Specific targeting of a pore-forming toxin (listeriolysin O) to LHRH-positive cancer cells using LHRH targeting peptide.

Conventional drug delivery systems have many limitations including cytotoxicity and affecting non-specific cells. Cell-targeting peptides (CTPs) as a potential class of targeting moiety have some advantages over previous targeting moieties such as monoclonal antibodies, offer additional benefits to design systems using CTPs. Here we have engineered listeriolysin O (LLO) pore-forming toxin by adding a luteinizing hormone-releasing hormone (LHRH) targeting peptide to its N-terminus. Two versions of the toxin, with and without targeting peptide, were sub-cloned into a bacterial expression plasmid. BL21 DE3 cells were used for induction of expression and recombinant proteins were purified using nickel-immobilized metal affinity chromatography column. In order to treat MDA-MB-231 and SKOV3 cell lines as LHRH receptor positive and negative cells, two mentioned LLO toxins were used to evaluate their cytotoxicity and specificity. Our results reveal that the IC50 of LLO toxin on MDA-MB-231 and SKOV3 cells was 0.32 and 0.41 µg/ml respectively. Furthermore, IC50 of fusion LHRH-LLO toxin on the cells was 0.88 and 19.55 µg/ml. Cytotoxicity of engineered LHRH-LLO toxin on negative cells was significantly 48-fold lower than wild-type LLO toxin. But this difference has been lowered to only 2.7-fold less cytotoxicity in positive cells. To the best of our knowledge, the current work as the first study regarding engineered toxin revealed that CDC family members could be used to target the specific cell-type.

Unlinked genetic loci control the reduced transcription of aminopeptidase N 1 and 3 in the European corn borer and determine tolerance to Bacillus thuringiensis Cry1Ab toxin.

Transgenic expression of Bacillus thuringiensis (Bt) crystalline (Cry) toxins by crop plants result in reduced insect feeding damage, but sustainability is threatened by the development of resistance traits in target insect populations. We investigated Bt toxin resistance trait in a laboratory colony of the European corn borer, Ostrinia nubilalis, selected for increased survival when exposed to Cry1Ab and correlated survival on Cry1Ab toxin with a constitutive ∼146.2 ± 17.3-fold reduction in midgut aminopeptidase N1 (apn1) transcript levels. A 7.1 ± 1.9-fold reduction apn3 transcript level was also correlated with Cry1Ab resistance. Quantitative trait locus (QTL) mapping identified a single major genome region controlling Cry1Ab resistance on linkage group 24 (LG24), and a minor QTL on LG27. Both QTL were independent of apn1 and apn3 loci on LG02. Positional mapping identified genetic markers that may assist in the identification of causal gene(s) within QTL intervals. This study indicates that genetic factor(s) may act in trans to reduce both apn1 and apn3 expression in Cry1Ab resistant O. nubilalis larvae, and suggest that gene regulatory pathways can influence Bt resistance traits. These findings show that gene interactions (epistasis) may influence Bt resistance in target insect populations.

Mutants of listeriolysin O for enhanced liposomal delivery of macromolecules.

Delivery of macromolecules into the cytosolic space of eukaryotic cells is a pressing challenge in biopharmaceutics. Macromolecules are often encapsulated into liposomes for protection and improved distribution, but the their size often induces endocytosis of the vehicle at the target site, leading to degradation of the cargo. Listeriolysin O is a key virulence factor of Listeria monocytogenes that forms pores in the endosomal membrane, ultimately allowing the bacterium to escape into the cytosol. This function of LLO has been used to improve cytosolic delivery of liposomally encapsulated macromolecules in a number of instances, but its innate toxicity and immunogenicity have prevented it from achieving widespread acceptance. Through site-directed mutagenesis, this study establishes a mutant of LLO (C484S) with enhanced activity, allowing for a reduction in the amount of LLO used for future applications in liposomal drug delivery.

Fate of Cry1Ab protein in agricultural systems under slurry management of cows fed genetically modified maize (Zea mays L.) MON810: a quantitative assessment.

The objective of the study was to track the fate of recombinant Cry1Ab protein in a liquid manure field trial when feeding GM maize MON810 to dairy cows. A validated ELISA was applied for quantification of Cry1Ab in the agricultural chain from GM maize plants, feed, liquid manure and soil to crops grown on manured fields. Starting with 23.7 µg of Cry1Ab g(-1) dry weight GM maize material, a rapid decline of Cry1Ab levels was observed as 2.6% and 0.9% of Cry1Ab from the GM plant were detected in feed and liquid manure, respectively. Half of this residual Cry1Ab persisted during slurry storage for 25 weeks. After application to experimental fields, final degradation of Cry1Ab to below detectable levels in soil was reported. Cry1Ab exhibited a higher rate of degradation compared to total protein in the agricultural processes. Immunoblotting revealed a degradation of the 65 kDa Cry1Ab into immunoreactive fragments of lower size in all analyzed materials.

Developmental and photosynthetic regulation of δ-endotoxin reveals that engineered sugarcane conferring resistance to 'dead heart' contains no toxins in cane juice.

