Insect chaperones Hsp70 and Hsp90 cooperatively enhance toxicity of Bacillus thuringiensis Cry1A toxins and counteract insect resistance.

Bacillus thuringiensis (Bt) produces different insecticidal proteins effective for pest control. Among them, Cry insecticidal proteins have been used in transgenic plants for the control of insect pests. However, evolution of resistance by insects endangers this technology. Previous work showed that the lepidopteran insect Plutella xylostella PxHsp90 chaperone enhanced the toxicity of Bt Cry1A protoxins by protecting them from degradation by the larval gut proteases and by enhancing binding of the protoxin to its receptors present in larval midgut cells. In this work, we show that PxHsp70 chaperone also protects Cry1Ab protoxin from gut proteases degradation, enhancing Cry1Ab toxicity. We also show that both PxHsp70 and PxHsp90 chaperones act cooperatively, increasing toxicity and the binding of Cry1Ab439D mutant, affected in binding to midgut receptors, to cadherin receptor. Also, insect chaperones recovered toxicity of Cry1Ac protein to a Cry1Ac-highly resistant P. xylostella population, NO-QAGE, that has a disruptive mutation in an ABCC2 transporter linked to Cry1Ac resistance. These data show that Bt hijacked an important cellular function for enhancing its infection capability, making use of insect cellular chaperones for enhancing Cry toxicity and for lowering the evolution of insect resistance to these toxins.

Toxic Determination of Cry11 Mutated Proteins Obtained Using Rational Design and Its Computational Analysis.

Cry11 proteins are toxic to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. Cry11Aa and Cry11Bb are protoxins, which when activated present their active-toxin form in two fragments between 30 and 35 kDa respectively. Previous studies conducted with Cry11Aa and Cry11Bb genes using DNA shuffling generated variant 8, which presented a deletion in the first 73 amino acids and one at position 572 and 9 substitutions including L553F and L556W. In this study, variant 8 mutants were constructed using site-directed mutagenesis, resulting in conversion of phenylalanine (F) and tryptophan (W) to leucine (L) at positions 553 and 556, respectively, producing the mutants 8F553L, 8W556L, and 8F553L/8W556L. Additionally, two mutants, A92D and C157R, derived from Cry11Bb were also generated. The proteins were expressed in the non-crystal strain BMB171 of Bacillus thuringiensis and subjected to median-lethal concentration (LC50) tests on first-instar larvae of A. aegypti. LC50 analysis showed that the 8F553L, 8W556L, 8F553L/8W556L, and C157R variants lost their toxic activity (>500 ng·mL-1), whereas the A92D protein presented a loss of toxicity of 11.4 times that of Cry11Bb. Cytotoxicity assays performed using variant 8, 8W556L and the controls Cry11Aa, Cry11Bb, and Cry-negative BMB171 on the colorectal cancer cell line SW480 reported 30-50% of cellular viability except for BMB171. Molecular dynamic simulations performed to identify whether the mutations at positions 553 and 556 were related to the stability and rigidity of the functional tertiary structure (domain III) of the Cry11Aa protein and variant 8 showed the importance of these mutations in specific regions for the toxic activity of Cry11 against A. aegypti. This generates pertinent knowledge for the design of Cry11 proteins and their biotechnological applications in vector-borne disease control and cancer cell lines.

Genomic-proteomic analysis of a novel Bacillus thuringiensis strain: toxicity against two lepidopteran pests, abundance of Cry1Ac5 toxin, and presence of InhA1 virulence factor.

