Differential proteome profiling of bacterial culture supernatants reveals candidates for the induction of oral immune priming in the red flour beetle.

Most organisms are host to symbionts and pathogens, which led to the evolution of immune strategies to prevent harm. Whilst the immune defences of vertebrates are classically divided into innate and adaptive, insects lack specialized cells involved in adaptive immunity, but have been shown to exhibit immune priming: the enhanced survival upon infection after a first exposure to the same pathogen or pathogen-derived components. An important piece of the puzzle are the pathogen-associated molecules that induce these immune priming responses. Here, we make use of the model system consisting of the red flour beetle (Tribolium castaneum) and its bacterial pathogen Bacillus thuringiensis, to compare the proteomes of culture supernatants of two closely related B. thuringiensis strains that either induce priming via the oral route, or not. Among the proteins that might be immunostimulatory to T. castaneum, we identify the Cry3Aa toxin, an important plasmid-encoded virulence factor of B. thuringiensis. In further priming-infection assays we test the relevance of Cry-carrying plasmids for immune priming. Our findings provide valuable insights for future studies to perform experiments on the mechanisms and evolution of immune priming.

Identification of two Bacillus thuringiensis Cry3Aa toxin-binding aminopeptidase N from Rhynchophorus ferrugineus (Coleoptera: Curculionidae).

Rhynchophorus ferrugineus is a quarantine pest that mainly damages plants in tropical regions, which are essential economic resources. Cry3Aa has been used to control coleopteran pests and is known to be toxic to R. ferrugineus. The binding of the Cry toxin to specific receptors on the target insect plays a crucial role in the toxicological mechanism of Cry toxins. However, in the case of R. ferrugineus, the nature and identity of the receptor proteins involved remain unknown. In the present study, pull-down assays and mass spectrometry were used to identify two proteins of aminopeptidase N proteins (RfAPN2a and RfAPN2b) in the larval midguts of R. ferrugineus. Cry3Aa was able to bind to RfAPN2a (Kd = 108.5 nM) and RfAPN2b (Kd = 68.2 nM), as well as midgut brush border membrane vesicles (Kd = 482.5 nM). In silico analysis of both RfAPN proteins included the signal peptide and anchored sites for glycosyl phosphatidyl inositol. In addition, RfAPN2a and RfAPN2b were expressed in the human embryonic kidney 293T cell line, and cytotoxicity assays showed that the transgenic cells were not susceptible to activated Cry3Aa. Our results show that RfAPN2a and RfAPN2b are Cry3Aa-binding proteins involved in the Cry3Aa toxicity of R. ferrugineus. This study deepens our understanding of the action mechanism of Cry3Aa in R. ferrugineus larvae.

Colorado potato beetle chymotrypsin genes are differentially regulated in larval midgut in response to the plant defense inducer hexanoic acid or the Bacillus thuringiensis Cry3Aa toxin.

When Colorado potato beetle larvae ingested potato plants treated with the plant defense inducer compound hexanoic acid, midgut chymotrypsin enzyme activity increased, and the corresponding chymotrypsin genes were differentially expressed, evidence of the larval digestive proteolytic system's plasticity. We previously reported increased susceptibility to Cry3Aa toxin in larvae fed hexanoic acid treated plants. Here we show that the most expressed chymotrypsin gene in larvae fed hexanoic acid treated plants, CTR6, was dramatically downregulated in Cry3Aa intoxicated larvae. lde-miR-965-5p and lde-miR-9a-5p microRNAs, predicted to target CTR6, might be involved in regulating the response to hexanoic acid but not to Cry3Aa toxin.

Validation of ADAM10 metalloprotease as a Bacillus thuringiensis Cry3Aa toxin functional receptor in Colorado potato beetle (Leptinotarsa decemlineata).

