Yasutaka Yoshizawa

The ATP‐binding cassette transporter subfamily C member 2 in Bombyx mori larvae is a functional receptor for Cry toxins from Bacillus thuringiensis

Bacillus thuringiensis is the most widely used biopesticide, and its Cry toxin genes are essential transgenes for the generation of insect‐resistant transgenic crops. Recent reports have suggested that ATP‐binding cassette transporter subfamily C2 (ABCC2) proteins are implicated in Cry intoxication, and that a single amino acid insertion results in high levels of resistance to Cry1 toxins. However, there is currently no available direct evidence of functional interactions between ABCC2 and Cry toxins. To address this important knowledge gap, we investigated the role of Bombyx mori ABCC2 (BmABCC2) or its mutant from a Cry1Ab‐resistant B. mori strain on Cry1A toxin action. When we expressed BmABCC2 ectopically on Sf9 cells, it served as a functional receptor, and the single amino acid insertion found in BmABCC2 from Cry1Ab‐resistant larvae resulted in lack of susceptibility to Cry1Ab and Cry1Ac. Using the same expression system, we found that Bo. mori cadherin‐like receptor (BtR175) conferred susceptibility to Cry1A toxins, albeit to a lower degree than BmABCC2. Coexpression of BtR175 and BmABCC2 resulted in the highest cell susceptibility to Cry1A, Cry1F, and even the phylogenetically distant Cry8Ca toxin, when compared with expression of either receptor alone. The susceptibility observed in the coexpressing cells and that in Bo. mori larvae are likely to be correlated, suggesting that BtR175 and BmABCC2 are important factors determining larval susceptibility. Our study demonstrates, for the first time, Cry toxin receptor functionality for ABCC2, and highlights the crucial role of this protein and cadherin in the mechanism of action of Cry toxin.

Identification and comparative analysis of three novel C-type lectins from the silkworm with functional implications in pathogen recognition

C-type lectins can act as pattern recognition receptors (PRRs) in innate immunity. Previously, we identified two C-type lectins from silkworm (Bombyx mori), BmLBP and BmMBP, as PRRs. In the present study, we identified three homologs of these lectins by searching the silkworm genome database. These novel B. mori low-expression lectins were designated BmLEL-1, BmLEL-2, and BmLEL-3. Although Western-blot analysis failed to detect BmLEL-1, -2, or -3 in plasma, affinity precipitation of larval plasma with various microorganisms revealed that BmLEL-1 and -2 bind to rough and smooth strains of Gram-negative bacteria, respectively. BmLEL-1, -2, and -3 were found to be expressed in testis and ovary, where BmLEL-2 expression was up-regulated after bacteria infection. These results indicate that the novel C-type lectins might play a role in the innate immunity in these tissues as PRRs. Here, we discuss the roles and members of the C-type lectins as primary PRRs in B. mori cellular immunity.

Analysis of the region for receptor binding and triggering of oligomerization on Bacillus thuringiensis Cry1Aa toxin

The determination of the receptor‐binding region of Cry toxins produced by Bacillus thuringiensis is expected to facilitate an improvement in their insecticidal ability through protein engineering. We analyzed the region on Cry1Aa molecules involved in interactions with the cadherin‐like protein receptor BtR175 using cysteine‐substituted mutant toxins and several synthetic peptides corresponding to the loops in domain 2. In addition, the region necessary to trigger oligomerization was analyzed using these mutant toxins. The mutant toxins were modified by two types of molecule, i.e. digested fragments of the Cry1Aa precursor with an average molecular mass of 2 kDa and 5‐iodoacetamidofluorescein, which has a molecular mass of 515 kDa. We examined whether these modifications interfere with the toxin–BtR175 interaction as a result of steric hindrance. 5‐Iodoacetamidofluorescein modification of R311C, N376C and G442C revealed steric hindrance effects, indicating that R311 on loop 1, N376 on loop 2 and G442 on loop 3 are on the contact face of the toxin–BtR175 interface when Cry1Aa binds to BtR175. Loop 2 is thought to interact with BtR175 directly, as a peptide corresponding to the N‐terminal half of loop 2, (365)LYRRIILG(372), has the potential to bind to BtR175 fragments. Meanwhile, mutant toxins with cysteine substitutions in loops 1 and 2 were oligomerized by the binding of digested fragments in the activation process without receptor interaction, and the wild‐type toxin formed oligomers by interaction with BtR175 fragments. These observations suggest that loops 1 and 2 form both a binding region and a sensor region, which triggers toxin oligomer formation.

Location of the Bombyx mori 175 kDa cadherin-like protein-binding site on Bacillus thuringiensis Cry1Aa toxin

To identify and gain a better understanding of the cadherin‐like receptor‐binding site on Bacillus thuringiensis Cry toxins, it is advantageous to use Cry1Aa toxin, because its 3D structure is known. Therefore, Cry1Aa toxin was used to examine the locations of cadherin‐like protein‐binding sites. Initial experiments examining the binding compatibility for Cry1Aa toxin of partial fragments of recombinant proteins of a 175 kDa cadherin‐like protein from Bombyx mori (BtR175) and another putative receptor for Cry1Aa toxin, aminopeptidase N1, from Bo. mori (BmAPN1), suggested that their binding sites are close to each other. Of the seven mAbs against Cry1Aa toxin, two mAbs were selected that block the binding site for BtR175 on Cry1Aa toxin: 2A11 and 2F9. Immunoblotting and alignment analyses of four Cry toxins revealed amino acids that included the epitope of mAb 2A11, and suggested that the area on Cry1Aa toxin blocked by the binding of mAb 2A11 is located in the region consisting of loops 2 and 3. Two Cry1Aa toxin mutants were constructed by substituting a Cys on the area blocked by the binding of mAb 2A11, and the small blocking molecule, N‐(9‐acridinyl)maleimide, was introduced at each Cys substitution to determine the BtR175‐binding site. Substitution of Tyr445 for Cys had a crippling effect on binding of Cry1Aa toxin to BtR175, suggesting that Tyr445 may be in or close to the BtR175‐binding site. Monoclonal antibodies that blocked the binding site for BtR175 on Cry1Aa toxin inhibited the toxicity of Cry1Aa toxin against Bo. mori, indicating that binding of Cry1Aa toxin to BtR175 is essential for the action of Cry1Aa toxin on the insect.