Bacillus thuringiensis (Bt) is a biological alternative to the indiscriminate use of chemical insecticides in agriculture. Due to resistance development on insect pests to Bt crops, isolating novel Bt strains is a strategy for screening new pesticidal proteins or strains containing toxin profile variety that can delay resistance. Besides, the combined genomic and proteomic approaches allow identifying pesticidal proteins and virulence factors accurately. Here, the genome of a novel Bt strain (Bt TOL651) was sequenced, and the proteins from the spore-crystal mixture were identified by proteomic analysis. Toxicity bioassays with the spore-crystal mixture against larvae of Diatraea saccharalis and Anticarsia gemmatalis, key pests of sugarcane and soybean, respectively, were performed. The toxicity of Bt TOL651 varies with the insect; A. gemmatalis (LC50 = 1.45 ng cm-2) is more susceptible than D. saccharalis (LC50 = 73.77 ng cm-2). Phylogenetic analysis of the gyrB gene indicates that TOL651 is related to Bt kenyae strains. The genomic analysis revealed the presence of cry1Aa18, cry1Ac5, cry1Ia44, and cry2Aa9 pesticidal genes. Virulence factor genes such as phospholipases (plcA, piplc), metalloproteases (inhA), hemolysins (cytK, hlyIII, hblA, hblC, hblD), and enterotoxins (nheA, nheB, nheC) were also identified. The combined use of the genomic and proteomic data indicated the expression of Cry1Aa18, Cry1Ac5, and Cry2Aa9 proteins, with Cry1Ac5 being the most abundant. InhA1 also was expressed and may contribute to Bt TOL651 pathogenicity. These results provide Bt TOL651 as a new tool for the biocontrol of lepidopteran pests.

In vivo and in silico comparison analyses of Cry toxin activities toward the sugarcane giant borer.

The sugarcane giant borer, Telchin licus licus, is an insect pest that causes significant losses in sugarcane crops and in the sugar-alcohol sector. Chemical and manual control methods are not effective. As an alternative, in the current study, we have screened Bacillus thuringiensis (Bt) Cry toxins with high toxicity against this insect. Bioassays were conducted to determine the activity of four Cry toxins (Cry1A (a, b, and c) and Cry2Aa) against neonate T. licus licus larvae. Notably, the Cry1A family toxins had the lowest LC50 values, in which Cry1Ac presented 2.1-fold higher activity than Cry1Aa, 1.7-fold larger than Cry1Ab, and 9.7-fold larger than Cry2Aa toxins. In silico analyses were performed as a perspective to understand putative interactions between T. licus licus receptors and Cry1A toxins. The molecular dynamics and docking analyses for three putative aminopeptidase N (APN) receptors (TlAPN1, TlAPN3, and TlAPN4) revealed evidence for the amino acids that may be involved in the toxin-receptor interactions. Notably, the properties of Cry1Ac point to an interaction site that increases the toxin's affinity for the receptor and likely potentiate toxicity. The interacting amino acid residues predicted for Cry1Ac in this work are probably those shared by the other Cry1A toxins for the same region of APNs. Thus, the presented data extend the existing knowledge of the effects of Cry toxins on T. licus licus and should be considered in further development of transgenic sugarcane plants resistant to this major occurring insect pest in sugarcane fields.

Computational study, synthesis and evaluation of active peptides derived from Parasporin-2 and spike protein from Alphacoronavirus against colorectal cancer cells.

Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 kDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and ß-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 µM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer-therapeutic agents.

Identification of Bacillus thuringiensis Strains for the Management of Lepidopteran Pests.

Bacillus thuringiensis (Bt)-based bioinsecticides and transgenic plants expressing proteins with insecticidal activity (Cry and Vip) have been successfully used in several integrated pest management programs worldwide. Lepidoptera comprise some of the most economically important insect pests of the major agricultural crops. In this study, the toxicity of 150 Bt strains was evaluated against Helicoverpa armigera (Hübner) larvae. Eight strains (426, 520B, 1636, 1641, 1644, 1648, 1657 and 1658) showed high insecticide activity against H. armigera and were therefore tested against Anticarsia gemmatalis (Hübner), Spodoptera cosmioides (Walker), Chrysodeixis includens (Walker), and Diatraea saccharalis (Fabricius) larvae. Our results showed that most of the Bt strains were also toxic to these lepidopteran species. The biochemical and molecular analyses of these strains revealed that they had a similar protein profile; however, their cry and vip gene contents were variable. In addition, the median lethal concentration (LC50) of the selected strains indicated that the strains 1636, 1641, and 1658 were the most effective against H. armigera, showing LC50 values of 185.02, 159.44, and 192.98 ng/cm2, respectively. Our results suggest that the selected Bt strains have great potential to control the lepidopteran pests H. armigera, A. gemmatalis, D. saccharalis, S. cosmioides, and C. includes.

Parasporin A13-2 of Bacillus thuringiensis Isolates from the Papaloapan Region (Mexico) Induce a Cytotoxic Effect by Late Apoptosis against Breast Cancer Cells.