Bacillus thuringiensis parasporal crystal proteins (Cry proteins) are insecticidal pore-forming toxins that bind to specific receptor molecules on the brush border membrane of susceptible insect midgut cells to exert their toxic action. In the Colorado potato beetle (CPB), a coleopteran pest, we previously proposed that interaction of Cry3Aa toxin with a CPB ADAM10 metalloprotease is an essential part of the mode of action of this toxin. Here, we annotated the gene sequence encoding an ADAM10 metalloprotease protein (CPB-ADAM10) in the CPB genome sequencing project, and using RNA interference gene silencing we demonstrated that CPB-ADAM10 is a Cry3Aa toxin functional receptor in CPB. Cry3Aa toxicity was significantly lower in CPB-ADAM10 silenced larvae and in vitro toxin pore-forming ability was greatly diminished in lipid planar bilayers fused with CPB brush border membrane vesicles (BBMVs) prepared from CPB-ADAM10 silenced larvae. In accordance with our previous data that indicated this toxin was a substrate of ADAM10 in CPB, Cry3Aa toxin membrane-associated proteolysis was altered when CPB BBMVs lacked ADAM10. The functional validation of CPB-ADAM10 as a Cry3Aa toxin receptor in CPB expands the already recognized role of ADAM10 as a pathogenicity determinant of pore-forming toxins in humans to an invertebrate species.

A Targeted and Protease-Activated Genetically Encoded Melittin-Containing Particle for the Treatment of Cutaneous and Visceral Leishmaniasis.

Intracellular infections are difficult to treat, as pathogens can take advantage of intracellular hiding, evade the immune system, and persist and multiply in host cells. One such intracellular parasite, Leishmania, is the causative agent of leishmaniasis, a neglected tropical disease (NTD), which disproportionately affects the world's most economically disadvantaged. Existing treatments have relied mostly on chemotherapeutic compounds that are becoming increasingly ineffective due to drug resistance, while the development of new therapeutics has been challenging due to the variety of clinical manifestations caused by different Leishmania species. The antimicrobial peptide melittin has been shown to be effective in vitro against a broad spectrum of Leishmania, including species that cause the most common form, cutaneous leishmaniasis, and the most deadly, visceral leishmaniasis. However, melittin's high hemolytic and cytotoxic activity toward host cells has limited its potential for clinical translation. Herein, we report a design strategy for producing a melittin-containing antileishmanial agent that not only enhances melittin's leishmanicidal potency but also abrogates its hemolytic and cytotoxic activity. This therapeutic construct can be directly produced in bacteria, significantly reducing its production cost critical for a NTD therapeutic. The designed melittin-containing fusion crystal incorporates a bioresponsive cathepsin linker that enables it to specifically release melittin in the phagolysosome of infected macrophages. Significantly, this targeted approach has been demonstrated to be efficacious in treating macrophages infected with L. amazonensis and L. donovani in cell-based models and in the corresponding cutaneous and visceral mouse models.

Prohibitin, an essential protein for Colorado potato beetle larval viability, is relevant to Bacillus thuringiensis Cry3Aa toxicity.

Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highly conserved family of proteins implicated in important cellular processes. We obtained the complete CPB prohibitin-1 DNA coding sequence of 828pb, in silico translated into a 276-amino acid protein. The analysis at the amino acid level showed that the protein contains a prohibitin-homology domain (Band7_prohibitin, cd03401) conserved among prohibitin proteins. A striking feature of the CPB identified prohibitin-1 is the predicted presence of cadherin elements, potential binding sites for Cry toxins described in other Bt susceptible insects. We also showed that CPB prohibitin-1 protein partitioned into both, detergent soluble and insoluble membrane fractions, as well as a prohibitin-2 homologous protein, previously reported to form functional complexes with prohibitin-1 in other organisms. Prohibitin complexes act as membrane scaffolds ensuring the recruitment of membrane proteases to facilitate substrate processing. Accordingly, sequestration of prohibitin-1 by an anti-prohibitin-1 antibody impaired the Cry3Aa toxin inhibition of the proteolytic cleavage of a fluorogenic synthetic substrate of an ADAM-like metalloprotease previously reported to proteolize this toxin. In this work, we also demonstrated that prohibitin-1 RNAi silencing in CPB larvae produced deleterious effects and together with a LD50 Cry3Aa toxin treatment resulted in a highly efficient short term response since 100% larval mortality was achieved just 5days after toxin challenge. Therefore, the combination of prohibitin RNAi and Cry toxin reveals as an effective strategy to improve crop protection.