The phosphoenolpyruvate (PEP) carboxylase is regulated at the levels of transcription and post-translation in C4 plants in light and abundantly accumulates in leaf mesophyll cells. We report here developmental and photosynthetic regulation of stably accumulated Bacillus thuringiensis δ-endotoxin under the control of PEP-C promoter in transgenic sugarcane. In young leaves of plants, the transprotein is accumulated to 39% of the levels in mature leaves (135 ng mg(-1)), and is induced with the cell development, from base to tip. Nevertheless, these levels are decreased up to 99.98% in non-photosynthetic cells as cane matures, from top to bottom, suggesting the photosynthesis regulation of δ-endotoxin in cane cells. Further, transgenic plants are highly resistant to 'dead heart'. In these studies, Scirpophaga nivela larvae causing 'dead heart' were killed within one hour of release to the transgenic plants. Therefore, this report may be regarded as the first report that provides a better strategy for developing transgenic sugarcane lines with absolute protection against invading larvae and no toxin residues in cane juice.

Safety assessment of Cry1Ab/Ac fusion protein.

Cry1ab/ac gene was fused by both the cry1ab gene (GenBank Accession No. X54939) and the cry1ac gene (GenBank Accession No. Y09787), which was widely used in genetically modified (GM) rice, cotton, maize and so on. In order to support the safety assessment of GM food or feed products containing Cry1Ab/Ac protein, sufficient quantities of Cry1Ab/Ac protein were produced in Escherichia coli for in vitro evaluation and animal studies. The Cry1Ab/Ac protein does not possess the characteristics associated with food toxins or allergens, i.e., it has no sequence homology with any known allergens or toxins, and no N-glycosylation sites, can be rapidly degraded in gastric and intestinal fluids, and is devoid of adverse effects in mice by gavage at a high dose level of 5g (Cry1Ab/Ac protein)/kg body weight. In conclusion, there is a reasonable certainty of no harm resulting from the inclusion of the Cry1Ab/Ac protein in human food or animal feed.

Emulsan-alginate beads for protein adsorption.

Emulsan-alginate beads were prepared and challenged using bovine serum albumin (BSA) to assess adsorption in comparison to alginate beads. BSA binding to the emulsan-alginate beads was improved over the alginate bead controls and protein adsorption was less sensitive to changes in ionic strength. BSA adsorption between pH 8.5 and 5.3 in alginate beads was 2-3-times lower compared to the emulsan-alginate beads in the same pH range. BSA adsorption and kinetic constants were at least 2-times higher for the emulsan-alginate beads compared to the alginate controls based on the Langmuir adsorption model. To further explore the utility of these novel emulsan-alginate bead systems, complex cell-free supernatants from some pathogenic microorganisms were exposed to the emulsan-alginate beads and increased protein adsorption was found when compared to controls. These trends were also confirmed with alpha-hemolysin toxicity studies. The data suggest that the protein-binding capacity of emulsan-alginate beads exceeds alginate controls, attributable to the unique binding features of emulsan.

Analysis of Cry1Ab toxin bioaccumulation in a food chain of Bt rice, an herbivore and a predator.

Lessons from organophosphorus pesticides, which could be bioaccumulated in non-target organisms at different trophic levels and caused unexpected negative impacts, necessitate a study of the possibility of biotransfer and bioaccumulation of Bacillus thuringiensis (Bt) insecticidal toxin(s) expressed in Bt plants. Using ELISA, we evaluated the transfer of Cry1Ab toxin in a food chain of Bt rice (KMD1 and KMD2), the target insect, Cnaphalocrocis medinalis, and its predator, Pirata subpiraticus. Cry1Ab was detected in C. medinalis and P. subpiraticus. However, the concentration of Cry1Ab detected from C. medinalis and P. subpiraticus did not increase as feeding or preying time increased. A binding study of Cry1Ab to the brush border membrane vesicle of C. medinalis and P. subpiraticus indicated that P. subpiraticus does not have binding receptors in its midgut to Cry1Ab, while C. medinalis does. Survivorship and fecundity of P. subpiraticus preying on Bt rice-fed C. medinalis were not significantly different from those preying on non-Bt rice-fed C. medinalis. Developmental time of P. subpiraticus was significantly longer when it preyed on Bt rice-fed C. medinalis than on non-Bt rice-fed prey. However, a 3-year field trial indicated that Bt rice did not significantly affect the density of P. subpiraticus.

Bt maize fed-prey mediated effect on fitness and digestive physiology of the ground predator Poecilus cupreus L. (Coleoptera: Carabidae).

