Ana Rodrigo-Simón, Silvia Caccia, and Juan Ferré* Departamento de Genética, Facultad de CC. Biológicas, Universidad de Valencia, Valencia, Spain

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2008, p Vol. 74, No /08/$ doi: /aem Copyright 2008, American Society for Microbiology. All Rights Reserved. Bacillus thuringiensis Cry1Ac Toxin-Binding and Pore-Forming Activity in Brush Border Membrane Vesicles Prepared from Anterior and Posterior Midgut Regions of Lepidopteran Larvae Ana Rodrigo-Simón, Silvia Caccia, and Juan Ferré* Departamento de Genética, Facultad de CC. Biológicas, Universidad de Valencia, Valencia, Spain Received 14 December 2007/Accepted 16 January 2008 It is generally accepted that Bacillus thuringiensis Cry toxins insert into the apical membrane of the larval midgut after binding to specific receptors, and there is evidence that the distribution of binding molecules along the midgut is not uniform. By use of the voltage-sensitive dye DiSC 3 (5) and 125 I-labeled Cry1Ac, we have measured the effect of Cry1Ac in terms of permeabilization capacity and of binding parameters on brush border membrane vesicles (BBMV) prepared from the anterior and the posterior regions of the larval midgut from two insect species, Manduca sexta and Helicoverpa armigera. The permeabilizing activity was significantly higher with BBMV from the posterior region than with the one observed in the anterior region in both insect species. Instead, 125 I-Cry1Ac bound specifically to BBMV from the two midgut regions, with no significant differences in the binding parameters between the anterior and posterior regions within an insect species. N-acetylgalactosamine inhibition patterns on pore formation and binding differed between anterior and posterior midgut regions and between species, providing evidence of a multifaceted involvement of the sugar in the Cry1Ac mode of action. The analysis of binding and pore formation in different midgut regions could be an effective method to study differences in the mode of action of Cry1Ac toxin in different species. The insecticidal activity of Bacillus thuringiensis proteins produced in parasporal crystals during sporulation (Cry proteins or Cry toxins) has been widely studied in lepidopteran insects, and there is relatively good evidence for the way the proteins act once ingested by a susceptible insect. There is general agreement in that the parasporal crystal dissolves after ingestion, and then the released protoxin is processed by gut proteases to an activated form; the active toxin crosses the peritrophic membrane and binds to specific receptors in the brush border membrane of midgut columnar cells and eventually leads to cell death (6, 13, 41, 43). The events that take place after binding are not yet clear, although the permeabilization of the membrane induced by toxin insertion and pore formation has long been proposed (34) and there is much evidence which supports this view (6, 13, 41, 43). In a model for the mode of action of Cry1A toxins, cadherin and aminopeptidase N (APN) receptors have a pivotal role (7): Cry1A toxins are proposed to bind to cadherin first, and then, after proteolytical modification of the bound toxin, a homo-oligomer is formed, which is transferred to APN and then inserts into the membrane. In support of this model, a recent study has shown that Cry1A-modified toxins, which spontaneously form the oligomer, can bypass the step of binding to cadherin to produce the toxic effect (46). Membranebound alkaline phosphatase also seems to play a role in the mode of action of Cry1A toxins, and it has been shown to be a Cry1Ac binding molecule in Manduca sexta (39, 42) and Heliothis virescens (14, 30). A recent model has challenged the * Corresponding author. Mailing address: Departamento de Genética, Facultad de CC. Biológicas, Dr. Moliner 50, Burjassot (Valencia), Spain. Phone: (34) Fax: (34) Juan.Ferre@uv.es. Published ahead of print on 25 January pore-forming model and proposes that only binding to cadherin is required to produce cell death; binding to APN or alkaline phosphatase is irrelevant in this model, and pore formation may be but a secondary effect of the toxic events triggered by toxin binding to cadherin (51, 52). It is important to note that evidence for the pore-forming model has been obtained mainly with in vitro experiments with artificial membranes or with brush border membrane vesicles (BBMV) (7, 19, 53), whereas the model of Zhang et al. has been developed from ex vivo observations with an insect ovarian cell line heterologously expressing the cadherin receptor. Immunochemical localization in midgut tissue sections has been applied to study the distribution of Cry1A receptors along the larval midgut. Early work by Bravo et al. (8) showed that some Cry proteins concentrated, after ingestion, in the anterior region of the midgut, whereas some others concentrated in the posterior region. Chen et al. (12) have shown the differential distribution in the M. sexta midgut of APN, alkaline phosphatase, and cadherin, and the bound Cry1A proteins. Aimanova et al. (1) also found an uneven distribution of the cadherin protein along the midgut regions of M. sexta and H. virescens. One study has used BBMV to show differences in the permeabilizing activity of Cry1Ac in different regions of the midgut of M. sexta (11) in the presence and in the absence of N-acetylgalactosamine (GalNAc). Cry1Ac has the peculiarity, not shared by other Cry1A proteins, of binding to APN through GalNAc residues through a lectin-like pocket in its domain III (10, 32, 33, 35). Cry1Ac binding to H. virescens membrane-bound alkaline phosphatase has also been shown to be dependent on the presence of an N-linked oligosaccharide containing a terminal GalNAc residue (30). Because of other similarities with APNs (both are glycosylphosphatidylinositol-anchored membrane proteins), it seems likely that membrane-bound alkaline phosphatases in 1710