Elucidating the genomic history of commercially used Bacillus thuringiensis subsp. tenebrionis strain NB176.

Bacillus thuringiensis subsp. tenebrionis (Btt) produces a coleopteran-specific crystal protoxin protein (Cry3Aa δ-endotoxin). After its discovery in 1982, the strain NB125 (DSM 5526) was eventually registered in 1990 to control the Colorado potato beetle (Leptinotarsa decemlineata). Gamma-irradiation of NB125 resulted in strain NB176-1 (DSM 5480) that exhibited higher cry3Aa production and became the active ingredient of the plant protection product Novodor® FC. Here, we report a comparative genome analysis of the parental strain NB125, its derivative NB176-1 and the current commercial production strain NB176. The entire genome sequences of the parental and derivative strains were deciphered by a hybrid de novo approach using short (Illumina) and long (Nanopore) read sequencing techniques. Genome assembly revealed a chromosome of 5.4 to 5.6 Mbp and six plasmids with a size range from 14.9 to 250.5 kbp for each strain. The major differences among the original NB125 and the derivative strains NB176-1 and NB176 were an additional copy of the cry3Aa gene, which translocated to another plasmid as well as a chromosomal deletion (~ 178 kbp) in NB176. The assembled genome sequences were further analyzed in silico for the presence of virulence and antimicrobial resistance (AMR) genes.

Cry3Aa Toxin Is Not Suitable to Control Lepidopteran Pest Spodoptera littoralis (Boisd.).

The toxicity of the Bacillus thuringiensis (Bt) toxin Cry3Aa-originally used against the main potato pest, the Colorado potato beetle, Leptinotarsa decemlineata-was verified on this species and then evaluated against the Egyptian armyworm, Spodoptera littoralis, which is a pest of several economically important plants. Larvae of S. littoralis were fed a semi-artificial diet supplemented either with a recombinant or with a natural Bt toxin Cry3Aa and with the genetically engineered (GE) potato of variety Superior NewLeaf (SNL) expressing Cry3Aa. Cry3Aa concentration in the diet and the content in the leaves were verified via ELISA (enzyme-linked immunosorbent assay) and HPLC (high-performance liquid chromatography) during and at the end of the experiments. The biological effectiveness of the coleopteran-specific Cry3Aa with previous reports of activity against S. littoralis was tested on five different populations of S. littoralis larvae by monitoring 13 parameters involving development from penultimate instar, weight, the efficiency of food conversion to biomass, ability to reproduce, and mortality. Although some occasional differences occurred between the Cry3Aa treatments and control, any key deleterious effects on S. littoralis in this study were not confirmed. We concluded that the Cry3Aa toxin appears to be non-toxic to S. littoralis, and its practical application against this pest is unsuitable.

Proteolytic Activation of Bacillus thuringiensis Cry3Aa Toxin in the Red Palm Weevil (Coleoptera: Curculionidae).

The red palm weevil (RPW), Rhynchophorus ferrugineus (Oliver) is an important pest of palms that causes significant damage by boring into and feeding within palm stem tissues. Here, we studied the proteolytic process of Cry3Aa in the RPW to understand the mechanism of Cry toxicity. The bioassays showed that Cry3Aa toxin is weakly toxic to the RPW. Proteolytic activation assays indicated the Cry3Aa protein is digested into smaller fragments than the 55-kDa activated fragments under different conditions. In particular, at higher mass ratios of gut protease and Cry3Aa protein (5:1, 2:1, and 1:1, respectively), and at 36.9°C for 16 h in a solution of pH 8.6, the Cry3Aa protoxin is over-digested by the gut proteases of weevil larvae. Moreover, the zymogram analysis of the gut proteases revealed the RPW larvae harbors intestinal digestive enzymes mainly composed of serine proteases. This study describes the proteolytic activation process of Cry3Aa in the midgut of RPW larvae.