The protein A13-2 was obtained from Bacillus thuringiensis strains isolated from the Papaloapan watershed region (Oaxaca, Mexico). The cytotoxic activity of parasporal inclusions was studied against breast cancer cell line (MCF-7) and normal cell (human peripheral blood mononuclear cells). The MTT, the formation of reactive species, nitric oxide, free cell DNA, and the type of death cellular were assessed. The protein A13-2 shows the highest cytotoxic activity against MCF-7 (13% cell viability at 6 µg/mL), the extracellular DNA increases, and it shows no stress for reactive species or nitric oxide. Besides, the A13-2 parasporin shows no toxicity to peripheral blood mononuclear cells, and it does not generate changes in nitric oxide levels or free cell DNA. Due to that, the cytotoxic effect of A13-2 was specific for MCF-7, and it does not affect normal cells. According to microscopy and flow cytometry, A13-2 parasporin leads to the death of MCF-7 cells by late apoptosis together with necrosis and without allowing the triggering of the survival mechanisms. When analyzed together, our results show for the first time that the A13-2 protein isolated from Mexican strains of B. thuringiensis preferentially kills MCF- 7 (cancer cells) over HEK 293 and PBMC cell lines (normal cells), thus representing a promising alternative for the treatment of cancer breast.

Design and characterization of a novel dimeric blood-brain barrier penetrating TNFα inhibitor.

Tumor necrosis factor-alpha (TNFα) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood-brain barrier (BBB). This research was aimed to design and characterize a novel TNFα inhibitor based on the angiopeptide-2 as a BBB shuttle molecule fused to the extracellular domain of human TNFα receptor 2 and a mutated vascular endothelial growth factor (VEGF) dimerization domain. This new chimeric protein (MTV) would be able to trigger receptor-mediated transcytosis across the BBB via low-density lipoprotein receptor-related protein-1 (LRP-1) and inhibit the cytotoxic effect of TNFα more efficiently because of its dimeric structure. Stably transformed CHO cells successfully expressed MTV, and its purification by Immobilized-Metal Affinity Chromatography (IMAC) rendered high purity degree. Mutated VEGF domain included in MTV did not show cell proliferation or angiogenic activities measured by scratch and aortic ring assays, which corroborate that the function of this domain is restricted to dimerization. The pairs MTV-TNFα (Kd 279 ± 40.9 nM) and MTV-LRP1 (Kd 399 ± 50.5 nM) showed high affinity by microscale thermophoresis, and a significant increase in cell survival was observed after blocking TNFα with MTV in a cell cytotoxicity assay. Also, the antibody staining in CHOK1 and bEnd3 cells demonstrated the adhesion of MTV to the LRP1 receptor located in the cell membrane. These results provide compelling evidence for the proper functioning of the three main domains of MTV individually, which encourage us to continue the research with this new molecule as a potential candidate for the systemic treatment of neurological disorders.

A trivalent TNF-R2 as a new tumor necrosis factor alpha-blocking molecule.

The neutralization of tumor necrosis factor alpha (TNFα) with biopharmaceuticals is a successful therapy for inflammatory diseases. Currently, one of the main TNFα-antagonists is Etanercept, a dimeric TNF-R2 ectodomain. Considering that TNFα and its receptors are homotrimers, we proposed that a trimeric TNF-R2 ectodomain could be an innovative TNFα-antagonist. Here, the 3cTNFR2 protein was designed by the fusion of the TNF-R2 ectodomain with the collagen XV trimerization domain. 3cTNFR2 was produced in HEK293 cells and purified by immobilized metal affinity chromatography. Monomers, dimers, and trimers of 3cTNFR2 were detected. The interaction 3cTNFR2-TNFα was assessed. By microscale thermophoresis, the KD value for the interaction was 4.17 ± 0.88 nM, and complexes with different molecular weights were detected by size exclusion chromatography-high performance liquid chromatography. Moreover, 3cTNFR2 neutralized the TNFα-induced cytotoxicity totally in vitro. Although more studies are required to evaluate the anti-inflammatory effect, the results suggest that 3cTNFR2 could be a TNFα-antagonist agent.

Probiotic Lactobacillus kefiri prevents endotoxin-induced preterm birth and stillbirth in mice.