2 VOL. 74, 2008 Cry1Ac INTERACTION WITH DIFFERENT MIDGUT REGIONS 1711 other insect species are also GalNAc-mediated Cry1Ac binding molecules. Cry1Ac also binds to Bombyx mori peritrophic membrane more efficiently than Cry1Aa does, and its binding is greatly reduced by preincubation with GalNAc (24). By making use of this property of Cry1Ac, one can dissect the binding of this protein by using GalNAc as an inhibitor and thus discriminate binding to GalNAc-bearing molecules (such as APN and alkaline phosphatase) from binding to other molecules in the brush border (such as cadherin) (11, 15) or the peritrophic membrane (24). Binding analyses of Cry toxins to lepidopteran BBMV have been carried out since 1988 (27) and have showed the importance of midgut receptors in the mode of action of Cry toxins. However, to our knowledge, binding studies have never been performed with BBMV prepared from different regions of the midgut. In the present work, we wanted to see whether the differences in permeabilization observed by Carroll et al. (11) in regions of M. sexta midgut would be also reflected at the level of toxin binding to BBMV. Furthermore, we have included a second insect species, also susceptible to Cry1Ac (2, 3), to determine how general the results obtained with M. sexta are. MATERIALS AND METHODS Cry1Ac toxin preparation. Cry1Ac was obtained from the recombinant B. thuringiensis EG11070 strain (Ecogen, Inc., Langhorne, PA) and was trypsinactivated and chromatographically purified as described previously (15). The protein concentration was determined by densitometry after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with Coomassie blue. Cry1Ac protein aliquots used for BBMV permeability assays (reported in fluorescence measurements) were dialyzed overnight at 4 C against 20 mm Tris-HCl, 150 mm CsCl at ph 8.6, with regenerated cellulose membranes (molecular mass cutoff of 7,000 Da; Pierce, Rockford, IL) in order to remove the Na present in the elution buffer used for chromatography purification. Preparation of BBMV. Last-instar larvae from laboratory colonies of M. sexta and H. armigera were excised, and the midguts were pulled out. Then the midgut was divided into three equidistant parts, and the central region was discarded (the reason being that if Cry1Ac receptors are distributed nonhomogeneously along the midgut, forming a gradient, the anterior and posterior regions would be the ones showing maximum differences). Tissue from the anterior and posterior regions was washed separately in MET buffer (250 mm mannitol, 17 mm Tris- HCl, and 5 mm EGTA, ph 7.5), blotted on filter paper, and frozen in liquid nitrogen in batches of about 5goftissue. Samples were stored in liquid nitrogen until used. BBMV for binding analyses and for enzyme assays were prepared following the Mg 2 differential precipitation method (49). BBMV were resuspended in one-half MET and stored at 80 C until required. For pore formation assays, BBMV were prepared according to the Ca 2 precipitation method (17). The final BBMV pellet was resuspended in a small volume of resuspension buffer (300 mm mannitol, 1 mm KCl, 10 mm HEPES-Tris, ph 7.2) to obtain a concentrated preparation of BBMV (final membrane protein concentration of 5 to 10 mg/ml). BBMV to be used in pore formation experiments were used within 2 h after preparation. The BBMV protein concentration was determined by the Bradford method (5) using bovine seroalbumine as standard. BBMV permeability assays. BBMV permeability to K was monitored by recording the fluorescence quenching of the voltage-sensitive cyanine dye 3,3 dipropylthiodicarbocyanine iodide, DiSC 3 (5). Vesicle membranes have a basal permeability to K ; thus, in the presence of a K inwardly directed electrochemical gradient, there is an influx of cations which leads to a release of the dye and thus to an increase in fluorescence. In the presence of pore-forming agents, there is a significantly higher influx and, thus, a significantly higher increase in fluorescence (44). Suitable amounts of M. sexta BBMV and H. armigera BBMV were preincubated for 30 and 45 min, respectively, at 4 C with Cry1Ac toxin (final concentration of 40 g/mg of BBMV proteins) or just with the toxin buffer (20 mm Tris-HCl at ph 8.6, 150 mm CsCl) in controls. After preincubation, the BBMV suspensions were transferred (at a final concentration of 22 g BBMV protein/ ml) into polyacryl cuvettes containing the same buffer used for BBMV resuspension to which 6 M DiSC 3 (5) (Molecular Probes, Società Italiana Chimici, Italy) had been added. Extravesicular increments of K concentrations were made by three successive additions of 2 M KCl to give final concentrations of 20, 40, and 60 mm KCl, respectively. Since intra- and extravesicular buffers had the same ionic composition, ph, and osmolarity and the apical membranes of lepidopteran columnar cells are almost impermeable to Cl, the membrane potential variations depended exclusively on the potassium movements across