Identification and Characterization of Aminopeptidase-N as a Binding Protein for Cry3Aa in the Midgut of Monochamus alternatus (Coleoptera: Cerambycidae).

Bacillus thuringiensis Cry proteins have been widely used over the past decades for many different insect pests, which are safe for users and the environment. The coleopteran-specific Cry3Aa toxin from B. thuringiensis exhibits toxicity to the larvae of Monochamus alternatus. Receptors play a key role in the mechanisms underlying the toxic action of Cry. However, the binding receptor for Cry3Aa has yet to be identified in the midgut of M. alternatus larvae. Therefore, the aim of this study was to identify the receptor for Cry3Aa toxin in the brush border membrane vesicles (BBMVs) of M. alternatus larvae. Our results indicate that the Cry3Aa toxin binds to the BBMVs (Kd = 247 nM) of M. alternatus via a 107 kDa aminopeptidase N (APN) (Kd = 57 nM). In silico analysis of the APN protein predicted that an 18 amino acid sequence in the N-terminal acted as a signal peptide, and that the Asn residue, located at position 918 in the C-terminus is an anchored site for glycosyl phosphatidyl inositol. Further analysis showed that M. alternatus APN exhibits 75% homology to the APN from Anoplophora glabripenis. Our work, therefore, confirmed that APN, which is localized in the BBMVs in the midgut of M. alternatus larvae, acts as a binding protein for Cry3Aa toxins.

Engineering of multiple trypsin/chymotrypsin sites in Cry3A to enhance its activity against Monochamus alternatus Hope larvae.

BACKGROUND: Bacillus thuringiensis Cry3 toxins exhibit specific toxicity against several coleopteran larvae. However, owing to its low toxicity to Monochamus alternatus, Cry3A toxin is not useful for managing M. alternatus larvae. Here we assessed the proteolytic activation of Cry3Aa toxin in M. alternatus larval midgut and increased its toxicity by molecular modification. RESULTS: Our results indicated that insufficient processing of Cry3Aa protoxin and non-specific enzymatic digestion of Cry3Aa toxin in the midgut of M. alternatus larvae led to low toxicity. The results of transcriptome analysis, enzymatic assay with fluorogenic substrates, and multiplex substrate profiling by mass spectrometry showed that the main digestive enzymes in M. alternatus larval midgut were trypsin-like proteases that preferentially cleaved peptides with arginine and lysine residues. Consequently, trypsin recognition sites were introduced into the Domain I of Cry3Aa protoxin in the loop regions between α-helix 3 and α-helix 4 to facilitate proteolytic activation. Multiple potential trypsin cleavage sites away from the helix sheet and functional regions in Cry3Aa proteins were also mutated to alanine to prevent non-specific enzymatic digestion. Bioassays indicated that a modified Cry3Aa-T toxin (K65A, K70A, K231A, K468A, and K596A) showed a 9.5-fold (LC50 = 12.3 µg/mL) increase in toxicity to M. alternatus larvae when compared to native Cry3Aa toxin. CONCLUSION: This study highlights an effective way to increase the toxicity of Cry3Aa toxin to M. alternatus, which may be suitable for managing the resistance of transgenic plants to other pests, including some of the most important pests in agriculture. © 2020 Society of Chemical Industry.

Proteomic insights into the immune response of the Colorado potato beetle larvae challenged with Bacillus thuringiensis.

Bacillus thuringiensis (Bt) toxins constitute effective, environmentally safe biopesticides. Nevertheless, insects' tolerance to Bt is influenced by environmental factors affecting immunity. To understand larval immune response in the devastating coleopteran insect pest Colorado potato beetle (CPB), we undertook a proteomic analysis of hemolymph of non-treated control larvae and larvae consuming non-lethal doses of spore-crystal mixtures containing the coleopteran-active Cry3Aa toxin. Results revealed lower amount of proteins involved in insect growth and higher amount of immune response-related proteins in challenged insects, sustaining the larval weight loss observed. Additionally, we found a potential regulatory role of the evolutionary conserved miR-8 in the insect's immune response relying on antimicrobial peptides (AMPs) production. Upon toxin challenge, different patterns of hemolymph AMPs expression and phenoloxidase activity were observed in CPB larvae reared on different Solanaceae plants. This suggests that diet and diet-associated insect midgut microbiota might modulate this insects' tolerance to non-lethal doses of Bt.