Preterm birth (PTB), defined as birth occurring before 37 weeks of pregnancy, affects 5-18% of pregnancies and is the leading cause of neonatal morbidity and mortality worldwide. Although PTB is considered a syndrome, infection-induced inflammation accounts for up to 50% of all cases. Despite the effort to reduce the incidence of PTB, it continues to rise worldwide and current approaches for preventing or treating PTB are largely unsatisfactory. Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. It is well known that probiotics can modulate the host immune system exerting a potent anti-inflammatory activity. The main aim of this work was to evaluate the capacity of the probiotic Lactobacillus kefiri (Lk48) to prevent preterm birth in mice. C57BL/6 female mice were treated with Lk48 or vehicle a week before and during pregnancy and were challenged with LPS (10 µg), a dose known to induce PTB on gestational day 16. Percentages of PTB as well as stillbirth were evaluated. We observed that oral administration of Lk48 significantly reduced the occurrence of LPS-induced PTB and stillbirth as well as improved post-natal development. This protective effect was associated with a reduction in leucocyte infiltration and reduced inflammation-induced damage in reproductive tissue. Besides, Lk48 treatment also modulated the diversity of vaginal microbiota. Our results demonstrated that prophylactic consumption of probiotic L. kefiri prevented LPS-induced PTB and still birth in mice and opens new avenues for exploring novel and promising strategies for preventing PTB in humans.

Regulated hypothermia in response to endotoxin in birds.

KEY POINTS: The costs associated with immune and thermal responses may exceed the benefits to the host during severe inflammation. In this case, regulated hypothermia instead of fever can occur in rodents as a beneficial strategy to conserve energy for vital functions with consequent tissue protection and hypoxia prevention. We tested the hypothesis that this phenomenon is not exclusive to mammals, but extends to the other endothermic group, birds. A decrease in metabolic rate without any failure in mitochondrial respiration, nor oxygen delivery, is the main evidence supporting the regulated nature of endotoxin-induced hypothermia in chicks. Thermolytic mechanisms such as tachypnea and cutaneous vasodilatation can also be recruited to facilitate body temperature decrease under lipopolysaccharide treatment, especially in the cold. Our findings bring a new perspective for evolutionary medicine studies on energy trade-off in host defence because regulated hypothermia may be a phenomenon spread among vertebrates facing a severe immune challenge. ABSTRACT: A switch from fever to regulated hypothermia can occur in mammals under circumstances of reduced physiological fitness (e.g. sepsis) to direct energy to defend vital systems. Birds in which the cost to resist a pathogen is additive to the highest metabolic rate and body temperature (Tb ) among vertebrates may also benefit from regulated hypothermia during systemic inflammation. Here, we show that the decrease in Tb observed during an immune challenge in birds is a regulated hypothermia, and not a result of metabolic failure. We investigated O2 consumption (thermogenesis index), ventilation (respiratory heat loss), skin temperature (sensible heat loss) and muscle mitochondrial respiration (thermogenic tissue) during Tb fall in chicken chicks challenged with endotoxin [lipopolysaccharide (LPS)]. Chicks injected with LPS were also tested regarding the capacity to raise O2 consumption to meet an increased demand driven by 2,4-dinitrophenol. LPS decreased Tb and the metabolic rate of chicks without affecting muscle uncoupled, coupled and non-coupled mitochondrial respiration. LPS-challenged chicks were indeed capable of increasing metabolic rate in response to 2,4-dinitrophenol, indicating no O2 delivery limitation. Additionally, chicks did not attempt to prevent Tb from falling during hypothermia but, instead, activated cutaneous and respiratory thermolytic mechanisms, providing an additional cooling force. These data provide the first evidence of the regulated nature of the hypothermic response to endotoxin in birds. Therefore, it changes the current understanding of bird's thermoregulation during severe inflammation, indicating that regulated hypothermia is either a convergent trait for endotherms or a conserved response among vertebrates, which adds a new perspective for evolutionary medicine research.

In vivo nanoscale analysis of the dynamic synergistic interaction of Bacillus thuringiensis Cry11Aa and Cyt1Aa toxins in Aedes aegypti.

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered "net like" structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.

