Activation Of Protoxin Research Articles

Performance insights into spray-dryer microencapsulated Bacillus thuringiensis cry pesticidal proteins with gum arabic and maltodextrin for effective pest control

Bacillus thuringiensis (Bt) produces crystals composed mainly of Cry pesticidal proteins with insecticidal activity against pests but are highly susceptible to degradation by abiotic factors. In this sense, encapsulation techniques are designed to improve their performance and lifetime. However, the effects of polymeric matrix encapsulation such as gum arabic and maltodextrin by spray-dryer in the mechanisms of action of Bt kurstaki and Bt aizawai are unknown. We analyzed crystal solubilization, protoxin activation, and receptor binding after microencapsulation and compared them with commercial non-encapsulated products. Microencapsulation did not alter protein crystal solubilization, providing 130 kDa (Cry1 protoxin) and 70 kDa (Cry2 protoxin). Activation with trypsin, chymotrypsin, and larval midgut juice was analyzed, showing that this step is highly efficient, and the protoxins were cleaved producing similar ~ 55 to 65 kDa activated proteins for both formulations. Binding assays with brush border membrane vesicles of Manduca sexta and Spodoptera frugiperda larvae provided a similar binding for both formulations. LC50 bioassays showed no significant differences between treatments but the microencapsulated treatment provided higher mortality against S. frugiperda when subjected to UV radiation. Microencapsulation did not affect the mechanism of action of Cry pesticidal proteins while enhancing protection against UV radiation. These data will contribute to the development of more efficient Bt biopesticide formulations.Key points• Microencapsulation did not affect the mechanisms of action of Cry pesticidal proteins produced by Bt.• Microencapsulation provided protection against UV radiation for Bt-based biopesticides.• The study’s findings can contribute to the development of more efficient Bt biopesticide formulations.Graphical

Tissue Specific Distribution and Activation of Sapindaceae Toxins in Horses Suffering from Atypical Myopathy.

Equine atypical myopathy is caused by hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG), the known protoxins of sycamore maple (Acer pseudoplatanus). Various tissues from five atypical myopathy cases were analyzed but only HGA was found. Whether deamination of MCPrG has already occurred in the intestine as the first stage of metabolization has not been investigated. Activation of the protoxins to methylenecyclopropylacetyl (MCPA)-CoA and methylenecyclopropylformyl (MCPF)-CoA, respectively, occurred mainly in the skeletal muscles, as evidenced by very high concentrations of MCPA-carnitine and MCPF-carnitine in this tissue. Inhibition of the acyl-CoA dehydrogenases of short- and medium-chain as well as branched-chain fatty acids by the toxins led to a strong increase in the corresponding acylcarnitines, again preferentially in skeletal muscles. An accumulation of the long-chain acylcarnitines beyond the level of the control samples could not be detected in the tissues. As a high amount of HGA was always found unmetabolized in the organs, we speculate that targeting the interruption of further metabolization might be a way to stop the progression of intoxication. Inhibition of the mitochondrial branched-chain amino acid aminotransferase, i.e., the first enzyme responsible for the activation of sycamore maple protoxins, could be a therapeutic approach.

Up-regulated serpin gene involved in Cry1Ac resistance in Helicoverpa armigera

Insect resistance to Bacillus thuringiensis (Bt) is a critical limiting factor for applying the Bt crops. Some studies indicated that decreased protoxin activation because of lower enzymatic activities of trypsin and chymotrypsin and increased expression of serpin might involve in Bt resistance. Our previous study identified an endogenous serpin could inhibit the midgut proteases to activate Cry1Ac and reduce the insecticide activity to Helicoverpa armigera. We hypothesis that up-regulated serpin involve in resistance via inhibiting enzymatic activities of trypsin and chymotrypsin to decrease protoxin activation. Herein, we found the serpin-e gene relative expression in midgut was significantly higher in the LF30 resistant strain than that in the susceptible strain during all developmental stages. Importantly, RNAi-mediated silencing of serpin-e gene expression caused 4.46-fold mortality changes in LF30 strain, but the trypsin and chymotrypsin proteases activities were only changed 0.79-fold and 2.22-fold. In addition, although proteases activities were significantly lower in LF30 strain than that in the susceptible strain, the resistance ratios of LF30 to Cry1Ac protoxin and to activated Cry1Ac toxin were no difference. The results indicated serpins caused insect resistance to Cry1Ac protoxins partly through inhibiting the trypsin and chymotrypsin proteases activities, but it also existed other mechanisms in LF30.

