Processing of Cry1Ab delta-endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: role in protoxin activation and toxin inactivation

Activation of Cry protoxins is carried out by midgut proteases. This process is important for toxicity and in some cases for specificity. Commercial proteases have been used for in vitro protoxin activation. In the case of Cry1A protoxins, trypsin digestion generates a toxic fragment of 60–65 kDa. Here, we have analyzed the in vitro and in vivo activation of Cry1Ab. We found differences in the processing of Cry1Ab protoxin by Manduca sexta and Spodoptera frugiperda midgut proteases as compared to trypsin. Midgut juice proteases produced two additional nicks at the N-terminal end removing helices 1 and 2a to produce a 58 kDa protein. A further cleavage within domain II splits the toxin into two fragments of 30 kDa. The resulting fragments were not separated, but instead coeluted with the 58 kDa monomer, in size-exclusion chromatography. To examine if this processing was involved in the activation or degradation of Cry1Ab toxin, binding, pore formation, and toxicity assays were performed. Pore formation assays showed that midgut juice treatment produced a more active toxin than trypsin treatment. In addition, it was determined that the 1 helix is dispensable for Cry1Ab activity. In contrast, the appearance of the 30 kDa fragments correlates with a decrease in pore formation and insecticidal activities. Our results suggest that the cleavage in domain II may be involved in toxin inactivation, and that the 30 kDa fragments are stable intermediates in the degradation pathway.     

Characterization and comparison of midgut proteases of Bacillus thuringiensis susceptible and resistant diamondback moth (Plutellidae: Lepidoptera)

The midgut proteases of the Bacillus thuringiensis resistant and susceptible populations of the diamondback moth, Plutella xylostella L. were characterized by using protease specific substrates and inhibitors. The midgut contained trypsin-like proteases of molecular weights of 97, 32, 29.5, 27.5, and 25 kDa. Of these five proteases, 29.5 kDa trypsin-like protease was the most predominant in activation of protoxins of Cry1Aa and Cry1Ab. The activation of Cry1Ab protoxin by midgut protease was fast (T(1/2) of 23-24 min) even at a protoxin:protease ratio of 250:1. The protoxin activation appeared to be multi-step process, and at least seven intermediates were observed before formation of a stable toxin of about 57.4 kDa from protoxin of about 133 kDa. Activation of Cry1Aa was faster than that of Cry1Ab on incubation of protoxins with midgut proteases and bovine trypsin. The protoxin and toxin forms of Cry proteins did not differ in toxicity towards larvae of P. xylostella. The differences in susceptibility of two populations to B. thuringiensis Cry1Ab were not due to midgut proteolytic activity. Further, the proteolytic patterns of Cry1A protoxins were similar in the resistant as well as susceptible populations of P. xylostella.     

Cadherin-like receptor binding facilitates proteolytic cleavage of helix alpha-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin

Cry toxins form lytic pores in the insect midgut cells. The role of receptor interaction in the process of protoxin activation was analyzed. Incubation of Cry1Ab protoxin with a single chain antibody that mimics the cadherin-like receptor and treatment with Manduca sexta midgut juice or trypsin, resulted in toxin preparations with high pore-forming activity in vitro. This activity correlates with the formation of a 250 kDa oligomer that lacks the helix alpha-1 of domain I. The oligomer, in contrast with the 60 kDa monomer, was capable of membrane insertion as judged by 8-anilino-1-naphthalenesulfonate binding. Cry1Ab protoxin was also activated to a 250 kDa oligomer by incubation with brush border membrane vesicles, presumably by the action of a membrane-associated protease. Finally, a model where receptor binding allows the efficient cleavage of alpha-1 and formation of a pre-pore oligomeric structure that is efficient in pore formation, is presented.     