Directed evolution of a genetically encoded immobilized lipase for the efficient production of biodiesel from waste cooking oil.

BACKGROUND: We have recently developed a one-step, genetically encoded immobilization approach based on fusion of a target enzyme to the self-crystallizing protein Cry3Aa, followed by direct production and isolation of the fusion crystals from Bacillus thuringiensis. Using this approach, Bacillus subtilis lipase A was genetically fused to Cry3Aa to produce a Cry3Aa-lipA catalyst capable of the facile conversion of coconut oil into biodiesel over 10 reaction cycles. Here, we investigate the fusion of another lipase to Cry3Aa with the goal of producing a catalyst suitable for the conversion of waste cooking oil into biodiesel. RESULTS: Genetic fusion of the Proteus mirabilis lipase (PML) to Cry3Aa allowed for the production of immobilized lipase crystals (Cry3Aa-PML) directly in bacterial cells. The fusion resulted in the loss of PML activity, however, and so taking advantage of its genetically encoded immobilization, directed evolution was performed on Cry3Aa-PML directly in its immobilized state in vivo. This novel strategy allowed for the selection of an immobilized PML mutant with 4.3-fold higher catalytic efficiency and improved stability. The resulting improved Cry3Aa-PML catalyst could be used to catalyze the conversion of waste cooking oil into biodiesel for at least 15 cycles with minimal loss in conversion efficiency. CONCLUSIONS: The genetically encoded nature of our Cry3Aa-fusion immobilization platform makes it possible to perform both directed evolution and screening of immobilized enzymes directly in vivo. This work is the first example of the use of directed evolution to optimize an enzyme in its immobilized state allowing for identification of a mutant that would unlikely have been identified from screening of its soluble form. We demonstrate that the resulting Cry3Aa-PML catalyst is suitable for the recyclable conversion of waste cooking oil into biodiesel.

Targeted delivery of antimicrobial peptide by Cry protein crystal to treat intramacrophage infection.

Antimicrobial peptides (AMPs) have recently attracted great attention due to their rapid action, broad spectrum of activity, and low propensity of resistance development. The successful application of AMPs in the treatment of intracellular infections, however, remains a challenge because of their low penetration efficiency into the pathogen's intracellular niche. Herein, we report that sub-micrometer-sized crystals of the protein Cry3Aa formed within Bacillus thuringiensis are readily and specifically taken up by macrophages. We demonstrate that these protein crystals efficiently encapsulate a known antileishmanial peptide, dermaseptin S1 (DS1), and thereby promote improved cellular uptake of DS1 and its lysosomal accumulation in macrophages. Notably, this targeted delivery of DS1 results in enhanced in vitro and in vivo antileishmanial activity, as well as reduced toxicity to the host macrophages. These findings suggest that the Cry3Aa crystal can be an effective delivery platform for AMPs to treat intramacrophage infections.

Saturation mutagenesis and self-inducible expression of trehalose synthase in Bacillus subtilis.