the membranes. Each experiment was tested three times or more with at least two independent BBMV preparations. DiSC 3 (5) fluorescence was measured in a Varian (Cary Eclipse) spectrofluorometer equipped with a thermostatized (25 C) four-cuvette holder, using excitation and emission wavelengths of 645 and 665 nm, respectively. For the GalNAc inhibition assays, 400 mm GalNAc was added to the preincubation reaction mixture and then the assays proceeded as indicated above. Enzyme assays. Alkaline phosphatase activity in BBMV was determined spectrophotometrically by using 4-nitrophenyl phosphate in diethanolamine buffer (Sigma, St. Louis, MO). The increase in absorbance at 405 nm was continuously read for 6 min at room temperature. The amount of BBMV to obtain an absorbance reading in the lineal range depended on the midgut region and the insect species. Nine and 15 g of proteins were used with BBMV from the anterior regions, whereas 0.3 and 1.5 g were used with BBMV from the posterior regions of M. sexta and H. armigera, respectively. Enzyme activities were calculated using an extinction coefficient of 18,450 M 1 cm 1 for 4-nitrophenol. Leucine aminopeptidase activity was determined by using 4 mm L-leucyl-pnitroanilide with 0.5 to 8 g BBMV proteins in 37.5 mm Tris-HCl, ph 7.5. Absorbance was measured at 410 nm during 3 min at room temperature. Enzyme activities were calculated using an extinction coefficient of 9,900 M 1 cm 1 for 4-nitroaniline. Cry1Ac labeling. Radiolabeling of Cry1Ac was performed using the chloramine-t method of incubating 20 g of protein with 0.25 mci of Na 125 I (GE Healthcare, Barcelona, Spain) (47). The specific radioactivity obtained was 1.42 mci/mg of Cry1Ac. Binding assays. Binding assays were carried out in a final volume of 0.1 ml in binding buffer (phosphate-buffered saline-0.1% bovine serum albumin). BBMV (4 or 2 g of membrane protein from H. armigera or M. sexta, respectively) were incubated for 1hatroom temperature with 320 pm 125 I-Cry1Ac. Competition experiments were carried out by adding increasing concentrations of unlabeled Cry1Ac and binding parameters (dissociation constant [K D ] and concentration of binding sites [R t ]) were estimated with the LIGAND computer program (40). These binding experiments were replicated at least twice. For the GalNAc inhibition assays, 125 I-Cry1Ac was preincubated with 25 mm GalNAc for 45 min at room temperature prior to the start of the binding assay with the addition of 10 g BBMV (15). Nonspecific binding was determined by adding a 1,000-fold excess of unlabeled Cry1Ac. Specific binding was estimated by subtracting the nonspecific binding from the total binding. These assays were replicated at least four times. RESULTS Cry1Ac permeabilization of BBMV from different midgut regions. As can be seen in Fig. 1 and 2, the intensity of fluorescence increases in the buffer control samples upon the addition of KCl. This is because BBMV have an intrinsic permeability to K, and this increase is taken as the negative control of these experiments. As a positive control, we used valinomycin, a K ionophore. Preliminary dose-response experiments indicated that 23 M valinomycin gave a maximal increase in fluorescence intensity, corresponding to the free influx of K (data not shown). When BBMV were preincubated with Cry1Ac, an increase of fluorescence intensity was observed, significantly different from that of the negative control, which indicates that pores are being formed (Fig. 1). In both insect species, 40 g Cry1Ac/mg BBMV protein caused maximal permeabilization of BBMV from the posterior region, comparable to that caused by valinomycin (Fig. 1B and D). This result is in contrast to its effect on the anterior region,

3 1712 RODRIGO-SIMÓN ETAL. APPL. ENVIRON. MICROBIOL. Downloaded from FIG. 1. Effect of Cry1Ac toxin on K permeability of BBMV from M. sexta anterior (A) or posterior (B) midgut regions and from H. armigera anterior (C) or posterior (D) midgut regions. BBMV, preloaded with 300 mm mannitol, 1 mm KCl, 10 mm HEPES-Tris at ph 7.2, were preincubated at 4 C with the buffer in which the toxin was dissolved (control and valinomycin samples) or with Cry1Ac (40 g/mg of BBMV proteins). After preincubation, BBMV were diluted in a cuvette with the resuspension buffer supplemented with 6 M DiSC 3 (5). The ionophore valinomycin was added to the cuvette simultaneously to the BBMV addition. KCl was added at the times indicated by the arrows to obtain extravesicular final concentrations of 20, 40, and 60 mm. Each trace represents the mean SE of at least three independent experiments. AU, arbitrary units of fluorescence. FIG. 2. Effect of GalNAc on Cry1Ac-induced K permeabilization of BBMV from M. sexta anterior (A) or posterior (B) midgut regions and from H. armigera anterior (C) or posterior (D) midgut regions. BBMV, preloaded with 300 mm mannitol, 1 mm KCl, 10 mm HEPES- Tris at ph 7.2, were preincubated at 4 C with the buffer in which the toxin was dissolved (control and valinomycin samples) or with Cry1Ac (40 g/mg of BBMV proteins) in the presence of 400 mm GalNAc. Other details are as described in the legend for Fig. 1. for which the permeabilizing capacity was lower than that of valinomycin, though significantly different from that of the negative control (Fig. 1A and C). GalNAc inhibition of Cry1Ac-induced permeabilization of BBMV. When GalNAc was added to the preincubation reaction mixture, K permeabilization by Cry1Ac in the posterior