We investigated the effects of a Bt maize hybrid on fitness and digestive physiology of the ground-dwelling predator Poecilus cupreus L., as compared with the near-isogenic hybrid. A tritrophic assay revealed that there was a great decline in the detection of Cry1Ab toxin through the trophic chain, the concentration of the toxin being 945, 349 and 37 ng g(-1) of fresh weight in Bt maize leaves, Spodoptera littoralis (Boisduval) larvae and P. cupreus larvae, respectively. Moreover, the toxin was only detected in 8% of the P. cupreus adults collected from fields growing Bt maize. Developmental time of both larvae and pupae of P. cupreus was not adversely affected by the Cry1Ab toxin via fed-prey. To elucidate potential detrimental effects due to a reduction in the quality of the prey, we assessed the digestive proteolytic activities of P. cupreus adults from a laboratory culture and insects collected in commercial Bt and non-Bt maize fields. Field-collected P. cupreus adults had higher proteolytic activities than those reared in the laboratory, whereas no significant differences were found between P. cupreus adults reared on Bt and non-Bt maize fed-S. littoralis or between P. cupreus adults collected in commercial Bt and non-Bt maize fields.

Bacillus thuringiensis Cry1Ac toxin-binding and pore-forming activity in brush border membrane vesicles prepared from anterior and posterior midgut regions of lepidopteran larvae.

It is generally accepted that Bacillus thuringiensis Cry toxins insert into the apical membrane of the larval midgut after binding to specific receptors, and there is evidence that the distribution of binding molecules along the midgut is not uniform. By use of the voltage-sensitive dye DiSC(3)(5) and (125)I-labeled Cry1Ac, we have measured the effect of Cry1Ac in terms of permeabilization capacity and of binding parameters on brush border membrane vesicles (BBMV) prepared from the anterior and the posterior regions of the larval midgut from two insect species, Manduca sexta and Helicoverpa armigera. The permeabilizing activity was significantly higher with BBMV from the posterior region than with the one observed in the anterior region in both insect species. Instead, (125)I-Cry1Ac bound specifically to BBMV from the two midgut regions, with no significant differences in the binding parameters between the anterior and posterior regions within an insect species. N-acetylgalactosamine inhibition patterns on pore formation and binding differed between anterior and posterior midgut regions and between species, providing evidence of a multifaceted involvement of the sugar in the Cry1Ac mode of action. The analysis of binding and pore formation in different midgut regions could be an effective method to study differences in the mode of action of Cry1Ac toxin in different species.

Perturbation of endothelial junction proteins by Staphylococcus aureus alpha-toxin: inhibition of endothelial gap formation by adrenomedullin.

Endothelial hyperpermeability is a hallmark of an inflammatory reaction and contributes to tissue damage in severe infections. Loss of endothelial cell-cell adhesion leads to intercellular gap formation allowing paracellular fluid flux. Although Staphylococcus aureus alpha-toxin significantly contributed to staphylococci disease, little is known about its mechanism of endothelial hyperpermeability induction. Here, we demonstrate that in a model of isolated perfused rat ileum discontinuation of capillary vascular-endothelial-cadherin (VE-cadherin) was observed after bolus application of S. aureus alpha-toxin being inhibited by the endogenous peptide adrenomedullin (ADM). In vitro, alpha-toxin exposure induced loss of immunoreactivity of VE-cadherin and occludin in human cultured umbilical vein endothelial cells. Likewise, ADM blocked alpha-toxin-related junctional protein disappearance from intercellular sites. Additionally, cyclic AMP elevation was shown to stabilize endothelial barrier function after alpha-toxin application. Although no RhoA activation was noted after endothelial alpha-toxin exposure, inhibition of Rho kinase and myosin light chain kinase blocked loss of immunoreactivity of VE-cadherin and occludin as well as intercellular gap formation. In summary, stabilization of endothelial junctional integrity as indicated by interendothelial immunostaining might be an interesting approach to stabilize endothelial barrier function in severe S. aureus infections.

Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize.

Proteins from the Cry 1 family, in particular Cry 1Ab, are commonly expressed in genetically modified Bt maize in order to control chewing insect pests. A sensitive chemiluminescent sandwich enzyme immunoassay for the detection of Cry1Ab protein from genetically modified Bt maize has been developed and validated. A Cry1Ab protein-specific antibody was immobilized on 96- or 384-well microtiter plates in order to capture the Cry1Ab toxin in the sample; the bound toxin was then detected by employing a second anti-Cry1Ab antibody and a horseradish peroxidase-labeled anti-antibody, followed by measurement of the enzyme activity with an enhanced chemiluminescent system. The chemiluminescent assay fulfilled all the requirements of accuracy and precision and exhibited limits of detection of a few pg mL(-1) Cry1Ab (3 or 5 pg mL(-1), depending on the assay format), which are significantly lower than that achievable using conventional colorimetric detection of peroxidase activity and also represent an improvement compared to previously developed Cry1Ab immunoassays. High-throughput analysis can be performed using the 384-well microtiter plate format immunoassay, which also allows one to reduce the consumption of samples and reagents. Validation of the assay, performed by analyzing certified reference materials, proved that the immunoassay is able to detect the presence of the Cry1Ab protein in certified reference samples containing as low as 0.1% of MON 810 genetically modified Bt maize. This value is below the threshold requiring mandatory labeling of foods containing genetically modified material according to the actual EU regulation.