Trehalose is a nonreducing disaccharide synthesized by trehalose synthase (TreS), which catalyzes the reversible interconversion of maltose and trehalose. We aimed to enhance the catalytic conversion of maltose to trehalose by saturation mutagenesis, and constructed a self-inducible TreS expression system by generating a robust Bacillus subtilis recombinant. We found that the conversion yield and enzymatic activity of TreS was enhanced by saturation mutations, especially by the combination of V407M and K490L mutations. At the same time, these saturation mutations were contributing to reducing by-products in the reaction. Compared to WT TreS, the conversion yield of maltose to trehalose was increased by 11.9%, and the kcat /Km toward trehalose was 1.33 times higher in the reaction catalyzed by treSV407M-K490L . treSV407M-K490L expression was further observed in the recombinant B. subtilis W800N(ΔσF ) under the influence of PsrfA , Pcry3Aa , and PsrfA-cry3Aa promoters without an inducer. It was shown that PsrfA-cry3Aa was evidently a stronger promoter for treSV407M-K490L expression, with the intracellular enzymatic activity of recombinant treSV407M-K490L being over 5,800 U/g at 35 hr in TB medium. These results suggested the combination of two mutations, V407M and K490L, was conducive for the production of trehalose. In addition, the self-inducible TreSV407M/K490L mutant in the B. subtilis host provides a low-cost choice for the industrial production of endotoxin-free trehalose with high yields.

A P-Glycoprotein Is Linked to Resistance to the Bacillus thuringiensis Cry3Aa Toxin in a Leaf Beetle.

Chrysomela tremula is a polyvoltine oligophagous leaf beetle responsible for massive attacks on poplar trees. This beetle is an important model for understanding mechanisms of resistance to Bacillus thuringiensis (Bt) insecticidal toxins, because a resistant C. tremula strain has been found that can survive and reproduce on transgenic poplar trees expressing high levels of the Cry3Aa Bt toxin. Resistance to Cry3Aa in this strain is recessive and is controlled by a single autosomal locus. We used a larval midgut transcriptome for C. tremula to search for candidate resistance genes. We discovered a mutation in an ABC protein, member of the B subfamily homologous to P-glycoprotein, which is genetically linked to Cry3Aa resistance in C. tremula. Cultured insect cells heterologously expressing this ABC protein swell and lyse when incubated with Cry3Aa toxin. In light of previous findings in Lepidoptera implicating A subfamily ABC proteins as receptors for Cry2A toxins and C subfamily proteins as receptors for Cry1A and Cry1C toxins, this result suggests that ABC proteins may be targets of insecticidal three-domain Bt toxins in Coleoptera as well.

RNAi induced knockdown of a cadherin-like protein (EF531715) does not affect toxicity of Cry34/35Ab1 or Cry3Aa to Diabrotica virgifera virgifera larvae (Coleoptera: Chrysomelidae).

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an important maize pest throughout most of the U.S. Corn Belt. Bacillus thuringiensis (Bt) insecticidal proteins including modified Cry3Aa and Cry34/35Ab1 have been expressed in transgenic maize to protect against WCR feeding damage. To date, there is limited information regarding the WCR midgut target sites for these proteins. In this study, we examined whether a cadherin-like gene from Diabrotica virgifera virgifera (DvvCad; GenBank accession # EF531715) associated with WCR larval midgut tissue is necessary for Cry3Aa or Cry34/35Ab1 toxicity. Experiments were designed to examine the sensitivity of WCR to trypsin activated Cry3Aa and Cry34/35Ab1 after oral feeding of the DvvCad dsRNA to knockdown gene expression. Quantitative real-time PCR confirmed that DvvCad mRNA transcript levels were reduced in larvae treated with cadherin dsRNA. Relative cadherin expression by immunoblot analysis and nano-liquid chromatography - mass spectrometry (nanoLC-MS) of WCR neonate brush border membrane vesicle (BBMV) preparations exposed to DvvCad dsRNA confirmed reduced cadherin expression when compared to BBMV from untreated larvae. However, the larval mortality and growth inhibition of WCR neonates exposed to cadherin dsRNA for two days followed by feeding exposure to either Cry3Aa or Cry34/35Ab1 for four days was not significantly different to that observed in insects exposed to either Cry3Aa or Cry34/35Ab1 alone. In combination, these results suggest that cadherin is unlikely to be involved in the toxicity of Cry3Aa or Cry34/35Ab1 to WCR.

Identification of Bacillus thuringiensis Cry3Aa toxin domain II loop 1 as the binding site of Tenebrio molitor cadherin repeat CR12.