Activation pattern and toxicity of the Cry11Bb1 toxin of Bacillus thuringiensis subsp. medellin

Bacillus thuringiensis protoxins undergo proteolytic processing in the midgut of susceptible insects to become active. The ability to process the Cry11Bb1 protoxin by trypsin and Culex quinquefasciatus larval gut extracts was tested. The protease activity indicated by the appearance of proteolytic products increased with an increment in pH, with the highest activity being observed at pH 10.6. A time course study showed the proteolysis of the 94-kDa Cry11Bb protein ending with the production of fragments of relative molecular mass of 30 and 35 kDa within 5 min. In vitro, gut proteases extract cleaved the solubilized toxin between Ser59 and Ile60 and between Ala395 and Asn396, generating a 30-kDa N-terminal and a 35-kDa C-terminal fragment, respectively. Similarly, mosquito larvae processed in vivo the parasporal inclusions, generating the same fragments as those observed in vitro. The Cry11Bb1 protoxin activated with trypsin or gut proteases showed larvicidal activity against C. quinquefasciatus first instar larvae. The data suggest that gut proteases participate in the activation of CryllBbl protoxin, generating at least two different fragments on which the activity could reside.     

Proteolytic processing of native Cry1Ab toxin by midgut extracts and purified trypsins from the Mediterranean corn borer Sesamia nonagrioides

The proteolytic processing of native Cry1Ab toxin by midgut extracts from the Mediterranean corn borer, Sesamia nonagrioides, takes place in successive steps. Several cuts occur until a 74 kDa protein is obtained; this is further digested to give rise to an active form of 69 kDa, which can be again processed to fragments of 67, 66 and 43 kDa. We have shown that three different trypsins (TI, TIIA and TIII) purified from the S. nonagrioides midgut were able to digest Cry1Ab protoxin to obtain the active form of 69 kDa. Interestingly, TI and TIII further hydrolyzed the 69 kDa protein to a fragment of slightly lower molecular mass (67 kDa), while TIIA was able to continue digestion to give fragments of 46 and 43 kDa. These results contrast with those obtained using bovine trypsin, in which the main product of Cry1Ab digestion is a 69 kDa protein. The digestion of the toxin with a "non-trypsin" fraction from S. nonagrioides midgut lumen, mostly containing chymotrypsins and elastases and free of trypsin-like activity, resulted in a different processing pattern, yielding fragments of 79, 77, 71, 69 and 51 kDa. Our results indicate that trypsins and other proteases are involved in the first steps of protoxin processing, but trypsins play the most important role in obtaining the 74 and 69 kDa proteins. All the digestion products, including the proteins of 46 and 43 kDa obtained from the digestion of Cry1Ab by TIIA, were toxic to neonate larvae, indicating that none of the tested proteases contribute to toxin degradation in a significant manner.     

棉铃虫和甜菜夜蛾中肠丝氨酸蛋白酶活性测定以及活化、降解Cry1Ac毒素分析

昆虫中肠对Bt原毒素活化与对活化毒素降解的变化被认为是害虫对Bt产生的机制之一,研究比较棉铃虫Helicoverpa armigera(Hübner)与甜菜夜蛾Spodoptera exigua(Hübner)的中肠液、BBMV蛋白酶的活性,通过SDS-PAGE分析2种昆虫对原毒素的活化速度与对活化毒素的降解速度。2种昆虫的中肠液蛋白酶活性均显著高于BBMV蛋白酶活性,中肠液与BBMV均能迅速活化原毒素并继续降解活化后的毒素,与中肠液相比,BBMV对原毒素的活化与对活化毒素的降解均慢于中肠液,甜菜夜蛾对毒素的活化与降解又慢于棉铃虫。另外,还测定抑制剂对中肠液蛋白酶活性的抑制作用,结果表明,各抑制剂对棉铃虫和甜菜夜蛾相应酶活性的抑制表现出相同的趋势,TLCK对丝氨酶蛋白酶具较好的抑制作用,而PMSF对胰蛋白酶的抑制作用次之,TPCK对胰凝乳蛋白酶的抑制作用较弱。     

棉铃虫和甜菜夜蛾中肠丝氨酸蛋白酶活性测定以及活化、降解CrylAc毒素分析

昆虫中肠对Bt原毒素活化与对活化毒素降解的变化被认为是害虫对Bt产生的机制之一,研究比较棉铃虫Helicoverpa armigern（Hǔbner）与甜菜夜蛾Spodoptera exigm（Hǔbner）的中肠液、BBMV蛋白酶的活性,通过SDS-PAGE分析2种昆虫对原毒素的活化速度与对活化毒素的降解速度。2种昆虫的中肠液蛋白酶活性均显著高于BBMV蛋白酶活性,中肠液与BBMV均能迅速活化原毒素并继续降解活化后的毒素,与中肠液相比,BBMV对原毒素的活化与对活化毒素的降解均慢于中肠液,甜菜夜蛾对毒素的活化与降解又慢于棉铃虫。另外,还测定抑制剂对中肠液蛋白酶活性的抑制作用,结果表明,各抑制剂对棉铃虫和甜菜夜蛾相应酶活性的抑制表现出相同的趋势,TLCK对丝氨酶蛋白酶具较好的抑制作用,而PMSF对胰蛋白酶的抑制作用次之,TPCK对胰凝乳蛋白酶的抑制作用较弱。     