region of both insect species was completely inhibited, giving values as the buffer control (Fig. 2B and D). The same complete inhibition was observed with BBMV from the anterior region of H. armigera (Fig. 2C); however, with BBMV from the anterior region of M. sexta, permeabilization was hardly inhibited by GalNAc (Fig. 2A), resembling the values obtained with Cry1Ac in the absence of GalNAc (Fig. 1A). Alkaline phosphatase and leucine aminopeptidase activities in different midgut regions. The activities of these two brush border membrane marker enzymes, which have been identified as midgut receptors for Cry1A toxins, were measured in the anterior and posterior regions of H. armigera and M. sexta. As shown in Table 1, both activities are significantly higher in the posterior region than in the anterior region in both species. A on November 20, 2018 by guest

4 VOL. 74, 2008 Cry1Ac INTERACTION WITH DIFFERENT MIDGUT REGIONS 1713 TABLE 1. Alkaline phosphatase and leucine aminopeptidase activities in different midgut regions of H. armigera and M. sexta Insect species Alkaline phosphatase Sp act a Leucine aminopeptidase Anterior Posterior Anterior Posterior H. armigera M. sexta a Expressed in mol/min per mg of protein. Results are means standard deviations of at least four replicates. TABLE 2. K D s and R t s for 125 I-Cry1Ac binding to BBMV from different midgut regions from H. armigera and M. sexta a Binding parameter Value for insect species H. armigera M. sexta Anterior Posterior Anterior Posterior K D (nm) R t (pmol/mg) a Results are shown as means standard errors. K D and R t values were calculated from at least two experiments. comparison between species showed that the specific activity of both enzymes in the posterior region was higher in M. sexta than in H. armigera, whereas the reversed situation applied to the anterior region. A comparison within a species between midgut regions showed that differences in specific enzyme activity were much smaller in H. armigera than in M. sexta (3-fold versus 100-fold, respectively, for both enzyme activities). Cry1Ac binding to different midgut regions. Specific binding of 125 I-Cry1Ac was found in both the anterior and the posterior regions of the midgut in the two insect species tested, as can be seen from the competition curves in Fig. 3. No apparent differences were observed between the competition curves within the same insect species, which is corroborated by the lack of significant differences (P 0.05) in the binding parameters between the anterior and the posterior regions (Table 2). Both insects bound Cry1Ac with similar affinities (they had similar K D values), whereas M. sexta showed higher R t values than H. armigera did. GalNAc inhibition of 125 I-Cry1Ac binding. Figure 4 shows the specific binding of 125 I-Cry1Ac to BBMV in the presence and in the absence of GalNAc represented as a percentage of the total radioactivity added. Considering the specific binding for each sample in the absence of GalNAc as 100%, GalNAc inhibition accounted for 11.6% and 68% of the specific binding in the anterior and posterior regions, respectively, of the H. armigera midgut. In M. sexta, the specific binding inhibition was 29% for the anterior region and 52% in the posterior region. GalNAc inhibition was statistically significant (P 0.05) in all samples except for the anterior midgut region of H. armigera. DISCUSSION It is known that in the presence of a transmembrane electrical potential, the fluorescence quenching of the voltagesensitive dye DiSC 3 (5) can be used to monitor the diffusional K movements across the vesicular membrane in insect midgut BBMV (9, 21, 37, 38). In the present study, we measured the ability of Cry1Ac to induce the formation of pores with K channel properties by recording the fluorescence signal variations of the dye DiSC 3 (5) with increasing inwardly directed K gradients. The results showed that the preincubation with Cry1Ac in both species affected the permeability in BBMV from the posterior region, leading to the complete dissipation of the K electrochemical gradient during the permeability assay. On the contrary, the effect of the toxin was lower in the BBMV from the anterior region than in BBMV from the posterior region. Furthermore, this permeabilizing activity was completely inhibited by GalNAc in the posterior regions of both insects and in the anterior region of H. armigera, showing that GalNAcbearing molecules (APN, alkaline phosphatase, or other) are involved in the permeabilization process in these regions. However, BBMV from the anterior region from M. sexta did not show any detectable inhibition of permeabilizing activity by GalNAc. Our results with M. sexta regarding pore formation and its inhibition by GalNAc are in good agreement with the results from the kinetics experiments performed by Carroll et al. (11), who used a more indirect method (light scattering) to measure permeabilization activity of Cry1Ac. However, in contrast to our results, these authors failed to obtain significant differences between both midgut regions when preincubating the toxin with BBMV. The main reason for this discrepancy FIG. 3. Binding of 125 I-Cry1Ac as a function of increasing concentrations of unlabeled Cry1Ac in H. armigera (A) and M. sexta (B). BBMV were prepared from the anterior (F) or the posterior region (E) of the midgut. Each data point represents the mean of two replicates, and the error bars show standard deviations.

