Part:BBa_K5066000
Cyt2Ba
Description
Bacillus thuringiensis toxins, or Bt toxins, are toxins that derive from Bt bacteria and are commonly used as insecticides as they can target specific insects without causing harm to other species. There are a wide variety of strains derived from a selection of Bt bacteria and each has similar effects but targets different species of insects. There are three main categories of the Bt toxin: Cry, Cyt, and Vip; there are also the Xpp, which were renamed from Cry.[1]
Usage and Biology
Cyt2Ba is a potent larvicide that works in the digestive tract of Aedes mosquitos. The Cyt protein’s reactivity to alkaline conditions in Aedes mosquito larvae’s midgut causes it to solubilize by increasing membrane permeability, and undergo proteolytic cleavage. This activates the protein, leading to its binding to unsaturated phospholipids present in the cell membrane of the larvae’s epithelial cells lining the alimentary canal. Such a disruption in an epithelial cell’s membranes changes its membrane permeability, hindering normal cell processes, and ultimately, cell lysis. Once this takes place on a large scale, larvae midgut tissues lose their function and cause symptoms in the Aedes mosquito larvae including starvation, electrolyte imbalance and eventually death.[2][3][4] Cyt2Ba is also used in composite part Cyt2Ba-p20-Xpp81Aa1 (BBa_K5066007) as it is one of the Bt toxins synergistic with basic part Xpp81Aa1[3]. The composite part is made up of Cyt2Ba, p20 (BBa_K5066008), Xpp81Aa1 (BBa_K5066001) and serves as an important launch point to future exploration of synergistic properties across Bt toxins.
Fig 1. Plasmid construct design in pET-28a
Characterization
The results from lanes 4 show that the IPTG has successfully inducted Cyt2Ba. Lane 3 has shown a faint band, indicating only a low protein concentration compared to the band on lane 4, which is vibrant and clear. Moreover, the band shows the correct size, 30KDa.
Growth curve studies of Cyt2Ba
Producing a collection of biolarvicidal toxin systems in the future where it becomes possible to synthesize any BioBricks of interest is ideal. The collection allows the rotation of insecticides preventing build-up of resistance in Aedes populations [5]. Even though the risk of developing resistance from the treatment of biolarvicidal toxins from Bacillus thuringiensis and Buthus martensii Karsch is limited, implementing integrated resistance management is still important for our project. It is pivotal to have a comprehensive understanding of the recombinant bacteria that harbor plasmids that we designed and built.
Therefore, a basic growth curve study was conducted to understand the growth trend of Cyt2Ba so that the growth rate of each unique biolarvicidal toxin and the ideal time to induce the bacterium can be determined to achieve maximum production of proteins from BL21(DE3).
Fig 3. Growth trend of Escherichia coli BL21(DE3)-pET28a-Cyt2Ba (with stop codon) for the optimal time to induce the bacterium to achieve maximum protein production.
The results of our raw data can be accessed here: https://docs.google.com/document/d/12cTsTOBVM_m1gROwZoAqAVQ6NDNmVKQHLPyWTKn1oTs/edit?usp=sharing
IPTG-inducted cultures showed an exceptionally slower growth rate than non-inducted cultures.
Analysis of Aedes mosquito larvae exposed to Cyt2Ba synthesised by our engineered bacteria
The larvicidal efficacy of Cyt2Ba against Aedes albopictus larvae was tested by exposing 10 larvae in a cup to Cyt2Ba by dissolving its serially diluted wet pellets. (Pellets containing recombinant biolarvicidal toxins were collected from BL21(DE3) bacterial cultures.) The larvicidal efficacy of toxins was compared with ddH2O as a control. (Note: control larvae were still alive when collected, whereas larvae exposed to larvicides were collected when they died.) The LC50 (lethal concentration required to kill 50% of the population) and morphology of larvae were examined and analyzed.
LC50
Morphology
Fig 5. Microscope amplification of control larvae
In Figure 5, features can be seen as annotated. An unbroken and food-filled midgut and continuous epithelium.