Soybean protease inhibitors increase Bacillus thuringiensis subs. israelensis toxicity against Hypothenemus hampei

Introduction. The coffee berry borer (Hypothenemus hampei Ferrari, CBB) is one of the most devastating pests on coffee plantations around the world. Although CBB is susceptible to the effect of δ–endotoxins of Bacillus thuringiensis subs. israelensis (Bti) at laboratory level, the efficacy of this control method is poor in the field, presumably due to the inactivation by digestive proteases different to those required for protoxin activation. Objective. To study whether the addition of a soybean flour extract enriched with protease inhibitors (PI), mixed with Bti crystals and spores (Bti-sc) in an artificial diet, could improve the toxicity of Bti against CBB. Materials and methods. This study was performed in San José, Costa Rica, between 2012 and 2013. A set of adult female CBB insects was exposed to a mixture containing different concentrations of a partially purified soybean meal extract with active PI and lyophilized Bti-sc, and were tested through a bioassay in artificial diet to estimate the sub-lethal concentration (LC50). The mortality results were validated by observing the dissected midgut, whose ultrastructure was analyzed by transmission electron microscopy. Results. The soybean extracts partially degraded the Bti-sc complex, it reduced its LC50 by almost four times (from 1.135 to 0.315 µg µl-1) and enhanced CBB mortality in a concentration-dependent manner. Histological analyses of the midgut confirmed this synergistic effect, since severe epithelial damage to the intestinal epithelium of CBB exposed to Bti-sc + PI was visualized compared to Bti-sc alone. Conclusions. The combination of a soybean extract enriched in PI and Bti-sc enhanced the mortality effect over CBB, which was confirmed by the midgut collapse. Soybean flour is a cost-effective supplement that could increase Bti effectiveness against CBB and delay the appearance of biological resistance.

Proteolysis activation of Cry1Ac and Cry2Ab protoxins by larval midgut juice proteases from Helicoverpa armigera.

Proteolytic processing of Bacillus thuringiensis (Bt) Cry protoxins by insect midgut proteases is critical to their insecticidal activities against target insects. Although transgenic Bt cotton expressing Cry1Ac and Cry2Ab proteins have been widely used for control of the cotton bollworm (Helicoverpa armigera) in the field, the proteolytic cleavage sites in the two protoxins targeted by H. armigera midgut proteases are still not clear. In this study, the proteolysis of Cry1Ac and Cry2Ab protoxins by midgut juice prepared from midgut tissue of H. armigera larvae was investigated. Cleavage of Cry1Ac protoxin by midgut proteases formed a major protein fragment of ~65 kDa, and N-terminal sequencing revealed that cleavage occurred at Arg28 in the fore-end of helix α-1 in domain I of Cry1Ac. Cleavage of Cry2Ab protoxin by midgut juice proteases produced a major protein fragment of ~50 kDa, and the cleavage occurred at Arg139 between helices α-3 and α-4 in domain I of Cry2Ab. The amino acids Arg28 of Cry1Ac and Arg139 of Cry2Ab were predicted as putative trypsin cleavage sites. Bioassay data showed that the toxicities (LC50s) of Cry1Ac and Cry2Ab protoxins were equivalent to those of their respective midgut juice-activated toxins in the susceptible SCD strain of H. armigera. Identification of the exact sites of N-terminal activation of Cry1Ac and Cry2Ab protoxins will provide a basis for a better understanding of the mode of action and resistance mechanisms based on aberrant activation of these protoxins in H. armigera.

Reduced Expression of a Novel Midgut Trypsin Gene Involved in Protoxin Activation Correlates with Cry1Ac Resistance in a Laboratory-Selected Strain of Plutella xylostella (L.).