5 1714 RODRIGO-SIMÓN ETAL. APPL. ENVIRON. MICROBIOL. FIG. 4. Specific binding of 125 I-Cry1Ac to BBMV from the anterior and posterior regions of the midgut from H. armigera and M. sexta in the presence (white bars) or in the absence (gray bars) of 25 mm GalNAc. Each point represents the mean of at least four determinations, and the error bars show standard deviations. Asterisks show the values which were significantly different from those for the control without GalNac (P 0.05), for which the percentage of inhibition is given. may be the presence of an alkaline ph inside the vesicles in the experiments by Caroll et al. (ph 9 was present in their study, in contrast to ph 7.2 in our study), which may have reasonably affected the integrity of the vesicles before starting the permeability experiment (18, 31). Moreover, the long preincubation time (1 h) at room temperature could further affect the quality of the vesicles used. These authors proposed that there must be a different mechanism in M. sexta that leads to pore formation in the anterior region different from the one that takes place in the posterior region. It is worth noting that this conclusion cannot be extended to H. armigera, which showed complete inhibition of both midgut parts, at least under the experimental conditions tested. The enzymatic assays indicated much higher APN activity in the posterior region than in the anterior region, in agreement with previous reports on APN activity in M. sexta (11, 48) and with the histochemical determination of APN1 distribution in the M. sexta midgut (12). In addition, we also found higher alkaline phosphatase activity in the posterior region in both species, which is in disagreement with the reported midgut distribution of membrane-bound alkaline phosphatase in M. sexta as assessed by immunolocalization (12). A possible explanation for this discrepancy may be the occurrence of more than one type of membrane-bound alkaline phosphatase in M. sexta. Our results for Cry1Ac-induced BBMV permeabilization and its GalNAc inhibition in the posterior parts of both insects would be perfectly explained by the accumulation of APN and/or alkaline phosphatase in the posterior region, although the involvement of additional molecules cannot be excluded. The GalNAc complete inhibition of the Cry1Ac permeabilizing activity in the anterior region of H. armigera could also be due to the presence of APN and/or alkaline phosphatase in this region. However, the lack of GalNAc inhibition in the anterior region of M. sexta midgut may reflect the occurrence of isozymes other than those found in the posterior part. Chen et al. (12), by the use of specific antibodies, determined the distribution of three Cry1A-binding proteins in the M. sexta midgut of second-instar larvae. The researchers showed that APN1 was concentrated in the posterior region, whereas the cadherin (Bt-R1) and membrane-bound alkaline phosphatase were more or less evenly distributed along the midgut. They also showed that in vitro binding to tissue sections of Cry1Ac, but not Cry1Aa or Cry1Ab, took place preferentially in the posterior region. The combination of our results and the observations of Chen et al. suggest that APN1 may be the key receptor involved in GalNAc-dependent pore formation, but they do not support (or reject) the involvement of alkaline phosphatase. Immunodetection of these receptors in B. mori has shown a similar distribution of APN along the midgut; however, in contrast to the situation in M. sexta, cadherin was found to be more abundant in the posterior region of B. mori (23). It seems that the distribution of these receptors varies depending on the species studied, in agreement with our observations of H. armigera. The pore-forming activity of Cry1Ac in the anterior region of M. sexta, which is not inhibited by GalNAc, must be independent of APN1. Therefore, in addition to cadherin as the main Cry1Ac-binding protein, other molecules different from APN1 must be involved in Cry1Ac insertion and pore formation in the anterior region. It was known that Cry1Ac binds with high affinity to binding sites in BBMV prepared from whole midguts from M. sexta (28, 47) and H. armigera (2, 15, 25). Our results using 125 I- Cry1Ac have shown that specific binding occurs in the two midgut regions from both insect species (Fig. 3). What was not so obvious was that 125 I-Cry1Ac binding was so quantitatively similar in both regions, i.e., we did not find significant differences in the binding parameters between the anterior and posterior regions within an insect species (Table 2). These results indicate that whatever the region which most contributes to the BBMV preparation, this should not affect binding parameters, at least in the two insect species tested. Given that APN is concentrated in the posterior region (determined not only by APN1 immunolocalization but also by enzyme activity), other Cry1Ac-binding molecules must compensate for this in the anterior region, with either their concentrations or their affinities for this toxin. Aimanova et al. (1), working with firstinstar larvae, have shown that cadherin was detected only in the anterior region of the H. virescens midgut and that although