Safety assessment of Mpp75Aa1.1, a new ETX_MTX2 protein from Brevibacillus laterosporus that controls western corn rootworm.

The recently discovered insecticidal protein Mpp75Aa1.1 from Brevibacillus laterosporus is a member of the ETX_MTX family of beta-pore forming proteins (ß-PFPs) expressed in genetically modified (GM) maize to control western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte). In this manuscript, bioinformatic analysis establishes that although Mpp75Aa1.1 shares varying degrees of similarity to members of the ETX_MTX2 protein family, it is unlikely to have any allergenic, toxic, or otherwise adverse biological effects. The safety of Mpp75Aa1.1 is further supported by a weight of evidence approach including evaluation of the history of safe use (HOSU) of ETX_MTX2 proteins and Breviballus laterosporus. Comparisons between purified Mpp75Aa1.1 protein and a poly-histidine-tagged (His-tagged) variant of the Mpp75Aa1.1 protein demonstrate that both forms of the protein are heat labile at temperatures at or above 55°C, degraded by gastrointestinal proteases within 0.5 min, and have no adverse effects in acute mouse oral toxicity studies at a dose level of 1920 or 2120 mg/kg body weight. These results support the use of His-tagged proteins as suitable surrogates for assessing the safety of their non-tagged parent proteins. Taken together, we report that Mpp75Aa1.1 is the first ETX-MTX2 insecticidal protein from B. laterosporus and displays a similar safety profile as typical Cry proteins from Bacillus thuringiensis.

Safety of the Bacillus thuringiensis-derived Cry1A.105 protein: Evidence that domain exchange preserves mode of action and safety.

The lepidopteran-active Cry1A.105 protein is a chimeric three-domain insecticidal toxin with distinct structural domains derived from the naturally occurring Cry1Ab, Cry1Ac and Cry1F proteins from the soil bacterium Bacillus thuringiensis (Bt). The X-ray crystal structure of the Cry1A.105 tryptic core at 3.0 Šresolution demonstrates its high structural similarity to the tryptic core of Cry1Ac. Bioinformatics analyses demonstrate that Cry1A.105 has no significant amino acid sequence similarity to known allergens or mammalian toxins, which is the same conclusion reached for its component domains. Like its intact donor proteins, Cry1A.105 was heat labile at temperatures ≥75 °C and degraded upon exposure to gastrointestinal proteases. No adverse effects were observed in mice when Cry1A.105 was dosed orally at 2451 mg/kg body weight. Therefore, the weight of evidence supports that Cry1A.105 is safe for human and animal consumption. These results support the conclusion that the safety of a chimeric protein for human or animal consumption can be evaluated in the context of the safety of its donor proteins.

Improving insect control protein activity for GM crops: A case study demonstrating that increased target insect potency can be achieved without impacting mammalian safety.

Many insect-protected crops express insecticidal crystal (Cry) proteins derived from the soil bacterium Bacillus thuringiensis (Bt), including both naturally-occurring Cry proteins and chimeric Cry proteins created through biotechnology. The Cry51Aa2 protein is a naturally-occurring Cry protein that was modified to increase its potency and expand its insect activity spectrum through amino acid sequence changes. The improved Cry51Aa2 variant, Cry51Aa2.834_16, and other developmental variants belong to the ETX_MTX2 family of proteins but share a low level of sequence similarity to other members of this family. This similarity is largely localized to the pore-forming and oligomerization protein domains, while sequence divergence is observed within the head domain that confers receptor binding specificity. The intact Cry51Aa2.834_16 protein was heat labile at temperatures ≥55 °C, and was rapidly degraded after exposure to the gastrointestinal protease pepsin. No acute oral toxicity was observed in mice for three protein variants of Cry51Aa2, including Cry51Aa2.834_16, at doses exceeding 1000 mg/kg body weight. The weight-of-evidence therefore supports the conclusion of safety for Cry51Aa2.834_16 and demonstrates that amino acid sequence modifications can be used to substantially increase insecticidal activity of a protein without an increased hazard to mammals.

The sequence, structural, and functional diversity within a protein family and implications for specificity and safety: The case for ETX_MTX2 insecticidal proteins.

The need for sustainable insect pest control is driving the investigation and discovery of insecticidal proteins outside of the typical 3-domain Cry protein family from Bacillus thuringiensis (Bt). Examples include Cry35 and Cry51 that belong to protein families (Toxin_10, ETX_MTX2) sharing a common ß-pore forming structure and function with known mammalian toxins such as epsilon toxin (ETX). Although ß-pore forming proteins are related to mammalian toxins, there are key differences in sequence and structure that lead to organism specificity that is useful in the weight-of-evidence approach for safety assessment. Despite low overall amino acid sequence identity among ETX_MTX2 proteins, sequence and structural similarities are found in the tail region responsible for the shared oligomerization and pore formation functions (causing the "relatedness"). Conversely, most of the sequence and structural diversity is located in the head region that is likely responsible for differential receptor binding and target species specificity (e.g., insecticidal vs. mammalian). Therefore, inclusion of a domain-based protein characterization approach that includes bioinformatic and functional comparisons of conserved and diverse domains will enhance the overall weight of evidence safety assessment of proteins including recently reported Cry51 protein variants (Cry51Aa1, Cry51Aa2, and Cry51Aa2.834_16).

Corn rootworm-active RNA DvSnf7: Repeat dose oral toxicology assessment in support of human and mammalian safety.

Genetically modified (GM) crops have been developed and commercialized that utilize double stranded RNAs (dsRNA) to suppress a target gene(s), producing virus resistance, nutritional and quality traits. MON 87411 is a GM maize variety that leverages dsRNAs to selectively control corn rootworm through production of a 240 base pair (bp) dsRNA fragment targeting for suppression the western corn rootworm (Diabrotica virgifera virgifera) Snf7 gene (DvSnf7). A bioinformatics assessment found that endogenous corn small RNAs matched ∼450 to 2300 unique RNA transcripts that likely code for proteins in rat, mouse, and human, demonstrating safe dsRNA consumption by mammals. Mice were administered DvSnf7 RNA (968 nucleotides, including the 240 bp DvSnf7 dsRNA) at 1, 10, or 100 mg/kg by oral gavage in a 28-day repeat dose toxicity study. No treatment-related effects were observed in body weights, food consumption, clinical observations, clinical chemistry, hematology, gross pathology, or histopathology endpoints. Therefore, the No Observed Adverse Effect Level (NOAEL) for DvSnf7 RNA was 100 mg/kg, the highest dose tested. These results demonstrate that dsRNA for insect control does not produce adverse health effects in mammals at oral doses millions to billions of times higher than anticipated human exposures and therefore poses negligible risk to mammals.

Transcriptomic Insights into the Life History of Bolidophytes, the Sister Lineage to Diatoms.

Diatoms are perhaps the most diverse lineage of eukaryotic algae, with their siliceous cell wall and diplontic life history often considered to have played important roles in their extraordinary diversification. The characteristic diminution of the diatom cell wall over the course of vegetative growth provides a reliable, intrinsic trigger for sexual reproduction, establishing a direct link between the evolution of their cell-wall and life-history features. It is unclear, however, whether the diplontic life cycle of diatoms represents an ancestral or derived trait. This uncertainty is based in part on our lack of understanding of the life cycle of the sister lineage to diatoms, which includes a mix of two free-living and separately classified forms: naked biflagellate unicells in the genus Bolidomonas and silicified forms in the order Parmales. These two forms might represent different life-history stages, although directly establishing such links can be difficult. We sequenced transcriptomes for Bolidomonas and two diatoms and found that ~0.1% of the coding regions in the two diploid diatoms are heterozygous, whereas Bolidomonas is virtually devoid of heterozygous alleles, consistent with expectations for a haploid genome. These results suggest that Bolidomonas is haploid and predict that parmaleans represent the diploid phase of a haplodiplontic life cycle. These data fill an important gap in our understanding of the origin of the diplontic life history of diatoms, which may represent an evolutionarily derived, adaptive feature.