BIOLOGICAL CONTROL
Bacillus thuringiensis (Bt) is a bacterium used as an alternative to chemical insecticides. Different strains of this bacterium display a narrow range of insect toxicity by producing different proteins (mainly in the Cry and Cyt families), which allows more selectivity when targeting insect populations. They are also innocuous to most other animals, which reduces environmental impact. One further advantage is that these proteins can be quickly degraded in the environment, in contrast to recalcitrant chemicals, reducing the window of effect and persistence.
Even though proteins from Bt have been used for many years to combat insect pests, many aspects of exactly how they act to damage insect midguts remain elusive. Research in the groups of Dr. Alejandra Bravo and Dr. Mario Soberón is focused on understanding the mode of action of these proteins, and this information has proven vital to genetic modification of the bacteria, biotechnological application in the field, and understanding and managing resistance traits in populations. |
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To answer this question, I used time course exposures of Aedes aegypti larvae to Cry11Aa and analyzed the transcriptome of the midgut throughout the intoxication. The trancriptomic analysis indicated up-regulation of genes in MAPK stress networks, as well as some previously undescribed changes in expression of pathways involved in endo/exocytosis, membrane lipid traffic, and cell junction maintenance. Bioinformatic analysis also showed clusters of genes with similar regulation profiles, which could be modulated by some master transcription factors.
Transcriptomic results were validated trough RT-qPCR, where strong correlation in gene expression was only found with larvae exposed to fully functional Cry11Aa. Mutant proteins that halt the mode of action at different steps did not recapture the response seen through transcriptomics. This suggests that binding of the protein to the midgut cells is not enough to trigger defense responses and formation of pores is necessary. |
I also collaborated with groups in Brazil to analyze the expression levels of Bt toxin receptors and other genes in resistant lines of the moth Spodoptera frugiperda, a voracious pest of multiple crops. This information will help determine if the resistance phenotype is linked to changes in gene expression or to mutations in the proteins that reduce binding of toxins to the midgut cells.
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Identification of determinants of specificity and synergyThe remarkable toxicity of Bti to mosquitoes stems from the synergistic effects between the individual proteins it produces, each one enhancing the toxicity of the other. Knowledge of the mechanism of how this happens is important when performing genetic modifications that could improve toxicity of Bti preparations. It was known that when Cyt1Aa interacts with Cry11Aa it facilitates structural changes that make pore formation and insertion in cell membranes more efficient. Despite having no known protein receptor and an affinity to membrane lipids, Cyt1Aa shows an in vivo toxicity range limited to mosquitoes, which is puzzling.
I investigated if the regions known to interact with Cry11Aa could also interact with the other proteins from Bti, and found that these sites are also important for toxic synergy between Cyt1Aa and Cry4 proteins, suggesting a common mechanism. I also compared different steps of processing, binding and insertion of Cyt1Aa in midgut membranes between Aedes aegypti and Maduca sexta, a species not susceptible to this protein. This allowed to determine which steps are not decisive in conferring insect specificity. I also determined that Cyt1Aa is capable of inserting in non-target membranes, though it is still unclear if this is followed by disruption of cell integrity or pore formation. |
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Selection of modified insect specificityPhage display is a powerful molecular biology technique to retrieve peptides with particular binding properties from massive libraries of variants, thereby reducing the work involved in mutagenesis and selection. We tested different phage constructs and systems to determine which platform was efficient in producing complete Bt proteins attached to phages. We eventually also compared this system to other high-throughput selection technologies (e.g. ribosome display).
I was involved in construction of Cyt1Aa vectors and testing of display efficiency. Our goal was to create a Cyt1Aa library that we could select against membranes of the non-target insect Manduca sexta. Variants of Cyt1Aa with increased binding to these membranes could potentially be used for synergy with Cry proteins toxic to lepidopteran species rather than mosquitoes. |