Bacillus thuringiensis (Bt) produce diverse insecticidal proteins to kill insect pests. Nevertheless, evolution of resistance to Bt toxins hampers the sustainable use of this technology. Previously, we identified down-regulation of a trypsin-like serine protease gene PxTryp_SPc1 in the midgut transcriptome and RNA-Seq data of a laboratory-selected Cry1Ac-resistant Plutella xylostella strain, SZ-R. We show here that reduced PxTryp_SPc1 expression significantly reduced caseinolytic and trypsin protease activities affecting Cry1Ac protoxin activation, thereby conferring higher resistance to Cry1Ac protoxin than activated toxin in SZ-R strain. Herein, the full-length cDNA sequence of PxTryp_SPc1 gene was cloned, and we found that it was mainly expressed in midgut tissue in all larval instars. Subsequently, we confirmed that the PxTryp_SPc1 gene was significantly decreased in SZ-R larval midgut and was further reduced when selected with high dose of Cry1Ac protoxin. Moreover, down-regulation of the PxTryp_SPc1 gene was genetically linked to resistance to Cry1Ac in the SZ-R strain. Finally, RNAi-mediated silencing of PxTryp_SPc1 gene expression decreased larval susceptibility to Cry1Ac protoxin in the susceptible DBM1Ac-S strain, supporting that low expression of PxTryp_SPc1 gene is involved in Cry1Ac resistance in P. xylostella. These findings contribute to understanding the role of midgut proteases in the mechanisms underlying insect resistance to Bt toxins.

Comprehensive analysis of Cry1Ac protoxin activation mediated by midgut proteases in susceptible and resistant Plutella xylostella (L.)

Insecticidal Cry toxins produced by Bacillus thuringiensis (Bt) have been widely used to control agricultural pests in both foliage sprays and transgenic crops. Nevertheless, rapid evolution of insect resistance to Cry toxins requires elucidation of the molecular mechanisms involved in Cry resistance. Two proposed models have been described to explain the toxicity of Cry proteins, the classic model states that Cry protoxin is activated by midgut proteases resulting in activated toxin that binds to receptors and forms a pore in the midgut cells triggering larval death, and the newly proposed dual model of the mode of action of Bt Cry toxins states that protoxin and activated toxins may have different mechanisms of action since several resistant strains to activated Cry toxins are still susceptible to the same Cry-protoxin. Protoxin activation by midgut proteases is a key step in both models. Herein, we evaluated Cry1Ac protoxin activation in a susceptible Plutella xylostella (L.) strain (DBM1Ac-S) and in the near-isogenic strain (NIL-R) with high field-evolved Cry1Ac resistance. Previous work showed that Cry1Ac resistance in NIL-R correlates with reduced binding to midgut receptors due to enhanced MAPK signaling pathway and down regulation of ABCC2 receptor. However, reduced midgut trypsin levels and altered midgut protease gene transcription were also observed in the Cry1Ac-resistant field isolated strain that is parent of the NIL-R strain. Therefore, we analyzed the midgut protease activities in both DBM1Ac-S and NIL-R strains. Detection of enzymatic activities showed that caseinolytic protease, trypsin and chymotrypsin activities were not significantly different between the susceptible and resistant strains. Furthermore, treatment with different trypsin or chymotrypsin inhibitors, such as Nα-tosyl-l-lysine chloromethyl ketone (TLCK) or Np-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not affect the susceptibility to Cry1Ac protoxin of the DBM1Ac-S and NIL-R larvae. Bioassay results indicated that the NIL-R larvae showed similar resistant levels to both Cry1Ac protoxin and trypsin-activated toxin. Taken together, our results demonstrated that high-level field-evolved Cry1Ac resistance in the NIL-R strain is independent of Cry1Ac protoxin activation and the specific protoxin mechanism of action. This discovery will strengthen our comprehensive understanding of the complex mechanistic basis of Bt resistance in different insects.

Activation of Bt Protoxin Cry1Ac in Resistant and Susceptible Cotton Bollworm.

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins.