Bacillus thuringiensis Cry1Ac toxin and protoxin do not provoke acute or chronic cytotoxicity on macrophages and leukocytes.

The bioinsecticidal Cry1Ac proteins (protoxin and toxin) are potent immunogens that can activate macrophages by inducing upregulation of costimulatory molecules, pro-inflammatory cytokines, and mitogen-activated protein kinase (MAPK) signaling pathways. Besides, by the oral route, Cry1Ac toxin is mildly allergenic and induces intestinal lymphoid hyperplasia in mice. Given the potential utility of Cry1Ac protoxin as an adjuvant, as well as the human consumption of Cry1Ac toxin in transgenic crops, it is necessary to more deeply evaluate the toxicological potential of these proteins in mammalian immune cells. Here, were used in vitro evaluations in leukocyte and macrophage cell lines to test the potential toxicity of various doses of Cry1Ac proteins, by means of Alamar Blue, MTT, Annexin V, and JC1 assays. Our results indicated that neither Cry1Ac protoxin nor toxin elicited acute toxic effects, after monitoring the cell activity for 4, 8, 10, and 24 h of exposure. By flow cytometry and confocal microscopy analysis, it was observed that neither Cry1Ac toxin nor protoxin generated mitochondrial damage or depolarization or induced apoptosis or necrosis. In conclusion, despite their immunostimulatory effects, it was demonstrated that Cry1Ac proteins did not have cytotoxic effects, even at high concentrations, in primary leukocytes or macrophages or cell lines.

Assessing the effects of an acute exposure to worst-case concentration of Cry proteins on zebrafish using the embryotoxicity test and proteomics analysis.

Cry1C, Cry1F and Cry1Ab are insecticidal proteins from Bacillus thuringiensis (Bt) which are expressed in transgenic crops. Given the entry of these proteins into aquatic environments, it is relevant to evaluate their impacts on aquatic organisms. In this work, we sought to evaluate the effects of Cry1C, Cry1F and Cry1Ab on zebrafish embryos and larvae of a predicted worst-case scenario concentration of these proteins (set to 1.1 mg/L). For that, we coupled a traditional toxicity approach (the zebrafish embryotoxicity test and dosage of enzymatic biomarkers) to gel free proteomics analysis. At the concentration tested, these proteins did not cause adverse effects in the zebrafish early life stages, either by verifying phenotypic endpoints of toxicity or alterations in representative enzymatic biomarkers (catalase, glutathione-S-tranferase and lactate-dehydrogenase). At the molecular level, the Cry proteins tested lead to very small changes in the proteome of zebrafish larvae. In a global way, these proteins upregulated the expression of vitellogenins. Besides that, Cry1C e Cry1F deregulated heterogeneous nuclear ribonucleoproteins (Hnrnpa0l and Hnrnpaba, respectively), implicated in mRNA processing and gene regulation. Overall, these data lead to the conclusion that Cry1C, Cry1F and Cry1Ab proteins, even at a very high concentration, have limited effects in the early stages of zebrafish life.

Bacillus thuringiensis Cry1Ab Domain III ß-16 Is Involved in Binding to Prohibitin, Which Correlates with Toxicity against Helicoverpa armigera (Lepidoptera: Noctuidae).