Identification of Henosepilachna vigintioctomaculata (Coleoptera: Coccinellidae) midgut putative receptor for Bacillus thuringiensis insecticidal Cry7Ab3 toxin

Bt WZ-9 strain, containing a single Cry7Ab3 toxin, had effective insecticidal activity against larvae of Henosepilachna vigintioctomaculata. By incubation with larvae midgut homogenate and trypsin in vitro, 130 kDa Cry7Ab3 protoxin was degraded into the ～75 kDa proteinase-resistant fragments. In vivo analysis, 130 kDa Cry7Ab3 protoxin was also processed into ～75 kDa fragment. Histopathological observations indicated that Cry7Ab3 ingestion by H. vigintioctomaculata larvae causes acceleration in the blebbing of the midgut epithelium cells into the gut lumen and eventual lysis of the epithelium cells resulting in larval death. A ligand blotting experiment demonstrated that Cry7Ab3 toxin bound a 220 kDa BBMV protein. This receptor protein was identified as cadherin by matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS). The cadherin protein may be the receptor of Cry7Ab3. The data obtained may contribute to a better understanding of the mechanism of Cry7Ab3 toxin against H. vigintioctomaculata larvae.     

Comparative study of Bacillus thuringiensis Cry1Ia and Cry1Aa delta-endotoxins: Activation process and toxicity against Prays oleae

Cry1Ia and Cry1Aa proteins exhibited toxicities against Prays oleae with LC₅₀ of 189 and 116 ng/cm², respectively. The ability to process Cry1Ia11 protoxin by trypsin, chymotrypsin and P. oleae larvae proteases was studied and compared to that of Cry1Aa11. After solubilization under high alkaline condition (50 mM NaOH), Cry1Aa11 was converted into a major fragment of 65 kDa, whereas Cry1Ia11 protoxin was completely degraded by P. oleae larvae proteases and trypsin and converted into a major fragment of 70 kDa by chymotrypsin. Using less proteases of P. oleae juice, the degradation of Cry1Ia11 was attenuated. When the solubilization (in 50 mM Na₂CO₃ pH 10.5 buffer) and activation were combined, Cry1Ia11 was converted into a proteolytic product of 70 kDa after 3 h of incubation with trypsin, chymotrypsin and P. oleae juice. These results suggest that the in vivo solubilization of Cry1Ia11 was assured by larval proteases after a swelling of the corresponding inclusion due to the alkalinity of the larval midgut.     

Proteolytic processing of Bacillus thuringiensis toxin Cry1Ab in rice brown planthopper, Nilaparvata lugens (Stål)

To understand the low toxicity of Cry toxins in planthoppers, proteolytic activation of Cry1Ab in Nilaparvata lugens was studied. The proteolytic processing of Cry1Ab protoxin by N. lugens midgut proteases was similar to that by trypsin activated Cry1Ab. The Cry1Ab processed with N. lugens midgut proteases was highly insecticidal against Plutella xylostella. However, Cry1Ab activated either by trypsin or the gut proteases of the brown planthopper showed low toxicity in N. lugens. Binding analysis showed that activated Cry1Ab bound to brush border membrane vesicles (BBMV) from N. lugens at a significantly lower level than to BBMV from P. xylostella.     

