ae. aegypti ALPs orAPNs are involved in Cry11Ba binding. Since the brushborder membranes of Ae. aegypti larvae have multiple formsof ALPs, we analyzed three ALPs (AaeALP1 to -3) andAaeAPN1 from Ae. aegypti for their ability to compete withCry11Ba binding. Binding to Aedes BBMV could be competedwith both AaeALP1 and AaeAPN1. We also demonstratedthat AaeALP1 more readily competes off the binding ofbiotinylated Cry11Ba toxin to BBMV than AaeALP2 or

FIG. 5. Loop mutants of Cry11Ba toxin retain their ability to bindthe G10 cadherin fragment. Binding of wild-type Cry11Ba (f) and thefour loop mutants �8-V256A/G257A/E258A (E), L1-R303A/E304A/N305A (‚), L3-N454A/K455A/L456A (F), and L1-H307A (�) isshown. The G10 cadherin fragment EC50 for binding to Cry11Ba is 25nM, and those for the four mutants were 38, 41, 40, and 66 nM,respectively. The EC50s were determined using Origin.

FIG. 6. Competition binding of biotinylated Cry11Ba toxin toAe. aegypti BBMV in the presence of unlabeled Cry11Ba or Cry4Batoxin. Results of competitive assays of binding of biotinylatedCry11Ba toxin to BBMV in the presence of Cry11Ba (F) or Cry4Ba(f) as a competitor were plotted by using Origin. The experimentwas repeated once with similar results. The concentration ofCry4Ba required to compete 50% of the binding of biotinylatedCry11Ba to BBMV was 42 nM.

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AaeALP3, suggesting that in Ae. aegypti, ALP1 could be moreimportant in the interaction with Cry11Ba than AaeALP2 orAaeALP3. In the case of An. gambiae, an AgAPN had a greaterbinding affinity to Cry11Ba than AgALP1 (6.4 nM versus 23.9nM) (25, 45).

To determine what domain II loop regions are involved inreceptor binding and toxicity, we analyzed the effects ofdifferent domain II loop mutations of Cry11Ba in bindingand toxicity. The L1-R303A/E304A/N305A mutant, whichlost almost all its toxicity against mosquito larvae in thebioassay, was found to retain an ability to bind Ae. aegyptiBBMV that was similar to that of the wild-type toxin (31).In the pore-forming model, Cry monomeric toxins first bindto the cadherin receptor, resulting in toxin oligomerization(8). The oligomeric Cry toxins then bind to GPI-anchoredreceptors, which leads to toxin insertion into the membrane(40). Our BBMV binding assays likely exemplify the bindingof monomeric toxins to receptor molecules on BBMV. Itmight be possible that all wild-type and mutant toxins canbind cadherin in their monomeric form. In this report, wedemonstrate that there are small differences in the bindingof the loop mutants analyzed and Cry11Ba toxin to the G10fragment. One interesting possibility is that such mutantsare unable to bind GPI-anchored receptors, such as ALP orAPN, in their oligomeric structure. Further investigationson binding of oligomeric structures of these mutants arerequired in order to understand the function of these criticalresidues.

It is known that receptors such as cadherin, ALP, andAPN (or, as determined more recently, �-amylase) are rec-ognized by different Cry toxins such as Cry4Ba and Cry11Aa(11, 12, 18, 21, 24, 25, 40). Hence, we analyzed the compet-itive binding interactions between Cry11Ba and Cry4Ba tox-ins in Ae. aegypti BBMV. The binding of biotinylatedCry11Ba to BBMV was competed more effectively withCry11Ba than with Cry4Ba. Since it has been reported that

both toxins are active against Ae. aegypti larvae, with LC50sof 470 ng/ml for Cry4Ba (13) and 19 ng/ml for Cry11Ba (15),it might be possible that the abilities of these Cry toxins tobind BBMVs are directly correlated with their levels oftoxicity. Indeed Cry4Ba has a lower affinity to Aedes BBMVthan Cry11Ba (S. Likitvivatanavong and S. S. Gill, unpub-lished data).

We previously developed a structural model of theCry11Ba toxin and identified exposed loop regions in do-main II, and we showed that loop �8, loop 1 (or �2-�3), andloop 3 (or �10-�11) are involved in toxicity and receptorbinding to BBMV of Ae. aegypti (31). For Cry4Ba, it hasbeen revealed that loops �6-�7, �8-�9, and �10-�11 play arole in Cry4Ba toxicity, and combinations of two-loop mu-tants (loops �6-�7/�8-�9, loops �6-�7/�10-�11, and loops�8-�9/�10-�11) exhibited reduced binding to apical mi-crovilli of the Ae. aegypti larval midgut (28, 41). However,there are no data on whether other loops of the Cry4Batoxin are involved in toxicity and binding activity. Since it isknown that various combinations of Cry toxins may helpreduce selection of resistance in insects (9, 43), the identi-fication of common binding sites of different Cry toxins mayfacilitate the development of insect control strategies thatwill reduce resistance selection in important insect pests.Thus, we conducted heterologous competition binding as-says to determine common binding regions for both toxins.Six synthetic peptides regions (�8, �2-�3, �4-�5, �6-�7,�8-�9, and �10-�11) corresponding to the loop regions ofCry4Ba (7) were used to compete individually with the bind-ing of biotinylated Cry11Ba to larval midgut BBMV. Fourpeptides corresponding to loops �8, �2-�3, �8-�9, and �10-�11 could significantly compete with the binding of biotin-ylated Cry11Ba to BBMV, whereas peptides correspondingto loops �4-�5 and �6-�7 showed negligible competition.These data suggest that Cry11Ba and Cry4Ba share bindingregions during their interaction with Ae. aegypti BBMV.

ACKNOWLEDGMENTS

We appreciate the technical assistance of Amy Evans and Subum Lee.This research was funded in part through a grant from the National

Institutes of Health (1R01 AI066014), grants from DGAPA/UNAM(IN218608 and IN210208-N) and CONACyT (U48631-Q), and theUniversity of California Agricultural Experiment Station.

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