Helicoverpa armigera is a major insect pest of several crops worldwide. This insect is susceptible to some Bacillus thuringiensis (Bt) Cry insecticidal proteins expressed in transgenic crops or used in biopesticides. Previously, we identified H. armigera prohibitin (HaPHB) as a Cry1Ac-binding protein. Here, we further analyzed the potential role of PHB as a Cry toxin receptor in comparison to cadherin (CAD), well recognized as a Cry1Ac receptor. HaPHB-2 midgut protein and HaCAD toxin-binding region (TBR) fragment from H. armigera were expressed in Escherichia coli cells, and binding assays with different Cry1 toxins were performed. We demonstrated that Cry1Ab, Cry1Ac, and Cry1Fa toxins bound to HaPHB-2 in a manner similar to that seen with HaCAD-TBR. Different Cry1Ab mutant toxins located in domain II (Cry1AbF371A and Cry1AbG439D) or domain III (Cry1AbL511A and Cry1AbN514A), which were previously characterized and found to be affected in receptor binding, were analyzed regarding their binding interaction with HaPHB-2 and toxicity against H. armigera One ß-16 mutant (Cry1AbN514A) showed increased binding to HaPHB-2 that correlated with 6-fold-higher toxicity against H. armigera, whereas the other ß-16 mutant (Cry1AbL511A) was affected in binding to HaPHB-2 and lost toxicity against H. armigera Our data indicate that ß-16 from domain III of Cry1Ab is involved in interactions with HaPHB-2 and in toxicity. This report identifies a region of Cry1Ab involved in binding to HaPHB-2 from a Lepidoptera insect, suggesting that this protein may participate as a novel receptor in the mechanism of action of the Cry1 toxins in H. armigeraIMPORTANCEHelicoverpa armigera is a polyphagous pest that feeds on important crops worldwide. This insect pest is sensitive to different Cry1 toxins from Bacillus thuringiensis In this study, we analyzed the potential role of PHB-2 as a Cry1 toxin receptor in comparison to CAD. We show that different Cry1 toxins bound to HaPHB-2 and HaCAD-TBR similarly and identify ß-16 from domain III of Cry1Ab as a binding region involved in the interaction with HaPHB-2 and in toxicity. This report characterized HaPHB-Cry1 binding interaction, providing novel insights into potential target sites for improving Cry1 toxicity against H. armigera.

Metabolic and Hematological Responses to Endotoxin-Induced Inflammation in Chicks Experiencing Embryonic 2,3,7,8-Tetrachlorodibenzodioxin Exposure.

Dioxin exposure during bird embryonic development disrupts immunity as well as mechanisms involved in energy metabolism, potentially affecting negatively acute-phase responses to pathogens. Thus, we hypothesized that embryonic exposure to 2,3,7,8-tetrachlorodibenzodioxin (TCDD) changes the metabolism and blood physiology of domestic chicks, affecting their physiological competence for responding to immune challenges. To test this hypothesis, we injected doses of 0, 1.5, and 3 ng TCDD/egg (based on survival experiments) on embryonic day 4 and then measured O2 consumption and CO2 production for metabolic rate, ventilation, and body temperature (TB ) in 5-d-old chicks. Then, chicks were injected with lipopolysaccharide (LPS, endotoxin) or saline prior to repeating the physiological measurements. A second chick group exposed to identical TCDD and LPS treatments had blood partial pressure of oxygen, partial pressure of carbon dioxide, pH, bicarbonate concentration, lactate concentration, osmolality, hemoglobin concentration, red blood cell concentration, and hematocrit, as well as TB , analyzed at 1 and 5 h after LPS injection. Metabolism in chicks embryonically exposed to 1.5 and 3 ng TCDD/egg was up to 37% higher, whereas body mass of chicks exposed to 3 ng TCDD/egg was approximately 6% lower. Chicks embryonically exposed to 3 ng TCDD/egg challenged with LPS showed a relative persistent hypometabolism accompanied by elimination of the normal hematological and osmotic responses to LPS. We conclude that embryonic exposure to TCDD affects posthatching metabolism as well as impairs metabolic, hematological, and osmotic responses to LPS. Environ Toxicol Chem 2020;39:2208-2220. © 2020 SETAC.

DNA damages induced by both endotoxin and exotoxin produced by cyanobacteria.

Cyanobacteria are prokaryotes involved in the contamination of aquatic environments since they release toxins that are highly potent and dangerous for living organisms. Prokaryotes produce endo and exotoxins, among others. Exotoxins are highly toxic, while endotoxins have milder toxic effects. The present study evaluated the cytotoxicogenetic potency of both toxins studying them in different concentrations of cyanobacterial biomasses (1 µg/L, 1.5 µg/L, 2 µg/L), to assess the amount of exotoxin present in the cultured medium in which the cyanobacteria were grown. For this evaluation, we used an extract taken from the medium in a concentration of 10%. Our results showed that genotoxic and mutagenic changes in Allium cepa could be observed in all of the varying concentrations of biomass (endotoxin action) and also in the medium induced with exotoxin. Even at low concentrations, these toxins were highly effective at triggering changes in the DNA molecules of organisms exposed to them. This information is highly significant when considering environmental contamination caused by cyanobacteria blooms, since the results of this study show that these toxins may not only kill organisms when found in high concentrations, but also induce mutations when found in low concentrations. Since these mutations are expressed later on in the organisms, it is impossible to associate the observed effect with the event that induced the damage.