Transcriptional profiling analysis of Spodoptera litura larvae challenged with Vip3Aa toxin and possible involvement of trypsin in the toxin activation

Vip proteins, a new group of insecticidal toxins produced by Bacillus thuringiensis, are effective against specific pests including Spodoptera litura. Here, we report construction of a transcriptome database of S. litura by de novo assembly along with detection of the transcriptional response of S. litura larvae to Vip3Aa toxin. In total, 56,498 unigenes with an N50 value of 1,853 bp were obtained. Results of transcriptome abundance showed that Vip3Aa toxin provoked a wide transcriptional response of the S. litura midgut. The differentially expressed genes were enriched for immunity-related, metabolic-related and Bt-related genes. Twenty-nine immunity-related genes, 102 metabolic-related genes and 62 Bt-related genes with differential expression were found. On the basis of transcriptional profiling analysis, we focus on the functional validation of trypsin which potentially participated in the activation of Vip3Aa protoxin. Zymogram analysis indicated that the presence of many proteases, including trypsin, in S. litura larvae midgut. Results of enzymolysis in vitro of Vip3Aa by trypsin, and bioassay and histopathology of the trypsin-digested Vip3Aa toxin showed that trypsin was possibly involved in the Vip3Aa activation. This study provides a transcriptome foundation for the identification and functional validation of the differentially expressed genes in an agricultural important pest, S. litura.

CHARACTERIZATION OF TRYPSIN-LIKE AND CHYMOTRYPSIN-LIKE SERINE PROTEASES FROM MIDGUT OF Mythimna separata Walker.

Two cDNA sequences encoding a trypsin-like and a chymotrypsin-like serine protease (MsT and MsCT, GenBank accession Nos. KP730443 and KP730444, respectively) were cloned from midgut of oriental armyworm, Mythimna separata Walker. Multiple alignments revealed that the deduced amino acid sequences of MsT and MsCT contained a serine protease catalytic motif GDSGGPL and catalytic triads (His, Asp, and Ser). Analyses of tissue and developmental expression of MsT and MsCT showed that they were mainly expressed in midguts and could be detected in first to sixth instar larvae, prepupal and pupal stages. Expressions of both MsT and MsCT were downregulated after 24 h of starvation and upregulated by subsequent insect refeeding. MsT expression in response to 20-hydroxyecdysone (20E) was dose dependent and upregulated after 24 h. However, MsCT expression in response to 20E was downregulated compared with controls. MsCT, but not MsT, transcripts were upregulated after 24 h of Cry1Ac protoxin exposure. These results suggested that MsT was most likely involved in food protein digestion and molting in M. separata whereas MsCT was most likely involved in food protein digestion and Bacillus thuringiensis (Bt) protoxin activation. RNA interference indicated that MsT and MsCT expression levels decreased 76.7 and 86.2% after treated with MsT and MsCT dsRNA, respectively. This study showed that M. separata expressed midgut proteases in line with known lepidopteran counterparts and contributed valuable sequence resource information regarding insect proteases.

Molecular characterisation of Bacillus thuringiensis strain MEB4 highly toxic to the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae).

Cry2 proteins play an essential role in current Bacillus thuringiensis (Bt) applications and in the prevention of insect resistance to Cry1A toxins. This paper reports on the screening and characterisation of novel Bt strains harbouring effective cry2A-type genes and higher insecticidal activity to Ephestia kuehniella. A total of 29 native Bt strains were screened to search for the potent strain against E. kuehniella. The plasmid pattern of the selected strains showed interesting variability. PCR-RFLP analysis of two amplified regions showed high sequence identity within the selected cry2A-type genes. SDS-PAGE and western blot analysis revealed the presence of Cry2Aa toxin only in the MEB4 and BLB240 strains. The activation of Cry2Aa protoxins by larval midgut juice, trypsin or chymotrypsin enzymes revealed significant differences in terms of proteolysis profiles. Interestingly, a 49 kDa band was detected in the proteolysis pattern of BLB240, suggesting the presence of a chymotrypsin cleavage site that might have affected its insecticidal activity. Further, bioassays demonstrated that MEB4 (103.08 ± 36 µg g(-1)) was more active than BLB240 (153.77 ± 45.65 µg g(-1)) against E. kuehniella. Based on its potent insecticidal activity, the MEB4 strain could be considered to be an effective alternative agent for the control of E. kuehniella.