Fig 6. Microscope amplification of midgut breakage in larvae exposed to Cyt2Ba
In Figure 6, there is a breakage in the larvae midgut. A clean spit in its epithelium is present, unlike in the control, where it is continuous. Despite the breakage, there is no observable damage to its surrounding body, meaning that the damage was unlikely caused by errors during the making of microscope slides and likely caused by the Cyt2Ba toxin.
Fig 7. Second microscope amplification of Midgut damage in larvae exposed to Cyt2Ba.
Figure 7 is an example of a larva that shows both external damage and internal damage. The area circled in white shows breakage similar to that in Figure 4, where it is purely internal and likely the action of the Cyt2Ba toxin. The area bracketed in black is external damage, likely from errors during the moving of the larva from its cups to its microscope slide. Additionally, both Figure 4 and 5 larvae have an absence of food in their midgut.
Deviating from the effects of Cyt2Ba on larvae which had been predicted by theory, some larvae showed different characteristics.
Fig 8. Third microscope amplification of Midgut damage in larvae exposed to Cyt2Ba.
The larva in Figure 8 shows characteristics not predicted by theory: darkening in the midgut. Deformities in the midgut are also a characteristic of midgut damage, but it is less certain that darkening is also a characteristic of midgut damage. The reasons for darkening will require additional research.
Larvae Scale
Larvae scale is a continuance of morphology; it's hypothesized that the potent recombinant larvicidal toxin Cyt2Ba would result in early mortality, and hence, the length of each larva was measured. The length of larvae exposed to Cyt2Ba and control groups were analyzed with ImageJ.
Fig 9. Control Larva length
Fig 10. Cyt2Ba Larva length
These larvicide assays were all carried out within the same time frame, but the larvae were mounted at different times. The control larvae were in the late stage three of their development during mounting, while most of the larvae from the treatment group were mounted at various time frames when they died. Therefore, the length of the larvae reflected the true length of their size in real life. The larvae were washed and preserved in absolute alcohol and mounted with the mounting solution. The difference observed in length, therefore suggests the relative efficacy of the toxins.
Fig 11. The relative length of larvae treated by Cyt2Ba and Xpp81Aa1 Compared to the control group. (For this registry, only Cyt2Ba is relevant)
The deviations of the results from Cyt2Ba larvicidal assays were taken relative to the control group. The values above and below the 0 point represent the extent to which a value deviates from the average control measurement (0.5338 cm). Cyt2Ba is represented by the pink values.
This graph shows that there is no significant difference between the bodies of the larvae exposed to Cyt2Ba and Xpp81Aa1, but relatively, the treated group was shorter in length compared to the control groups. It was deduced that the shorter length was due to the early mortality experienced by the treatment groups compared to the control groups.
The biolarvicidal assay started with larvae of the same stage, stage 3, and the control group was mounted 2 days after the test. Due to the larvae’s early mortality, they were unable to grow past the stage at which they were upon death. This allows us to see that the effects of Cyt2Ba and Xpp81Aa1 manifested in the form of early death, showing that the larvicides indeed impact larvae mortality.
Immune responses of Aedes albopictus larvae by qPCR
Furthermore, to determine whether the mortality that we observed from the biolarvicidal assay is solely due to the mechanisms exerted by each of the toxins, such as the cytolytic effects of Cyt2Ba, it is necessary to rule out the effect of E.coli in the death of mosquito larvae.
Measurements
The experiment was designed to observe the effect of Cyt2Ba toxins on Ae.albopictus larvae in comparison to control (negative control), BL21(DE3)-empty vector (control), and treatment groups. BL21(DE3)-empty vector is used to investigate whether the mortality observed was due to the cytotoxicity effect or bacterial infection of the E.coli or solely due to the potency of the Cyt2Ba.