it could be detected in M. sexta in both regions, it was concentrated in the anterior region in this insect. GalNAc inhibition of Cry1Ac binding has also shown a different pattern in M. sexta and in H. armigera (Fig. 4). In the former, this sugar inhibited binding in the anterior and posterior regions, although relatively more inhibition occurred in the posterior region. In contrast, in H. armigera, binding was only significantly inhibited in the posterior region. The inhibition found in both insects in the posterior region is in agreement with the differential distribution of APN. The binding component not inhibited by GalNAc indicates the contribution of other types of molecules in the binding of Cry1Ac. The inhibition of Cry1Ac binding in the anterior region of M. sexta seems to indicate that this region possesses GalNAc-dependent Cry1Ac-binding molecules which do not take part in the pore formation process since GalNAc did not inhibit pore formation in this region. In H. armigera, the Cry1Ac binding inhibition in the anterior region is not statistically significant. However, given that GalNAc inhibits pore formation in that

6 VOL. 74, 2008 Cry1Ac INTERACTION WITH DIFFERENT MIDGUT REGIONS 1715 region, it follows that GalNAc must be inhibiting binding to some receptor molecules. Since the reduction in binding is so small (Fig. 4), the GalNAc-bearing molecules must represent a minor part of the binding molecules in that region. Taken together, the results suggest that all Cry1Ac permeabilizing activity in BBMV from the posterior region of the midgut of the two insect species studied is due to Cry1Ac binding to GalNAc residues, probably from APN and alkaline phosphatase, and the same applies to BBMV from the anterior region in H. armigera. However, another type of GalNAcindependent binding takes place in the anterior region of M. sexta, which also leads to membrane permeabilization. This is the reason why Carroll et al. (11) proposed that Cry1Ac is acting on the anterior region of M. sexta midgut by a different mechanism from the posterior region. The lack of differences between the two midgut regions in terms of specific binding of Cry1Ac in the two species, together with the different GalNAc binding-inhibition patterns, indicates that there is a distinct distribution of Cry1Ac-binding proteins, some GalNAc dependent and some not, and in turn, some of the GalNAc-dependent Cry1Ac-binding proteins do not seem to be involved in membrane permeabilization (those of the anterior region of M. sexta midgut). It is important to note that despite the GalNAc-mediated binding and pore-forming activity of Cry1Ac observed in vitro, two independent groups obtained evidence that Cry1Ac binding through GalNAc residues does not have a major effect on in vivo toxicity (10, 29). They showed that Cry1Ac mutants, unable to bind to GalNAc residues and to purified APN from M. sexta, had impaired pore-forming capacity and reduced binding affinity for M. sexta BBMV; however, their toxicity to this insect remained practically unaffected. The binding of Cry1Ac to midgut proteins through GalNAc residues may mask other toxin-receptor interactions, in binding and permeabilization analysis, with more relevance to the in vivo toxicity. There is an increasing body of evidence showing that not all binding to BBMV is effective and that there are binding molecules in the brush border that do not contribute to the toxicity of Cry toxins. For example, there are some B. thuringiensisresistant strains with high levels of resistance to Cry1Aa, Cry1Ab, and Cry1Ac toxins, but not to other B. thuringiensis proteins, which have lost binding to Cry1Aa or Cry1Ab, but not to Cry1Ac (4, 20, 22, 26, 36, 45, 50). This type of highly selective resistance is typical of alterations in the binding site and we expect an alteration in a shared receptor to be the cause of resistance (16). These cases have been explained by researchers proposing either different binding molecules or different epitopes/binding sites, one shared by all Cry1A toxins but also others that only Cry1Ac would bind. These unique Cry1Ac binding sites may very likely be the GalNAc residues. It is therefore important to be able to distinguish between the Cry1Ac binding which is dependent on GalNAc residues (which seems to be futile for in vivo toxicity) and the GalNAcindependent one (which may be effective). The approach used in this work, although indirect, has succeeded in revealing the complex distribution of Cry1Ac binding molecules along the larval midgut regions of two lepidopteran species. In addition to the interest of the work per se, the differential distribution of GalNAc-dependent and -independent Cry1Ac-binding molecules, and of GalNAc-dependent and -independent Cry1Ac-mediated pore formation, can have practical applications. One could use different regions of the midgut to prepare BBMV enriched with different types of binding molecules and then use GalNAc to dissect further binding and/or pore-forming activity. Depending on the insect species studied, the analysis of binding and pore formation in different midgut regions could be an effective method to study differences in susceptibility to Cry1Ac toxin in different species and also to study the molecular basis of Cry1Ac resistance in some resistant insect strains minimizing interference from futile binding. ACKNOWLEDGMENTS We thank B. Giordana and M. G. Leonardi for their encouragement and scientific support of S.C. This research was supported by the Spanish Ministry of Education and Science (grants AGL C03-01 and AGL ) and the Generalitat Valenciana (grant GRUPOS ). REFERENCES 1. Aimanova, K. G., M. Zhuang, and S. S. Gill Expression of Cry1Ac cadherin receptors in insect midgut and cell lines. J. Invertebr. Pathol. 