Selection and characterization of Bacillus thuringiensis strains toxic against pyralid stored‐product pests

AbstractIn this study, we present the selection and the characterization of Bacillus thuringiensis strains toxic against pyralid pests of stored products. Among 201 new B. thuringiensis strains isolated from different countries, two strains (BLB249 and BLB384) showed greater toxicity than the commercialized strain B. thuringiensis kurstaki HD1 against Ephestia kuehniella larvae. Morphological, molecular and biochemical investigations revealed that these strains were similar to HD1 but presented different cry gene content. Additional bioassays revealed that only strain BLB249 displayed higher toxicity than HD1 against Plodia interpunctella larvae. The study of Cry protoxin activation by midgut proteases of E. kuehniella and P. interpunctella larvae supported that higher toxicity of BLB249 and BLB384 strains compared to HD1 was not due to differential protoxin activation. Moreover, the toxic strains produced δ‐endotoxins and spores in similar amounts to HD1. Interestingly, the δ‐endotoxin production and the yield of BUPM26 strain were 32.87% and 35.12% greater than that of HD1. The potent insecticidal activities of BLB249 and BLB384 strains and the high level of δ‐endotoxin production by BUPM26 strain make them excellent candidates for use against E. kuehniella and P. interpunctella larvae.

Cis-mediated down-regulation of a trypsin gene associated with Bt resistance in cotton bollworm.

Transgenic plants producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are useful for pest control, but their efficacy is reduced when pests evolve resistance. Here we examined the mechanism of resistance to Bt toxin Cry1Ac in the laboratory-selected LF5 strain of the cotton bollworm, Helicoverpa armigera. This strain had 110-fold resistance to Cry1Ac protoxin and 39-fold resistance to Cry1Ac activated toxin. Evaluation of five trypsin genes revealed 99% reduced transcription of one trypsin gene (HaTryR) was associated with resistance. Silencing of this gene with RNA interference in susceptible larvae increased their survival on diets containing Cry1Ac. Bioassays of progeny from crosses revealed that resistance to Cry1Ac was genetically linked with HaTryR. We identified mutations in the promoter region of HaTryR in the resistant strain. In transfected insect cell lines, transcription was lower when driven by the resistant promoter compared with the susceptible promoter, implicating cis-mediated down-regulation of HaTryR transcription as a mechanism of resistance. The results suggest that H. armigera can adapt to Bt toxin Cry1Ac by decreased expression of trypsin. Because trypsin activation of protoxin is a critical step in toxicity, transgenic plants with activated toxins rather than protoxins might increase the durability of Bt crops.

Enhancement of the T-cell armamentarium as a cell-based therapy for prostate cancer.

Prostate cancer is frequently characterized by a large inflammatory infiltrate that includes T cells. Although T cells traffic to cancer lesions in large numbers, they are unable to generate a therapeutic response because of the immunosuppressive microenvironment. Therefore, arming T cells with a cytotoxic agent that is capable of killing cancer cells independent of these immunosuppressive signals is a rational approach to enhance their potency. Essentially, the T cells would serve as a cell-based vector, or "Trojan Horse," to selectively deliver a protoxin to disseminated prostate cancer lesions. The selective delivery of a protoxin using T cells represents an ideal method to maximize their therapeutic potency through a "field effect." Because systemically infused T cells are expected to traffic to sites of inflammation other than cancer, an additional level of specificity may be needed to prevent toxicity to nontarget tissues. Toward this goal, genetic engineering can be used to make protoxin expression dependent upon T-cell recognition of the prostate-specific membrane antigen by a chimeric antigen receptor. Furthermore, selective activation of the protoxin using a tissue- or tumor-specific protease, such as PSA, can promote further specificity. Thus, T-cell potency can be enhanced by targeted protoxin secretion and greater specificity achieved using combinatorial antigen recognition and protoxin activation.