Hence, real-time PCR, also known as quantitative PCR (qPCR), was run to confirm our hypothesis. This is a method used to determine the concentration of a target DNA[6]. To carry out qPCR, the mRNA from the larvae was first extracted and reverse-transcribed into cDNA[7]. Subsequently, qPCR was performed to examine the expression levels of the target immune genes, including Gambicin, Rel1, Rel2, and Defensin of the larvae. Primer sequences can be accessed from this link: https://docs.google.com/document/d/1vuQBWPxt6SqvY4GTTdZnFptT6rTMivvPFxrSpYhxPjI/edit?usp=drive_link
These key innate immune genes of Ae. albopictus larvae were selected to investigate the responses regulated by the larvae after exposure to the control, BL21(DE3)-empty vector, and treatment groups such as BL21-Cyt2Ba, BL21-BmK IT1 and BL21(DE3)-Xpp81Aa1.
Innate immune genes in Aedes albopictus
Gambicin and Defensin are antimicrobial peptides. It is reported that Gambicin is regulated by various immune pathways such as Toll, JAK-STAT, and ImD in Aedes mosquitoes[8]. Defensin is an inducible immune gene response to viruses and bacteria [9]; it is an acute phase response protein gene [10]. In addition, we studied two innate immune pathways, Toll and immune deficiency (IMD) pathways. These pathways are the first defense line against pathogens in Ae. albopictus larvae. The genes we selected were homologous members of NF-κB factors such as Relish-1 (Rel1) and Relish-2 (Rel2), both of which are downstream transcription activators of the Toll and Immune Deficiency (IMD) pathways [11].
Experiment design
Larvae that were exposed to BL21-Cyt2Ba with IPTG induction at 20°C overnight (B1) (Figure 20A) and IPTG induction at 37°C for 4hr (B2) (Figure 20B), compared to water control, and BL21(DE3)-empty vector were collected at 24h and 48h post-exposure. The fold expression of Gambicin, Defensin, Rel1, and Rel2 in response to biolarvicidal toxins was measured relative to the housekeeping gene, S7.
Results and discussion
In this experiment, two different bacteria cultures were used and cultured in different conditions, which were 37℃ and 20℃. Different culturing conditions were explored to investigate whether yields would differ at different conditions. In this experiment, two different bacteria cultures were cultured in different conditions and used to conduct biolarvicidal assays to investigate the immune response of larvae to Cyt2Ba. The two conditions that were tested include IPTG induction at 20°C overnight and IPTG induction at 37°C for 4hr.
Fig 12. Expression levels of Gambicin relative to the housekeeping gene S7 (A & B) Larvae exposed to biolarvicidal toxins with IPTG induction at 20°C overnight, and (C & D) IPTG induction at 37°C for 4 hours, respectively, compared to the control group treated with ddH2O and BL21(DE3)-empty vector at 24 hours and 48 hours
Fig 13. Expression levels of Rel1 relative to the housekeeping gene S7 (A & B) Larvae exposed to biolarvicidal toxins with IPTG induction at 20°C overnight, and (C & D) IPTG induction at 37°C for 4 hours, respectively, compared to the control group treated with ddH2O and BL21(DE3)-empty vector at 24 hours and 48 hours
Fig 14. Expression levels of Rel2 relative to the housekeeping gene S7 (A & B) Larvae exposed to biolarvicidal toxins with IPTG induction at 20°C overnight, and (C & D) IPTG induction at 37°C for 4 hours, respectively, compared to the control group treated with ddH2O and BL21(DE3)-empty vector at 24 hours and 48 hours
Fig 15. Expression levels of Defensin relative to the housekeeping gene S7 (A & B) Larvae exposed to biolarvicidal toxins with IPTG induction at 20°C overnight, and (C & D) IPTG induction at 37°C for 4 hours, respectively, compared to the control group treated with ddH2O and BL21(DE3)-empty vector at 24 hours and 48 hours
For Cyt2Ba produced at 20°C overnight, there was a lower regulation for the Rel1 gene compared to water only and lower expression of Gambicin compared to the control group. It should be acknowledged that there is a limitation to this particular study because biolarvicides were not compared to BL21(DE3) for biolarvicidal toxins produced at 20°C overnight. There was a shortage of mosquito larvae so it was not possible to conduct additional groups during this period of time, hence, cytotoxicity effect of BL21(DE3) could not be compared.