92: Akhurst, R. J., W. James, L. J. Bird, and C. Beard Resistance to the Cry1Ac -endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). J. Econ. Entomol. 96: Avilla, C., E. Vargas-Osuna, J. González-Cabrera, J. Ferré, and J. E. González- Zamora Toxicity of several -endotoxins of Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) from Spain. J. Invertebr. Pathol. 90: Ballester, V., F. Granero, B. E. Tabashnik, T. Malvar, and J. Ferré Integrative model for binding of Bacillus thuringiensis toxins in susceptible and resistant larvae of the diamondback moth (Plutella xylostella). Appl. Environ. Microbiol. 65: Bradford, M. M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. J. 72: Bravo, A., S. S. Gill, and M. Soberón Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49: Bravo, A., I. Gómez, J. Conde, C. Muñoz-Garay, J. Sánchez, R. Miranda, M. Zhuang, S. S. Gill, and M. Soberón Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochim. Biophys. Acta 1667: Bravo, A., S. Jansens, and M. Peferoen Immunocytochemical localization of Bacillus thuringiensis insecticidal crystal proteins in intoxicated insects. J. Invertebr. Pathol. 60: Bravo, A., R. Miranda, I. Gómez, and M. Soberón Pore formation activity of Cry1Ab toxin from Bacillus thuringiensis in an improved membrane vesicle preparation from Manduca sexta midgut cell microvilli. Biochim. Biophys. Acta 1562: Burton, S. L., D. J. Ellar, J. Li, and D. J. Derbyshire N-acetylgalactosamine on the putative insect receptor aminopeptidase N is recognised by a site on the domain III lectin-like fold of a Bacillus thuringiensis insecticidal toxin. J. Mol. Biol. 287: Carroll, J., M. G. Wolfersberger, and D. J. Ellar The Bacillus thuringiensis Cry1Ac toxin-induced permeability change in Manduca sexta midgut brush border membrane vesicles proceeds by more than one mechanism. J. Cell Sci. 110: Chen, J., M. R. Brown, G. Hua, and M. J. Adang Comparison of the localization of Bacillus thuringiensis Cry1A -endotoxins and their binding proteins in larval midgut of tobacco hornworm Manduca sexta. Cell Tissue Res. 321: de Maagd, R. A., A. Bravo, C. Berry, N. Crickmore, and H. E. Schnepf Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annu. Rev. Genet. 37: English, L. H., and T. L. Readdy Endotoxin inhibits a phosphatase in midgut epithelial membranes of Heliothis virescens. Insect Biochem. 19: Estela, A., B. Escriche, and J. Ferré Interaction of Bacillus thuringiensis toxin with larval midgut binding sites of Helicoverpa armigera (Lepidoptera: Noctuidae). Appl. Environ. Microbiol. 70: Ferré, J., and J. Van Rie Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Annu. Rev. Entomol. 47:

7 1716 RODRIGO-SIMÓN ETAL. APPL. ENVIRON. MICROBIOL. 17. Giordana, B., V. F. Sacchi, and G. M. Hanozet Intestinal amino acid absorption in lepidopteran larvae. Biochim. Biophys. Acta 692: Giordana, B., M. G. Leonardi, M. Casartelli, P. Consonni, and P. Parenti K -neutral amino acid symport of Bombyx mori larval midgut: a system operative in extreme conditions. Am. J. Physiol. 43:R1361 R Gómez, I., J. Sánchez, R. Miranda, A. Bravo, and M. Soberón Cadherin-like receptor binding facilitates proteolytic cleavage of helix alpha-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin. FEBS Lett. 513: González-Cabrera, J., B. Escriche, B. E. Tabashnik, and J. Ferré Binding of Bacillus thuringiensis toxins in resistant and susceptible strains of pink bollworm (Pectinophora gossypiella). Insect Biochem. Mol. Biol. 33: González-Cabrera, J., G. P. Farinós, S. Caccia, M. Díaz-Mendoza, P. Castañera, M. G. Leonardi, B. Giordana, and J. Ferré Toxicity and mode of action of Bacillus thuringiensis Cry proteins in the Mediterranean corn borer, Sesamia nonagrioides (Lefebvre). Appl. Environ. Microbiol. 72: González-Cabrera, J., S. Herrero, and J. Ferré High genetic variability for resistance to Bacillus thuringiensis toxins in a single population of diamondback moth. Appl. Environ. Microbiol. 67: Hara, H., S. Atsumi, K. Yaoi, K. Nakanishi, S. Higurashi, N. Miura, H. Tabunoki, and R. Sato A cadherin-like protein functions as a receptor for Bacillus thuringiensis Cry1Aa and Cry1Ac toxins on midgut epithelial cells of Bombyx mori larvae. FEBS Lett. 538: Hayakawa, T., Y. Shitomi, K. Miyamoto, and H. Hori GalNAc pretreatment inhibits trapping of Bacillus thuringiensis Cry1Ac on the peritrophic membrane of Bombyx mori. FEBS Lett. 576: Hernández, C. S., A. Rodrigo., and J. Ferré Lyophilization of lepidopteran midguts: a preserving method for Bacillus thuringiensis toxin binding studies J. Invertebr. Pathol. 85: Herrero, S., B. Oppert, and J. Ferré Different mechanisms of resistance to Bacillus thuringiensis toxins in the Indianmeal moth. Appl. Environ. Microbiol. 