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Wageningen UR iGEM 2016

To improve on existing methods, BeeT should affect Varroa mites only. This means it should be harmless to bees, humans and other organisms. In our search for a suitable toxin we looked into Cry toxins, also named Bt toxins, which are produced by B. thuringiensis. Due to interaction with species specific receptors in the gut membrane, these toxins are known to be very species specific. To find a suitable Cry toxin we tried isolating B. thuringiensis from dead mites, as well as mutating an existing Cry toxin. For testing the potential toxins that were obtained, we employed an in vitro toxicity assay with brush border membrane vesicles made from Varroa mites. The vesicles contain fluorophores. When a Cry toxin is applied that is toxic to Varroa mites it binds the receptors on the vesicles, rupturing them and releasing the fluorophores.

In Vitro Assay

A High Throughput Method to Test Cry Toxin Activity

Cry toxins have been found to have an activity towards insects of different phylogenetic orders. In short, a functional Cry protein is only active when specific binding occurs to the gut membrane of the target insect. In more detail, these receptors have been found in the gut epithelium cells, where the Cry proteins polymerize binding to its membrane receptor forming pores (holes). After the formation of these ion channels, the osmotic pressure is disturbed resulting in cell death of the target host¹².
Although a number of Cry toxins have been (partly) characterized, a toxin specific to Varrao destructor has yet to be found. For this reason, we developed an in vitro toxicity test in order to be able to efficiently screen Cry toxins on their activity against V. destructor. From the V. destructor, brush border membrane vesicles (BBMVs) were made and loaded with the fluorophore 6-carboxyfluorescein to test the pore formation ability of Cry proteins, see Figure 1a. 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 ³⁴.

Figure 1. Schematic overview of the function of the in vitro assay to the activity of Cry proteins. When active Cry toxins are mixed with BBMVs incorporated with fluorophores (a), they will bind to their receptors and create pores into the membrane (b). The thereby induced leaking of fluorophores results in an increase in fluorescence (c).

BBMVs are composed of phospholipid double layers originating from midgut membrane cells. This membrane therefore should also contain the membrane proteins of the midgut membrane. After incorporation of 6-carboxyfluorescein into the BBMVs the presence of an active Cry toxin can principally be detected as followed: the Cry toxins will bind to their highly specific receptors ⁵ and consequently create holes into the phospholipid double membrane layer, see Figure 1b. After this event, the fluorophores leak out of the vesicles due to diffusion, which can be measured as an increase in fluorescence. This method has been proven to work for the activity of Cry3Aa on Leptinotarsa decemlineata. ⁶. Rausell et al. only demonstrated how the magnitude of the fluorescence increase links to toxicity, but this method has its limitations. BBMVs have the disadvantage of being unstable and undergoing degradation⁷. In other words, measurements on BBMVs can be influenced by spontaneous breaking of the vesicles. In our project, we took this into account by relating toxicity activity to a kinetic value.

The increase of fluorescence due to fluorophore leaking can be attributed to dequenching Dequenching refers to any process which increases the fluorescence intensity of a given substance. . 6-Carboxyfluorescein is a self-quenching fluorophore. At a high concentration 6-carboxyfluorescein forms dimers. Because of dimer-dimer and dimer-monomer interactions, the fluorescence of a carboxyfluorescein molecule decreases drastically⁸. The vesicles created in our study contain a high concentration of fluorophores, which results in self-quenching behavior. When the fluorophores leak out of the vesicles, the local concentration of fluorophores decreases. Hence, dequenching occurs and the fluorescence increases. This effect is illustrated in Figure 1c.

Analysis of Brush Border Membrane Vesicles

To visualize the presence of BBMVs, images were made with the use of TEMTransmission Electron Microscopy . This was done in collaboration with the iGEM team from Delft

Figure 2. TEM images of BBMVs. The images of v. destructor were made by the iGEM team from Delft.
(a) T. molitor (b) V. destructor

Figure 2 shows the vesicles - round shapes with sizes between the 100 nm and 300 nm. To gain more information about the composition of the samples that are visualized above, Dynamic light scattering was performed. The dynamic light scattering results give information about the size distribution of the particles that are present in the solution.

Figure 3. The size distribution of particles in solution of BBMVs obtained from Tenebrio molitor and Varroa destructor calculated with data obtained by Dynamic Light Scattering.

Looking at both transmission electron microscopy results and dynamic light scattering results, the T. molitor samples have a large population of vesicles that vary their size around 200 nm. Looking at the V. destructor samples, the population around this size is smaller. To learn more about the proteins in these samples, click here.