The qPCR data of Cyt2Ba produced at 37°C for 4 hours demonstrated that the majority of the transcripts of immune genes were not regulated after the larvae were exposed to the toxins. The transcripts of Gambicin, Rel1, and Rel2 of BL21(DE3)-empty vector were significantly lower than the control group for the 24-hour group. This data indicated that the mortality observed in the larvicidal assay was not due to the introduction of BL21(DE3). Groups treated with BL21-Cyt2Ba have lower expression of immune gene transcripts.
In conclusion, the qPCR study showed that the potency observed from Cyt2Ba was due to the toxicity and mechanism of the cytolytic effect of Cyt2Ba.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 399
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 399
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 399
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 399
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 399
- 1000COMPATIBLE WITH RFC[1000]
Reference
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[2] Bravo, A., Likitvivatanavong, S., Gill, S. S., & Soberón, M. (2011). Bacillus thuringiensis: A story of a successful bioinsecticide. Insect Biochemistry and Molecular Biology, 41(7), 423–431. https://doi.org/10.1016/j.ibmb.2011.02.006
[3] Wu, J., Wei, L., He, J., Fu, K., Li, X., Jia, L., Wang, R., & Zhang, W. (2021). Characterization of a novel Bacillus thuringiensis toxin active against Aedes aegypti larvae. Acta Tropica, 223, 106088. https://doi.org/10.1016/j.actatropica.2021.106088
[4] Gu, J.-B., Dong, Y.-Q., Peng, H.-J., & Chen, X.-G. (2010). A Recombinant AeDNA Containing the Insect-Specific Toxin, BmK IT1, Displayed an Increasing Pathogenicity on Aedes albopictus. American Journal of Tropical Medicine and Hygiene, 83(3), 614–623. https://doi.org/10.4269/ajtmh.2010.10-0074
[5] Dusfour, I., Vontas, J., David, J. P., Weetman, D., Fonseca, D. M., Corbel, V., ... & Chandre, F. (2019). Management of insecticide resistance in the major Aedes vectors of arboviruses: Advances and challenges. PLOS Neglected Tropical Diseases, 13(10), e0007615.
[6] Dymond, J. S. (2013). Explanatory Chapter: quantitative PCR. Methods in Enzymology, 529, 279–289. https://doi.org/10.1016/b978-0-12-418687-3.00023-9
[7] Gene Expression Analysis Using Real-Time PCR | Thermo Fisher Scientific - CA. (2024). Thermofisher.com. https://www.thermofisher.com/tw/zt/home/life-science/pcr/real-time-pcr/real-time-pcr-applications/gene-expression-using-real-time-pcr.html
[8] Zhang, R., Zhu, Y., Pang, X., Xiao, X., Zhang, R., & Cheng, G. (2017). Regulation of antimicrobial peptides in Aedes aegypti Aag2 cells. Frontiers in Cellular and Infection Microbiology, 7, 22. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291090/
[9] Zhao, L., Alto, B. W., Smartt, C. T., & Shin, D. (2018). Transcription profiling for defensins of Aedes aegypti (Diptera: Culicidae) during development and in response to infection with Chikungunya and Zika viruses. Journal of Medical Entomology, 55(1), 78-89. https://pubmed.ncbi.nlm.nih.gov/28968775/
[10] Cho, W. L., Fu, T. F., Chiou, J. Y., & Chen, C. C. (1997). Molecular characterization of a defensin gene from the mosquito, Aedes aegypti. Insect Biochemistry and Molecular Biology, 27(5), 351-358. https://pubmed.ncbi.nlm.nih.gov/9219362/
[11] Zou, Z., Souza-Neto, J., Xi, Z., Kokoza, V., Shin, S. W., Dimopoulos, G., & Raikhel, A. (2011). Transcriptome analysis of Aedes aegypti transgenic mosquitoes with altered immunity. PLOS Pathogens, 7(11), e1002394. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3219725/
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