INCREASE IN LARVAL GUT PROTEOLYTIC ACTIVITIES AND Bti RESISTANCE IN THE DENGUE FEVER MOSQUITO

The bioinsecticide Bacillus thuringiensis var. israelensis (Bti) is increasingly used worldwide for mosquito control. Although no established resistance to Bti has been described in the field so far, a resistant Aedes aegypti strain (LiTOX strain) was selected in the laboratory using field-collected leaf litter containing Bti toxins. This selected strain exhibits a moderate resistance level to Bti, but a high resistance level to individual Cry toxins. As Bti contains four different toxins, generalist resistance mechanisms affecting mosquito tolerance to different toxins were expected in the resistant strain. In the present work, we show that the resistant strain exhibits an increase of various gut proteolytic activities including trypsins, leucine-aminopeptidases, and carboxypeptidase A activities. These elevated proteolytic activities resulted in a faster activation of Cry4Aa protoxins while Cry4Ba or Cry11Aa were not affected. These results suggest that changes in proteolytic activities may contribute to Bti resistance in mosquitoes together with other mechanisms.

Characterization of cDNAs Encoding Serine Proteases and Their Transcriptional Responses to Cry1Ab Protoxin in the Gut of Ostrinia nubilalis Larvae

Serine proteases, such as trypsin and chymotrypsin, are the primary digestive enzymes in lepidopteran larvae, and are also involved in Bacillus thuringiensis (Bt) protoxin activation and protoxin/toxin degradation. We isolated and sequenced 34 cDNAs putatively encoding trypsins, chymotrypsins and their homologs from the European corn borer (Ostrinia nubilalis) larval gut. Our analyses of the cDNA-deduced amino acid sequences indicated that 12 were putative trypsins, 12 were putative chymotrypsins, and the remaining 10 were trypsin and chymotrypsin homologs that lack one or more conserved residues of typical trypsins and chymotrypsins. Reverse transcription PCR analysis indicated that all genes were highly expressed in gut tissues, but one group of phylogenetically-related trypsin genes, OnTry-G2, was highly expressed in larval foregut and midgut, whereas another group, OnTry-G3, was highly expressed in the midgut and hindgut. Real-time quantitative PCR analysis indicated that several trypsin genes (OnTry5 and OnTry6) were significantly up-regulated in the gut of third-instar larvae after feeding on Cry1Ab protoxin from 2 to 24 h, whereas one trypsin (OnTry2) was down-regulated at all time points. Four chymotrypsin and chymotrypsin homolog genes (OnCTP2, OnCTP5, OnCTP12 and OnCTP13) were up-regulated at least 2-fold in the gut of the larvae after feeding on Cry1Ab protoxin for 24 h. Our data represent the first in-depth study of gut transcripts encoding expanded families of protease genes in O. nubilalis larvae and demonstrate differential expression of protease genes that may be related to Cry1Ab intoxication and/or resistance.

Cloning eleven midgut trypsin cDNAs and evaluating the interaction of proteinase inhibitors with Cry1Ac against the tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae)

Midgut trypsins are associated with Bt protoxin activation and toxin degradation. Proteinase inhibitors have potential insecticidal toxicity against a wide range of insect species. This study was conducted to evaluate the interaction of proteinase inhibitors with Bt toxin and to examine midgut trypsin gene profile of Heliothis virescens. A sublethal dose (15ppb) of Cry1Ac, 0.75% soybean trypsin inhibitor, and 0.1% and 0.2% N-α-tosyl-L-lysine chloromethyl ketone significantly suppressed midgut proteinase activities, and resulted in reductions in larval and pupal size and mass. The treatment with inhibitor+Bt suppressed approximately 65% more larval body mass and 21% more enzymatic activities than the inhibitor-only or Bt-only. Eleven trypsin-like cDNAs were sequenced from the midgut of H. virescens. All trypsins contained three catalytic center residues (H73, D153, and S231), substrate specificity determinant residues (D225, G250, and G261), and six cysteines for disulfide bridges. These putative trypsins were separated into three distinct groups, indicating the diverse proteinases evolved in this polyphagous insect. These results indicated that the insecticidal activity of proteinase inhibitors may be used to enhance Bt toxicity and delay resistance development.