Oligomerization is a key step for Bacillus thuringiensis Cyt1Aa insecticidal activity but not for toxicity against red blood cells.

Bacillus thuringiensis (Bt) Cyt1Aa toxin shows toxicity to mosquitoes, to certain coleopteran pests and also to red blood cells (RBC). However, its mode of action in the different target cells is not well defined. This protein is a single α-ß domain pore-forming toxin, where a ß sheet is wrapped by two α-helices layers. The Cyt1Aa α-helix hairpin in the N-terminal has been proposed to be involved in initial membrane binding and oligomerization, while the ß sheet inserts into the membrane to form a pore that lyze the cells. To determine the role of the N-terminal α-helix hairpin region of Cyt1Aa in its mode of action, we characterized different single point mutations located in helices α-1 and α-2. Eight cysteine substitutions in different residues were produced in Bt, and we found that three of them: Cyt1AaA65C, Cyt1AaL85C and Cyt1AaN89C, lost insecticidal toxicity against Aedes aegypti larvae but retained similar or increased hemolytic activity towards rabbit RBC. Analysis of toxin binding and oligomerization using Ae. aegypti midgut brush border membrane vesicles showed that the three Cyt1Aa mutants non-toxic to Ae. aegypti were affected in oligomerization. However, these mutants were still hemolytic. Our data shows that oligomerization of Cyt1Aa toxin is essential for its toxicity to Ae. aegypti but not for its toxicity against RBC indicating that the mode of action of Cyt1Aa is different in these distinct target membranes.

Development of an Efficient Protocol to Obtain Transgenic Coffee, Coffea arabica L., Expressing the Cry10Aa Toxin of Bacillus thuringiensis.

This report presents an efficient protocol of the stable genetic transformation of coffee plants expressing the Cry10Aa protein of Bacillus thuringiensis. Embryogenic cell lines with a high potential of propagation, somatic embryo maturation, and germination were used. Gene expression analysis of cytokinin signaling, homedomains, auxin responsive factor, and the master regulators of somatic embryogenesis genes involved in somatic embryo maturation were evaluated. Plasmid pMDC85 containing the cry10Aa gene was introduced into a Typica cultivar of C. arabica L. by biobalistic transformation. Transformation efficiency of 16.7% was achieved, according to the number of embryogenic aggregates and transgenic lines developed. Stable transformation was proven by hygromycin-resistant embryogenic lines, green fluorescent protein (GFP) expression, quantitative analyses of Cry10Aa by mass spectrometry, Western blot, ELISA, and Southern blot analyses. Cry10Aa showed variable expression levels in somatic embryos and the leaf tissue of transgenic plants, ranging from 76% to 90% of coverage of the protein by mass spectrometry and from 3.25 to 13.88 µg/g fresh tissue, with ELISA. qPCR-based 2-ΔΔCt trials revealed high transcription levels of cry10Aa in somatic embryos and leaf tissue. This is the first report about the stable transformation and expression of the Cry10Aa protein in coffee plants with the potential for controlling the coffee berry borer.

Endothelial Cells Exhibit Two Waves of P-selectin Surface Aggregation Under Endotoxic and Oxidative Conditions.

Sepsis is a clinical syndrome characterized by the presence of circulating microbial endotoxins and oxidative stress. Endotoxin and oxidative stress activate endothelial cells via a convergent signaling pathway (TLR4/MyD88/PI3 K/PLCÉ£/NF-B) that stimulates both the transcription of SELP gene (which encodes for human P-selectin) and the release of P-selectin from Weibel-Palade bodies (WPBs). However, time course pattern of P-selectin surface aggregation has not been established in endothelial cells under 24 h of endotoxic or oxidative stress. Our study shows that P-selectin has at least two waves of aggregation at the cell surface: one 10 min and the other 12 h after endotoxic or oxidative stress. The first wave depends exclusively on WPB delivery to the cell membrane, while the second depends on P-selectin translation machinery, ER-Golgi sorting, and WPB surface delivery. Understanding adhesion molecule dynamics in endothelial cells could provide further molecular insights to develop diagnostic or therapeutic tools to aid in the management of sepsis.