Effect of Insect Larval Midgut Proteases on the Activity of Bacillus thuringiensis Cry Toxins

To test the possibility that proteolytic cleavage by midgut juice enzymes could enhance or inhibit the activity of Bacillus thuringiensis insecticidal toxins, once activated, the effects of different toxins on the membrane potential of the epithelial cells of isolated Manduca sexta midguts in the presence and absence of midgut juice were measured. While midgut juice had little effect on the activity of Cry1Aa, Cry1Ac, Cry1Ca, Cry1Ea, and R233A, a mutant of Cry1Aa from which one of the four salt bridges linking domains I and II of the toxin was eliminated, it greatly increased the activity of Cry1Ab. In addition, when tested in the presence of a cocktail of protease inhibitors or when boiled, midgut juice retained almost completely its capacity to enhance Cry1Ab activity, suggesting that proteases were not responsible for the stimulation. On the other hand, in the absence of midgut juice, the cocktail of protease inhibitors also enhanced the activity of Cry1Ab, suggesting that proteolytic cleavage by membrane proteases could render the toxin less effective. The lower toxicity of R233A, despite a similar in vitro pore-forming ability, compared with Cry1Aa, cannot be accounted for by an increased susceptibility to midgut proteases. Although these assays were performed under conditions approaching those found in the larval midgut, the depolarizing activities of the toxins correlated only partially with their toxicities.     

Prey mediated effects of Bt maize on fitness and digestive physiology of the red spider mite predator Stethorus punctillum Weise (Coleoptera: Coccinellidae)

The present study investigated prey-mediated effects of two maize varieties expressing a truncated Cry1Ab, Compa CB (event Bt176) and DKC7565 (event MON810), on the biology of the ladybird Stethorus punctillum. Although immuno-assays demonstrated the presence of Cry1Ab in both prey and predator collected from commercial maize-growing fields, neither transgenic variety had any negative effects on survival of the predator, nor on the developmental time through to adulthood. Furthermore, no subsequent effects on ladybird fecundity were observed. As a prerequisite to studying the interaction of ladybird proteases with Cry1Ab, proteases were characterised using a range of natural and synthetic substrates with diagnostic inhibitors. These results demonstrated that this predator utilises both serine and cysteine proteases for digestion. In vitro studies demonstrated that T. urticae were not able to process or hydrolyze Cry1Ab, suggesting that the toxin passes through the prey to the third trophic level undegraded, thus presumably retaining its insecticidal properties. In contrast, S. punctillum was able to activate the 130 kDa protoxin into the 65 kDa fragment; a fragment of similar size was also obtained with bovine trypsin, which is known to cleave the protoxin to the active form. Thus, despite a potential hazard to the ladybird of Bt-expressing maize (since the predator was both exposed to, and able to proteolytically cleave the toxin, at least in vitro), no deleterious effects were observed.     

Effect of insect larval midgut proteases on the activity of Bacillus thuringiensis Cry toxins

To test the possibility that proteolytic cleavage by midgut juice enzymes could enhance or inhibit the activity of Bacillus thuringiensis insecticidal toxins, once activated, the effects of different toxins on the membrane potential of the epithelial cells of isolated Manduca sexta midguts in the presence and absence of midgut juice were measured. While midgut juice had little effect on the activity of Cry1Aa, Cry1Ac, Cry1Ca, Cry1Ea, and R233A, a mutant of Cry1Aa from which one of the four salt bridges linking domains I and II of the toxin was eliminated, it greatly increased the activity of Cry1Ab. In addition, when tested in the presence of a cocktail of protease inhibitors or when boiled, midgut juice retained almost completely its capacity to enhance Cry1Ab activity, suggesting that proteases were not responsible for the stimulation. On the other hand, in the absence of midgut juice, the cocktail of protease inhibitors also enhanced the activity of Cry1Ab, suggesting that proteolytic cleavage by membrane proteases could render the toxin less effective. The lower toxicity of R233A, despite a similar in vitro pore-forming ability, compared with Cry1Aa, cannot be accounted for by an increased susceptibility to midgut proteases. Although these assays were performed under conditions approaching those found in the larval midgut, the depolarizing activities of the toxins correlated only partially with their toxicities.     

Protease activities in the midgut of Western corn rootworm (Diabrotica virgifera virgifera LeConte)

The Western corn rootworm is one of the most economically important pests in corn. One possibility for controlling this pest is the cultivation of transgenic corn expressing Bacillus thuringiensis (Bt) toxins, such as Cry3A, Cry34Ab1/Cry35Ab1, and Cry3Bb1. However, widespread cultivation of the resulting Bt corn may result in the development of resistant pest populations. The Bt toxins are processed by proteases in the midgut of susceptible insects. Thus, protease activity studies were conducted using the midgut juice (pH 5.75) from third instars larvae of the susceptible Western corn rootworm. As a result, the activities of the serine endopeptidases trypsin, chymotrypsin, elastase, cathepsin G, plasmin, and thrombin; the cysteine endopeptidases cathepsin L, papain, cathepsin B, and cathepsin H; the aspartic endopeptidase pepsin; the metallo endopeptidase saccharolysin; the exopeptidase aminopeptidase, and the omegapeptidase acylaminoacylpeptidase were detected. These results are of basic interest but also lead to reference systems for the identification of protease-mediated resistance mechanisms in potentially resistant individuals.     