RNAi‐mediated knockdown of a Spodoptera frugiperda trypsin‐like serine‐protease gene reduces susceptibility to a Bacillus thuringiensis Cry1Ca1 protoxin

SfT6 has been identified in a subtracted cDNA library of Spodoptera frugiperda larval midgut transcripts as a serine-protease gene downregulated within 24 h of intoxication with Bacillus thuringiensis Cry1Ca1 protein. In the present study, the specific role of SfT6 during Cry1Ca1 intoxication was investigated by RT-PCR and in vivo RNA interference. Quantitative real-time RT-PCR analysis showed SfT6 mRNA levels in the midgut tissue were significantly reduced after injecting or feeding 4th-instar larvae with specific long-size dsRNA. Gut juice-mediated in vitro protoxin activation and susceptibility for Cry1Ca1 were investigated in Sft6-knockdown larvae and compared with control treated with nonspecific dsRNA. Our results demonstrate SfT6 plays a determinant role in Cry1Ca1 toxicity against S. frugiperda since a decreased expression caused a reduced protoxin activation by larval gut juice and reduced susceptibility of insects to toxin in bioassays. We propose SfT6 downregulation occurring at the early stages of Cry1Ca1 intoxication is part of a complex and multifaceted defensive mechanism triggered in the insect gut to withstand B. thuringiensis pathogenesis.

Targeting the cancer stroma with a fibroblast activation protein-activated promelittin protoxin

Fibroblast-Activation Protein-α (FAP) is a membrane-bound serine protease that is expressed on the surface of reactive stromal fibroblasts present within the majority of human epithelial tumors but is not expressed by normal tissues. FAP is a postprolyl peptidase that differs from other dipeptidyl prolyl peptidases such as diprolylpeptidase 4 in that it also has gelatinase and collagenase endopeptidase activity. Therefore, FAP represents a potential pan-tumor target whose enzymatic activity can be exploited for the intratumoral activation of prodrugs and protoxins. To evaluate FAP as a tumor-specific target, putative FAP-selective peptide protoxins were constructed through modification of the prodomain of melittin, a 26 amino acid amphipathic cytolytic peptide that is the main toxic component in the venom of the common European honeybee Apis milefera. Melittin is synthesized as promelittin, containing a 22 amino acid NH(2)-terminal prodomain rich in the amino acids proline and alanine. In this study, peptides containing truncated melittin prodomain sequences were tested on erythrocytes to determine the optimal prodomain length for inhibiting cytolytic activity. Once optimized, modified promelittin peptides were generated in which previously identified FAP substrate sequences were introduced into the prodomain. Peptide protoxins were identified that were efficiently activated by FAP and selectively toxic to FAP-expressing cell lines with an IC(50) value in the low micromolar range that is similar to melittin. Intratumoral injection of an FAP-activated protoxin produced significant lysis and growth inhibition of human breast and prostate cancer xenografts with minimal toxicity to the host animal.

A new Tunisian strain of Bacillus thuringiensis kurstaki having high insecticidal activity and δ-endotoxin yield

BLB1 is a new Bacillus thuringiensis kurstaki strain, isolated from a Tunisian soil sample. Assay of toxicity of BLB1 crystal proteins resulted in an LC50 of 70.32 ng of toxin per mg of flour against third instar Ephestia kuehniella with confidence limits of (31.6-109.04 ng). This LC50 is less than that of the commercial strains HD1 used as a reference. The characterization of this strain by scanning transmission electron microscopy, analysis of its cry genes content by PCR-sequencing, and analysis of its delta-endotoxin patterns demonstrate that it belongs to the same subgroup than HD1, but ruled out the involvement of cry gene content or protoxin activation in the hypertoxicity of this strain. Taking into account the delta-endotoxin/spore ratio for each strain, and by allowing the estimation of the production level per spore, it might be concluded that BLB1 production is the highest, when compared with that of HD1. On the basis of its toxicity, BLB1 could be considered as a strain of great interest and would allow the production of quantities of bioinsecticides at low cost.