67: Hofmann, C., P. Lüthy, R. Hutter, and V. Pliska Binding of the -endotoxin from Bacillus thuringiensis to brush-border membrane vesicles of the cabbage butterfly (Pieris brassicae). Eur. J. Biochem. 173: Hofmann, C., H. Vanderbruggen, H. Höfte, J. Van Rie, S. Jansens, and H. Van Mellaert Specificity of Bacillus thuringiensis -endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts. Proc. Natl. Acad. Sci. USA 85: Jenkins, J. L., M. K. Lee, S. Sangadala, M. J. Adang, and D. H. Dean Binding of Bacillus thuringiensis Cry1Ac toxin to Manduca sexta aminopeptidase-n receptor is not directly related to toxicity. FEBS Lett. 462: Jurat-Fuentes, J. L., and M. J. Adang Characterization of a Cry1Acreceptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae. Eur. J. Biochem. 271: Klip, A., S. Grinstein, T. Marti, and G. Semenza Transient opening of brush border membrane vesicles in alkaline media. FEBS Lett. 105: Knight, P. J., J. Carroll, and J. D. Ellar Analysis of glycan structures on the 120 kda aminopeptidase N of Manduca sexta and their interactions with Bacillus thuringiensis Cry1Ac toxin. Insect Biochem. Mol. Biol. 34: Knight, P. J., N. Crickmore, and D. J. Ellar The receptor for Bacillus thuringiensis CryIA(c) -endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N. J. Mol. Microbiol. 11: Knowles, B. H., and D. J. Ellar Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis -endotoxins with different insect specificity. Biochim. Biophys. Acta 924: Knowles, B. H., P. J. Knight, and D. J. Ellar N-acetyl galactosamine is part of the receptor in insect gut epithelia that recognizes an insecticidal protein from Bacillus thuringiensis. Proc. R. Soc. London 245: Lee, M. K., F. Rajamohan, F. Gould, and D. H. Dean Resistance to Bacillus thuringiensis Cry1a -endotoxins in a laboratory-selected Heliothis virescens strain is related to receptor alteration. Appl. Environ. Microbiol. 61: Leonardi, M. G., S. Caccia, J. González-Cabrera, J. Ferré, and B. Giordana Leucine transport is affected by Bacillus thuringiensis Cry1 toxins in BBMV from Ostrinia nubilalis Hb (Lepidoptera: Pyralidae) and Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae) midgut. J. Membr. Biol. 214: Leonardi, M. G., P. Parenti, M. Casartelli, and B. Giordana Bacillus thuringiensis Cry1Aa -endotoxin affects the K /amino acid symport in Bombyx mori larval midgut. J. Membr. Biol. 159: McNall, R. J., and M. J. Adang Identification of novel Bacillus thuringiensis Cry1Ac binding proteins in Manduca sexta midgut through proteomic analysis. Insect Biochem. Mol. Biol. 33: Munson, P., and D. Rodbard LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107: Pigott, C. R., and D. J. Ellar Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol. Mol. Biol. Rev. 71: Sangadala, S., F. S. Walters, L. H. English, and M. J. Adang A mixture of Manduca sexta aminopeptidase and phosphatase enhances Bacillus thuringiensis insecticidal CryIA(c) toxin binding and 86Rb ( ) -K efflux in vitro. J. Biol. Chem. 269: Schnepf, E., N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D. R. Zeigler, and D. H. Dean Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62: Sims, P. J., A. S. Waggoner, C. H. Wang, and J. F. Hoffman Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry 13: Siqueira, H. A. A., J. González-Cabrera, J. Ferré, R. Flannagan, and B. D. Siegfried Analyses of Cry1Ab binding in resistant and susceptible strains of the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae). Appl. Environ. Microbiol. 72: Soberón, M., L. Pardo-López, I. López, I. Gómez, B. E. Tabashnik, and A. Bravo Engineering modified Bt toxins to counter insect resistance. Science 318: Van Rie, J., S. Jansens, H. Höfte, D. Degheele, and H. Van Mellaert Specificity of Bacillus thuringiensis -endotoxins: importance of specific receptors on the brush border membrane of the mid-gut of target insects. Eur. J. Biochem. 186: Wolfersberger, M. G Localization of amino acid absorption systems in larval midgut of the tobacco hornworm Mancuda sexta. J. Insect Physiol. 42: Wolfersberger, M. G., P. Lüthy, P. Maurer, P. Parenti, V. F. Sacchi, B. Giordana, and G. M. Hanozet Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Comp. Biochem. Physiol. 86A: Wright, D. J., M. Iqbal, F. Granero, and J. Ferré A change in a single midgut receptor in the diamondback moth (Plutella xylostella) is only in part responsible for field resistance to Bacillus thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai. Appl. Environ. Microbiol. 63: Zhang, X., M. Candas, N. B. Griko, L. Rose-Young, and L. A. Bulla, Jr Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R(1) expressed in insect cells. Cell Death Differ. 12: Zhang, X., M. Candas, N. B. Griko, R. Taussig, and L. A. Bulla, Jr A mechanism of cell death involving an adenylyl cyclase/pka signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis. Proc. Natl. Acad. Sci. USA 103: Zhuang, M., D. I. Oltean, I. Gómez, A. K. Pullikuth, M. Soberón, A. Bravo, and S. S. Gill Heliothis virescens and Manduca sexta lipid rafts are involved in Cry1A toxin binding to the midgut epithelium and subsequent pore formation. J. Biol. Chem. 277: