Progress 07/01/13 to 02/28/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems: 1. Changes and problems in Objective 1 The original objective 1 was to discovery “TEP” peptides using in-vivo phage screen assay. This objective remains the same with some changes. One of the major changes to the objective 1 is that Manduca sexta larva was used to replace the proposed Spodoptera exigua larva for the phage selection assay. S. exigua larva were explored and it was found that its small size caused two problems: one is that it is difficult to collect the haemolymph without haemolymph contamination, the other is decontamination procedure was not efficient to this small size body. Therefore phage selection assay using S. exigua frequently resulted in phage plaques in plates showing surface contaminated phages and those recovered from haemolymph. M. sexta larva have relatively large-size body and can be efficiently cleaned to avoid surface phage contamination. Another major change to this objective is the strategy to identify TEPs. Originally we proposed to discover some “TEP” motifs based on the large amount of accumulation of the “TEP” sequence recovered from the insect haemolymph in the in-vivo phage screen assay. However, it can be very difficult to discover a common motif because the phage library can have up to 4.1x1015 possible different phage particles with different variable regions of PIII. Therefore in different phage in vivo screen assays, we deal with different phage “libraries”. Identification of a common motif from different “libraries” and this astronomical number of possible PIII variable region sequences is not efficient and would require a huge workload that would be very time-consuming. Therefore instead, we changed our “TEP” discovery strategy to use multiple rounds of phage in-vivo screens. The rationale is that if some phages are truly more capable to penetrate insect guts than others, multiple rounds of screens will tend to specifically select those phages to be recovered from the haemolymph and they will become more and more dominant in the recovered phage pool in further rounds of screening. This strategy helped us more quickly identify some “TEP” sequences and move forward. 2. Changes and problems in Objective 2 The original objective 2 was to discover “TEPs” using other species of insects in the in-vivo phage screen assays and identify “TEPs” with broad insect spectrum. This objective was changed to objective 3 as described in this report due to the change in strategy to the TEP insect spectrum study. One major change to the TEP insect spectrum study is that the insect spectrum study of TEPs will be performed in the multiple insect per os bioassays with the TEP fused toxins using the identified TEP sequence from the work in objective 1 instead of repeating the work in objective 1 on multiple insect species. During the work for objective 1, multiple TEP phages were identified and more will continue to be identified, some of them may have broader spectrum in insect gut penetration. Screening and evaluation from the identified TEP sequences is a more efficient way to identify the TEPs with broad insect gut penetration spectrum than phage screening from a phage library with 4.1x1015 possible different phage particles. Therefore, the objective 2 now is to produce enough TEP fusion peptides using both the chemical synthesis and yeast expression systems for objective 3. In the original proposal, chemical synthesis is proposed to produce the TEP-toxin fusion peptides. However, the chemical synthesis has some disadvantages. Vestaron toxins are only active when they have three correctly paired disulfide bonds (ICK motif), which can not be guaranteed in the chemical synthesis. Secondly, Vestaron previously had difficulty in chemically synthesizing hybrid-ACTX-Hv1a, which is planned to be used in the TEP fusion. This is why chemical synthesis will prioritize the omega-ACTX-Hv1a peptide. Finally, the chemical synthesis of peptide is very expensive and cannot produce enough amounts to allow us to perform the desired insect per os bioassays for both TEP performance study and TEP insect spectrum study. At this time we only were able to synthesize two TEP fused peptides with 20 mg each. This limited peptide supplies limited our capability to perform insect per os bioassays in what type of bioassays and how many replications. Besides the chemical synthesis pathway, we also change the peptide production strategy to use a yeast expression system to express the TEP fused toxin peptide. First, the yeast expression system is known to provide the toxin correct folding. Secondly, Vestaron has proven capability to produce the hybrid-ACTX-Hv1a at as high as 6 g/L yield in yeast, and has experience producing cell-penetrating-peptide fused hybrid-ACTX-Hv1a as high as 1.5 g/L yield in yeast. Third, the yeast expression strain generation is about a month per cycle, but each cycle can generate multiple TEP-Hybrid fusion strains. 3. Changes and problems in Objective 3 The original objective 3 was to produce TEP fusion peptides and test in insect bioassays. Now it became Objective 2 and part of object 3 in this report. In this final report, objective 3 is to use the TEP fusion peptide produced as described in objective 2 above to evaluate whether TEP peptides improve the toxin activities orally in the insect per os bioassays and study the TEP insect spectrum. A reliable insect per os bioassay development was a time-consuming process and different species of insects may need a different appropriate bioassay. We spent almost a month to develop Manduca droplet feeding bioassay. We then tried the droplet bioassay in T. ni. and Spodoptera exigua, but the assay reliability was not to our satisfaction due to the high control mortality and weak food dye efficiency in those bioassays. Hybrid+2-ACTX-Hv1a was not effectively suitable for this assay because Manduca is not very susceptible to hybrid toxin and hybrid toxin crashed out from bait solution at the presence of the food dye when its concentration exceeded 3 mM. SCR is more susceptible to hybrid toxin and the diet incorportation bioassay (DIB) is well suitable for SCR. However, the DIB assay consumed large amount of peptides, for example, hybrid+2 treatment at 2 PPT dose in DIB will consume 50 mg peptide per replication. Due to the limited peptide supplies, we only were able to perform limited insect per os bioassays at this stage. These difficulties limited our capability to screen more insects. Therefore, by the end of this project we only were able to test the peptides in two species of insects instead of originally proposed 6 species. What opportunities for training and professional development has the project provided? Vestaron has hired an undergraduate student from Western Michigan University, Sarah Ghekiere, to work as an intern for assistance of this project. Sarah's research activities towards this project were mentored by the project director, Dr. Lin Bao. Sarah has been trained broadly in entomology, phage display technology, protein biochemistry, molecular biology, microbiology and protein expression, etc., and has learned how to perform scientific research, from strategy to detailed tactics. Sarah’s research activities for this project resulted in an undergraduate research paper and three credits for her graduation from WMU at the end of 2013. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Each year global food loss caused by insect damage is estimated $100 billion, including approximately 14% of total crop loss and 20% of global stored food grain damage. However, the world faces fewer options for crop insect pest control due to de-registration of multiple classes of synthetic insecticides for their adverse impact to human health and environmental safety and development of insect resistance to currently marketed insecticides. Vestaron insecticide peptide technology provides solutions for both, which are specific to insects and not toxic to vertebrate, and won’t accumulate in the environments. However, these insecticidal peptides are not readily bioavailable to pests by ingestion because insect gut wall barriers prevent these large molecules from reaching their target sites. Therefore the insecticidal efficacies of these insecticidal peptides are 3-4 magnitudes less than the synthetic insecticides. This SBIR project is to develop a technology (TEP technology) to break the insect gut wall barrier and improve the insecticidal efficacy of the peptides. This TEP technology not only provides clean, safe, effective spray-on peptide insecticide products, but also provides opportunities to GMO plant market, a $5.4B market worldwide. This SBIR project proposed to identify Translocation Enchancement Peptides (TEPs) using in-vivo phage screen assays to help improve the efficacy of the insecticidal peptides. For this purpose, three objectives have been proposed originally and modified during the process of the project. The progress is described below. 1. Objective 1: Identify TEPs using in vivo phage screen technology. Rigorous efforts were taken to establish a reliable in vivo phage screen assay by feeding phage particles from phage libraries to Manduca sexta larva and establishing a phage decontamination protocol to differentiate the phage particles recovered from the larva haemolymph from those from the larva surface contamination. Then we moved quickly from the primary in-vivo phage screening to the current 4th round of screening. A total of 816 different phages were recovered from the primary in-vivo phage screening, and 192 of them have been through multiple rounds of screening. Thus far, 158 different “TEP” sequences were identified from the first round of screening, 79 “TEP” sequences were identified from the 2nd round, 33 “TEP” sequences were identified from the 3rd round, and 8 “TEP” sequences were identified from the 4th round. Three of these “TEP” sequences, P23TEP1, P23TEP2, and P26TEP1 have been selected for fusion peptide production and further insect bioassay evaluation. 2. Objective 2: Produce TEP fused insecticidal toxin peptides chemically and recombinantly. As originally planned, Vestaron contracted Bachem for chemical synthesis of TEP fused Omega-ACTX-Hv1a peptides with 20 mg each as follows. P23TEP1-omega-1 is P23TEP1::omega fusion. P23TEP1-omega-2 is P23TEP1::GGG::omega fusion. TEP fused hybrid+2-ACTX-Hv1a peptides were produced using Vestarons’ standard yeast expression system. The TEP fusion expression vectors were generated using K. lactis expression vector, pKLAC1, from New England Biolabs and then transformed into a K. lactis strain. At least 48 K. lactis transformants for expression of each of the TEP fusions were screened for identification of the production strains, which were then processed in the 2 L or 5 L fermentation to produce the desired TEP-hybrid fusion peptides. The fermentation beer, which contained the desired peptide, was then concentrated through Tangential Flow Filtration process. Finally rpHPLC was adopted to purify the TEP-hybrid+2 fusion peptides from the concentrated fermentation beer. Thus far 7 TEP fusion peptide expression yeast strains have been generated and used to produce the TEP fused hybrid+2 peptides as described below. P23TEP1-H2-1 is P23TEP1::Hybrid+2 fusion. 536.7 mg has been purified by HPLC. P23TEP1-H2-2 is P23TEP1::GGG::Hybrid+2 fusion. 504.4 mg has been purified by HPLC. P23TEP1-H2-3 is P23TEP1::G::Hybrid+2 fusion. 357.8 mg has been purified by HPLC. P23TEP1-H2-4 is Hybrid+2::P23TEP1 fusion. The production of this peptide by yeast fermentation is still in process. P23TEP2-H2-2 is P23TEP2::GGG::Hybrid+2 fusion. 466.8 mg has been purified by HPLC. P23TEP2-H2-5 is Hybrid+2::GGG::P23TEP2 fusion. 131.1 mg has been purified by HPLC. P26TEP1-H2-2 is P26TEP1::GGG::Hybrid+2 fusion. 138 mg has been purified by HPLC. 3. Objective 3: Evaluate TEP fusion peptides in the insect bioassays and their insect spectrum. The fusion peptides’ insecticidal activities were first evaluated using the Vestaron gold-standard housefly injection bioassay. In the housefly injection bioassays, the synthetic P23TEP1 fused omega peptides had 3 – 4 fold activity reduction comparing to Omega-ACTX-Hv1a. And all the TEP-Hybrid+2 fusion peptides produced from yeast fermentation had 3-6 fold reduction in insecticidal activity against houseflies compared with hybrid+2-ACTX-Hv1a. These results indicated the TEP peptides have some adverse impact on the toxin activities, no matter which TEP sequence is chosen, whether the linker exists or not, and no matter which fusion orientation is chosen. It appears that this activity reduction is simply related to the increased peptide size, which may cause more difficulty in penetrating the “blood-brain-barrier” of insects. To evaluate whether TEP peptides improve the insecticidal toxin efficacy orally, synthetic TEP-omega fusions were tested in the Manduca larva droplet feeding bioassay. In 4 replications of these experiments at toxin dose of 2mM, Omega-ACTX-Hv1a consistently caused 17 ± 2% mortality, P23TEP1-omega-1 caused 21 ± 20% mortality, equivalent to omega toxin; and P23TEP1-omega-2 caused 11 ± 11% mortality, slightly less active than omega toxin. Therefore, P23TEP1 did not improve the omega toxin potency in the Manduca droplet feeding bioassay. The recombinant TEP-Hybrid+2 fusions were tested in the Southern Corn Rootworm diet incorporation bioassays. By day 5 of this experiment, untreated control group only had 6% mortality, 43.8 µM of hybrid+2-ACTX-Hv1a (or 200 ppm) treatment resulted in 19% mortality, while SCR mortalities reached 56%, 50%, 63% and 56% in the treatments of P23TEP2-H2-2, P23TEP1-H2-1, P23TEP1-H2-2 and P23TEP1-H2-3 peptides at 43.8 µM respectively. In other words, all the tested TEP-Hybrid+2 fusion peptides appeared to be more active than the hybrid+2 itself in the SCR DIB bioassay. In summary, in the insect per os bioassay assay tests using two orders of insects (Lepidoptera and Coleoptera) in this project so far; TEP fusion peptides appeared to improve the oral availability of the peptides. In the Manduca droplet feeding bioassay, the TEP fusion toxins had similar insecticidal activities to the toxin itself. However, the direct delivery to the haemolymph in the housefly injection bioassay showed that the TEP fusions had 3-6 fold activity reduction compared to the non-fusion toxin, indicating possible more difficulty in insect “blood-brain-barrier” penetration due to the increased size of peptides. Therefore, in the Manduca droplet feeding bioassay the TEP fusion peptides may have better insect gut penetration than the toxin, but their activities were held back due to difficulties in the “blood-brain-barrier” penetration and possible relatively insusceptible Manduca. Later when the more susceptible insect, SCR, was used in the feeding bioassay, TEP fusion peptides showed improved activities. Though more insect species need to be tested and more replications of the per os bioassay need to be accumulated, these preliminary insect per os bioassay data seemed to indicate that TEPs improve the capability of the toxin to penetrate the insect gut across different orders of insects.
Publications
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