Response of midgut epithelial cells to Cry1Aa is toxin-dependent and depends on the interplay between toxic action and the host apoptotic response

Cry1Aa is an insecticidal protein produced by Bacilllus thuringiensis. To elucidate the mechanisms of cell/individual death and healing in the midgut epithelium, Bombyx mori larva were given different concentrations of Cry1Aa, and sections from the midgut were examined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. In the lethal condition, most midgut columnar cells were observed to swell and burst without being stained by TUNEL 4 h after intoxication, and the epithelial layer collapsed by 24 h. In the sublethal to nonlethal conditions, midgut columnar cells did not swell, but their nuclei were intensely stained by TUNEL at 4–24 h after intoxication. These apoptotic cells dropped out of the epithelial layer, and the epithelial layer was finally renewed by newly mature columnar cells within 72 h after intoxication. These results suggest that the reaction, which occurs in the midgut of intoxicated insects, is determined by the interplay between passive toxic action and the active host healing response, in a toxin concentration‐dependent manner. To investigate the signal pathways regulating apoptosis and the proliferation of the intoxicated epithelial layer, the expression of several genes was further examined by quantitative real‐time PCR. Bm‐Apaf1 was the most upregulated gene postintoxication.

Localization of the serine protease homolog BmSPH-1 in nodules of E. coli-injected Bombyx mori larvae and functional analysis of its role in nodule melanization.

The molecular mechanisms underlying nodule formation and melanization, an important pathogen defense mechanism in insects, are poorly understood. In this study, we investigated the role of BmSPH-1, a catalytically inactive Bombyx mori serine protease homolog, in nodule melanization induced by injection of Escherichia coli cells into the B. mori larval hemocoel. Addition of the melanization substrate L-3,4-dihydroxyphenylalanine (DOPA) to newly formed nodules prompted nodule melanization, confirming that nodules contain activated prophenoloxidase needed for melanization. Immunoprecipitation and immunoblot studies demonstrated that BmSPH-1 interacts with BmLBP, a C-type lectin that binds Gram-negative bacteria, and that BmSPH-1 is present in a truncated, putatively activated form at the E. coli cell surface in nodules. Pretreatment of larvae with anti-BmSPH-1 serum inhibited nodule melanization in E. coli-injected larvae. These results suggest that BmSPH-1 regulates nodule melanization and is recruited into nodules from the hemolymph by BmLBP.

Ligand carrier protein genes expressed in larval chemosensory organs of Bombyx mori.

Expressed sequence tags (ESTs) of the maxillary galea of the silkworm were analyzed to identify proteins involved in food selection systems. From the 1251 redundant genes of the ESTs, we identified 7 odorant-binding protein-like genes (bmObpL), 6 takeout-like genes (bmToL), and 6 chemosensory protein genes (bmCsp). Quantitative RT-PCR analysis indicated that bmObpL1, bmObpL2, bmObpL3, bmObpL5, bmToL1, bmToL3, and bmorCsp15 were predominantly expressed in the larval oral appendages, such as the maxilla, labrum, labium and antenna. Immunocytochemical analysis indicated that the proteins of bmObpL1, bmObpL3, and bmToL1 were localized in the gustatory chemosensilla on the maxillary galea and olfactory sensilla in the antenna. The proteins encoded by bmObpL1 and bmObpL3 were detected in the gustatory chemosensilla of the epipharynx. However, bmObpL1 and bmToL1 were also detected in tactile hairs and in the epidermis of several chemosensory organs. The bmObpL2 protein was localized inside and in the epidermis around the chemosensilla, tactile hairs, and wide surface of the epipharynx. From these results, bmObpL3 is the most likely to have a dedicated role in chemoreception in the silkworm, Bombyx mori.

Immunohistochemical analysis of the role of hemocytin in nodule formation in the larvae of the silkworm, Bombyx mori.

Abstract Hemocytin, a multidomain protein from Bombyx mori L. (Lepidoptera: Bombycidae), is an ortholog of von Willebrand factor and is expected to be a major mediator of hemocyte aggregation. Antiserum was generated against hemocytin, and immune staining of hemocytes, hemolymph, and nodules was performed. Hemocytin was observed in steady-state hemocytes but not in plasma, even after bacterial injection. When hemolymph was smeared on glass slides, hemocytin-containing fibrous structures formed a cellular network mainly consisting of granulocytes and oenocytoids. Hemocytin was stained only in the granules of the granulocytes. When nodule-like aggregates formed 30 sec after bacterial injection, both granulocytes and bacterial cells were observed binding to hemocytin-containing fibrous structures. When nodule sections were stained with antiserum, hemocytin was seen in the matrix of the nodules surrounding the hemocytes. These data suggest that hemocytin plays a major role in nodule formation as a component of the sticky fibrous structure exocytosed from granulocytes.

Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.