Results

In this section we like to present you the main results of the BeeT project. BeeT is engineered to produce a toxin specific for Varroa destructor, produce the toxin on the right time and incapable of escaping the hive alive. To accomplish this, we performed multiple experiments and created different models. The outcome is in short shown on this page.

In order to improve on existing methods, BeeT should effect Varroa mites only. To accomplish this we decided to make use of Cry toxins. These toxins are naturally produced by Bacillus thuringiensis and because of this also known as BT toxins. A functional Cry toxin is only effective when specific binding occurs to the gut membrane of the target organism. Hereafter, the Cry toxins will form pores into the cell membrane, which results in cell death. As cell death occurs, the gut membrane becomes porous. Consequently, the organism dies. ¹ To find a Cry toxin active against V. destructor we engineered our own toxins and we searched in nature for one as well.

Due to the parasitic nature of Varroa mites, testing Cry toxins proved to be very problematic. To overcome this problem we developed an in vitro test for Cry toxins. Out of the membranes of the target organism, brush border membrane vesicles (BBMVs) were made and incorporated with 6-carboxyfluorescein. A functional Cry toxins will create pores into the BBMVs, which then results in the leaking of fluorophores out of the BBMVs. Due to self-quenching behaviour of 6-carboxyfluorescein, this can be measured as an increase in fluorescence. As a proof of principle, BBMVs from the gut of Tenebrio molitor were made and loaded with 6-carboxyfluorescein to test the pore formation ability of Cry3Aa, which is known to be toxic to T. molitor larvae ³. Figure 1a shows how the fluorescence increases of BBMVs incorporated with fluorophores in the presence and absence of Cry3Aa. A kinetic value could be coupled to this process. These values for multiple measurements for BBMVs in the presence and absence of Cry3Aa are shown in Figure 1b. From this can be concluded that the presence of a functional Cry protein results can be measured.

Figure 1. (a) The fluorescence of a two solutions with BBMVs obtained from T. molitor incorporated with fluorophores was measured over time. One in the presence and one in the absence of Cry3Aa. (b) The reaction rate constants for 6 individual measurements were calculated with the equation: fluorescence_t=fluorescence_(t=∞)-fluoresence_(t=∞)∙e^(-k∙t)+fluorescence_(t=0).

To engineer a Cry toxin active against V. destructor we took the Cry3Aa toxin as a starting point. This Cry toxin consists of three domains, from which one is responsible for the binding. In 1996, Rajamohan et al. demonstrated that mutations in the binding sites of Cry toxins can both decrease and enhance the specificity of a toxin towards its target organism². Three putative binding sites have been identified after analysing the 3D structure of the binding domain of Cry3Aa. The putative sites were changed with random mutagenesis and the adapted proteins were cloned into E. coli. 144 Cry proteins were produced and tested for activity on BBMVs from V. destructor as described previously. From these measurements 24 candidates were selected to test further. The results are shown in Figure 2. From these results, it can be concluded that the third binding site (amino acids 410-416) seems to be a good candidate for future engineering and specificity adaptation of this particular Cry toxin. Due to the relatively high deviation in reaction speed for the toxins 3.3.3 and 3.3.7, these should not be taken into account, as they are rather inconclusive. This leaves us with one proper candidate – the toxin mutant Cry3.3.5.

Figure 2. Heatmap of the reaction constants relative to the blank. A higher value indicates a higher toxicity and specificity of the tested toxin.

In order to find the right specific binding motif, phage display was performed. Phages with a binding motif on their exterior were exposed to the gut membrane of V. destructor. Hereafter, the bound phages were isolated and analysed. The filamentous bacteriophage M13 was used with a 12-mer library (The Ph.D.™-12 Phage Display Peptide Library). The phages were fed to Varroa mites and exposed to BBMVs originating from Varroa mites. The recovered phages were isolated and sequenced. The consensus sequences of the binding motif of the 12-mer both in vivo and in vitro are shown in Figure 3.

Figure 3. Consensus sequence of recovered phages in (a) in vivo phage display, (b) in vitro phage display, and (c) the combined results. The legend shows which amino acid has which properties. The letters “N” and “C” in the graph indicate the N-terminus and the C-terminus of the protein respectively.

Alongside creating a Cry toxin ourselves, we searched in nature for one as well. We gathered 800 death Varroa mites and looked for B. thuringiensis or related species inside these mites that might have been the cause the death. Figure 4 shows the morphology of B. thuringiensis and two found strains. Five out of 106 isolates were identified as Bacillus-like species. One strain, not B. thuringiensis, showed the presence of a large overexpressed protein and was sent for sequencing. We are waiting with excitement for the results.

Figure 4. Microscopy images of Coomassie-stained isolates, 1000x magnification with a Zeiss Axio Scope.A1 brightfield microscope. (a) B. thuringiensis HD350. The red arrow points to a Cry toxin, the green arrow to a spore and the yellow arrow to a vegetative cell. (b) Isolate 62, a coccus. Most isolates had this morphology. (c) Isolate 82, showing Bacillus-like morphology.

A constant low level of Cry toxin can facilitate resistances¹¹. That is why when BeeT spreads through the hive, the toxin production should be regulated. We created two main systems that regulate the toxin production. One is a system designed with riboswitches that promote toxin production when Varroa mites are present. The other system, that works in parallel with the riboswitches, uses quorum sensing to start toxin production only when the concentration BeeT is high.

Riboswitches are pieces of mRNA that can regulate gene expression depending on whether it is bound to a ligand. The ligands for the riboswitches that were used here were guanine and vitamin B12. Both substances indicate the presence of Varroa mites. 95% Of the mite faeces consist of guanine. Vitamin B12 is present in the haemolymph of the honey bees, which is the food source of Varroa mites. Both riboswitches are successfully built into a construct in a way that when the ligand is present, toxin can be produced. Furthermore, they have been tested with RFP as reported gene in the presence of different concentrations of their corresponding ligand. The results for the vitamin B12 riboswitch are shown in Figure 5. As can be seen here, when the concentration vitamin B12 increases, the RFP production increases as well.

Figure 5. Escherichia coli with the vitamin B12 riboswitch coupled to a RFP output was grown overnight in the presence of different concentration vitamin B12. (a) The fluorescence divided by OD over time is shown for different concentrations vitamin B12. (b) The relation between the fluorescence divided by OD after 12 hours incubations and different concentrations B12 can be seen.

The second regulatory system uses quorum sensing. A quorum sensing mechanism enables the bacteria to regulate their expression based on their density. We adopted the lux system originating from Vibrio fischeri and demonstrated this system’s functionality using a newly constructed GFP reporter, which can been seen in Figure 6. When the cell density increases, the cells will sense each other’s autoinducers. These induce via a complex production of more autoinducers and production of GFP.

Figure 6. Fluorescence and absorbance data for E. coli quorum sensing strains. The continuous line represents the fluorescence divided by OD600. The dashed line represents the absorbance at 600 nm. Whereas the red and green line represent quorum sensing strains, the purple strain has a reporter plasmid only. For both strains every value displayed is the average of at least three technical replicates and for each, the line displayed is one of three biological repeats all showing a similar pattern.

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Here all the nice results regarding the light kill switch and biocontainment will be shown.

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Testing BeeT in ~~a Beehive~~ BEEHAVE

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Beehave model and a nice conclusion

Team:Wageningen UR/Results

Results

Results

Testing BeeT in a Beehive BEEHAVE