Production of Chymotrypsin-Resistant Bacillus thuringiensis Cry2Aa1 δ-Endotoxin by Protein Engineering

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when ¹²⁵I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.     

Production of chymotrypsin-resistant Bacillus thuringiensis Cry2Aa1 delta-endotoxin by protein engineering.

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when (125)I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.     

Resistance to Bt maize in Mythimna unipuncta (Lepidoptera: Noctuidae) is mediated by alteration in Cry1Ab protein activation

Bt maize cultivars based on the event MON810 (expressing Cry1Ab) have shown high efficacy for controlling corn borers. However, their efficiency for controlling some secondary lepidopteran pests such as Mythimna unipuncta has been questioned, raising concerns about potential outbreaks and its economic consequences. We have selected a resistant strain (MR) of M. unipuncta, which is capable of completing its life cycle on Bt maize and displays a similar performance when feeding on both Bt and non-Bt maize. The proteolytic activation of the protoxin and the binding of active toxin to brush border membrane vesicles were investigated in the resistant and a control strain. A reduction in the activity of proteolytic enzymes, which correlates with impaired capacity of midgut extracts to activate the Cry1Ab protoxin has been observed in the resistant strain. Moreover, resistance in larvae of the MR strain was reverted when treated with Cry1Ab toxin activated with midgut juice from the control strain. All these data indicate that resistance in the MR strain is mediated by alteration of toxin activation rather than to an increase in the proteolytic degradation of the protein. By contrast, binding assays performed with biotin labelled Cry1Ab suggest that binding to midgut receptors does not play a major role in the resistance to Bt maize. Our results emphasize the risk of development of resistance in field populations of M. unipuncta and the need to consider this secondary pest in ongoing resistance management programs to avoid the likely negative agronomic and environmental consequences.     

昆虫中肠液性质对苏云金芽孢杆菌伴孢晶体毒力的影响

综述了昆虫中肠液性质对苏云金芽孢杆菌Bacillus thuringiensis伴孢晶体毒力的影响。中肠液的酸碱度和蛋白酶是影响伴孢晶体溶解与原毒素活化的两大因素。中肠液的酸碱度不仅影响到伴孢晶体的溶解速度,还影响到各种蛋白酶的活性表现;而蛋白酶则直接参与了原毒素的活化,其组成与活性影响着原毒素的活化速度和杀虫专一性。因中肠液蛋白水解能力过高而导致原毒素的过度降解是某些昆虫对苏云金芽孢杆菌低度敏感的主要原因,而中肠液对原毒素活化能力的降低则与昆虫抗性的形成有关。此外,中肠液的沉淀作用及其它生理生化特性也影响着原毒素毒力的正常发挥。     

Differential Activity and Activation of Bacillus thuringiensis Insecticidal Proteins in Diamondback Moth, Plutella xylostella

Whole-crystal preparations from strains HD-1 and HD-133, activated Cry1Ab and Cry1C toxins as well as Cry1Aa, Cry1Ac, Cry1D, and Cry2Aa protoxins were tested for toxicity to 2nd-instar larvae of the diamondback moth, Plutella xylostella. Mortality data recorded after 2 and 5 days provided different results that were related to differential rates of solubilization, activation, and degradation of insecticidal crystal proteins. The two most active proteins are Cry1Ab and Cry1C, which are both present in HD-133. The Cry1Ab protoxin is activated within 2 days, whereas activation of the Cry1C protoxin occurs between 2 and 5 days. HD-133 is more active than HD-1 immediately after infection and remains toxic over 5 days owing to the sequential activation of its crystal components. Solubility properties of crystals and rates of activation of protoxins influence the overall toxicity of HD-1 and HD-133 to the diamondback moth. Received: 30 March 1999 / Accepted: 3 May 1999