Bacillus thuringiensis Cry toxins exert their toxic effect by specific recognition of larval midgut proteins leading to oligomerization of the toxin, membrane insertion and pore formation. The exposed domain II loop regions of Cry toxins have been shown to be involved in receptor binding. Insect cadherins have shown to be functionally involved in toxin binding facilitating toxin oligomerization. Here, we isolated a VHH (VHHA5) antibody by phage display that binds Cry3Aa loop 1 and competed with the binding of Cry3Aa to Tenebrio molitor brush border membranes. VHHA5 also competed with the binding of Cry3Aa to a cadherin fragment (CR12) that was previously shown to be involved in binding and toxicity of Cry3Aa, indicating that Cry3Aa binds CR12 through domain II loop 1. Moreover, we show that a loop 1 mutant, previously characterized to have increased toxicity to T. molitor, displayed a correlative enhanced binding affinity to T. molitor CR12 and to VHHA5. These results show that Cry3Aa domain II loop 1 is a binding site of CR12 T. molitor cadherin.

Presence and significance of Bacillus thuringiensis Cry proteins associated with the Andean weevil Premnotrypes vorax (Coleoptera: Curculionidae)

The Andean weevil Premnotrypes vorax represents an important cause of damage to Colombian potato crops. Due to the impact of this plague on the economy of the country, we searched for new alternatives for its biological control, based on the entomopathogenic bacteria Bacillus thuringiensis. A total of 300 B. thuringiensis strains obtained from potato plantations infested with P. vorax were analyzed through crystal morphology, SDS-PAGE, PCR and bioassays. We used site- directed mutagenesis to modify the Cry3Aa protein. Most of the B. thuringiensis isolates had a bipyramidal crystal morphology. SDS-PAGE analyses had seven strains groups with σ-endotoxins from 35 to 135 kDa. The genes cry 2 and cry 1 were significantly more frequent in the P. vorax habitat (PCR analyses). Three mutant toxins, 1 (D354E), 2 (R345A, ∆Y350, ∆Y351), and 3 (Q482A, S484A, R485A), were analyzed to assess their activity against P. vorax larvae. Toxicity was low, or absent, against P. vorax for isolates, wild type cry 3Aa and cry 3Aa mutants. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against other insect pests of agricultural importance, and for designing Cry proteins with improved insecticidal toxicity. Rev. Biol. Trop. 57 (4): 1235-1243. Epub 2009 December 01.

El gorgojo andino Premnotrypes vorax es una causa importante de daño en los cultivos colombianos de este tubérculo. Debido al impacto que esta plaga tiene sobre la economía del país, nos interesamos en buscar alternativas nuevas para el control biológico de P. vorax, basadas en la bacteria entomopatógena Bacillus thuringiensis. Se recolectaron un total de 300 cepas de B. thuringiensis a partir de plantaciones de papa infestadas con P. vorax, las cuales fueron analizadas por medio de la morfología del cristal, SDS-PAGE, PCR y ensayos biológicos. La mayoría de los aislamientos de B. thuringiensis presentaron cristales bipiramidales. Los análisis de SDS-PAGE indicaron la presencia de siete grupos de cepas con σ- endotoxinas que variaban entre 35 a 135 kDa. Las pruebas con PCR demostraron que los genes cry 2 y cry 1 fueron significativamente más frecuentes en el medioambiente de P. vorax. Además, se utilizó la mutagénesis sitio-dirigida para modificar la proteína Cry3Aa. Se analizaron tres toxinas mutantes, 1 (D354E), 2 (R345A, ∆Y350, ∆Y351), y 3 (Q482A, S484A, R485A), para determinar su actividad contra larvas de P. vorax. Los ensayos de toxicidad señalaron escasa, o nula, actividad hacia P. vorax tanto para las cepas, la toxina Cry3Aa de referencia y las proteínas Cry3Aa mutantes. La caracterización genética de la colección puede proveer oportunidades para la selección de cepas que pueden evaluarse por medio de bioensayos contra otros insectos-plaga de importancia agrícola, y para el diseño de proteínas Cry con actividad toxica mejorada.