Fluorophore Leaking Experiments

The hypothesis is that due to addition of a detergent, the phospholipid bilayer will rearrange. This process will subsequently give the fluorophores the chance to escape their enclosed space and disperse in the solution. The dispersion of the fluorophores into the solution would then cause an increase in fluorescence as described previously.

To determine whether these BBMVs were capable of releasing fluorophores and whether this release could be measured, the following was done: to BBMVs containing carboxyfluorescein, 1 volume percent of SDS (sodium dodecyl sulphate) was added to disturb their stability. Meanwhile the fluorescence was measured over time.

Looking at Figure 4, after addition of SDS (detergent), an increase in fluorescence is indeed visible. However, the same increase in fluorescence can be observed over time with vesicles that are in an environment without SDS. Two possible explanations for this phenomenon are that BBMVs are already porous or are not stable under the circumstances during measurement. Both processes would result in an increase in fluorescence over time. In order to compare induced fluorophore leaking from standard fluorophore leaking, the speed of the carboxyfluorescein leaking should be calculated and compared.

Figure 4. (a) The fluorescence of a solution with BBMVs incorporated with fluorophores and a solution with free fluorophores was measured over time. After 330 seconds, detergent was added. (b) The fluorescence of a two solutions with BBMVs incorporated with fluorophores was measured over time. To one sample detergent was added. Both graphs were made from data obtained from BBMVs from T. molitor. For both graphs a similar effect was visible with V. destructor BBMVs.

Obtaining Kinetic Values

To determine whether a specific Cry protein can induce fluorophore release, Cry3Aa was Isolated from B. thuringiensis and tested on BBMVs obtained from T. molitor. Activated Cry3Aa gives a clear band on a gel of a 68 kDa protein after SDS-PAGE was performed, as described by Rausell et al.⁶. The SDS-PAGE gel of the protein extract from B. thuringiensis, shown in Figure 4, shows such a band of approximately this size.

Figure 4. SDS-PAGE gel. M represents the marker lane. In the other three lanes, protein extracts of B. thuringiensis are loaded. Lane 1 has an undiluted sample, lane 2 a 10x diluted sample and lane 3 a 100x diluted sample.

T. molitor BBMVs were incubated with and without Cry3Aa and the fluorescence was measured overtime see Figure 6. From this graph it can be concluded that the presence of Cry3Aa results in a faster increase in fluorescent and therefore in a faster fluorophore release. To determine how large the role of induced leaking is, the kinetic properties of both measurements have to be compared. In order to compare these properties, it should be known which kinetic rules the fluorophore leaking follows. Plotting the natural logarithm of the concentration of fluorophores entrapped in vesicles against time, gives a straight line, see Figure 6. The concentration fluorophores entrapped in vesicles is expressed as the difference of the maximum fluorescence and the fluorescence at a certain time point. The observed straight line in this graph tells us that first order reaction rules can be applied on the measurement⁹. This means that the following formula can be applied to the obtained data:
$r e a c t i o n r a t e = k_{f} \cdot [e n t r a p p e d f l u o r o p h o r e s]$
The reaction rate constant, k_f, determines how fast the reaction goes. This constant can be used to compare one reaction with another. To find this constant the following formula was plotted through directly obtained data:
$f l u o r e s e n c e_{t} = f l u o r e s e n c e_{t = \infty} - f l u o r e s e n c e_{t = \infty} \cdot e^{- k \cdot t} + f l u o r e s e n c e_{t = 0}$
The plotted formulas are visible in figure 8 and 9 as well.

Figure 6. 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 ofCry3Aa. (a) shows the fluorescence against time, whereas (b) shows the natural logarithm of the maximum fluorescence minus the fluorescence at a certain time point against the time.

The measurements for both samples were done in 6-fold. Representative measurements of each sample are shown in Figure 6. All data can be found here. For all measurements, the reaction rate constants were determined as explained above and are shown in Figure 9. The figure shows that in the presence of Cry3Aa, the k_f values are higher and thus the fluorophores are released faster. Overall, fluorophore leaking in the presence of Cry3Aa is accelerated with a factor of 1.59 compared to fluorophore leaking in the absence of Cry3Aa. Unless one of the other few proteins still present in the sample would unexpectedly interfere with the experiment, Cry3Aa induces leakage of carboxyfluorescein from T. molitor BBMVs.

Figure 7. A boxplot shows the k_f values that represent the velocity at which fluorophore release occurs for BBMVs obtained from T. molitor in the presence and absence of Cry3Aa.

A library of Cry3Aa proteins with mutations in the three different binding sites was made to create a Cry toxin that is potentially toxic to V. destructor. When one of these mutant Cry3Aa proteins is toxic to V. destructor, the toxin should induce leakage of carboxyfluorescein from V. destructor BBMVs, and therefore have a higher k_f value than the negative control. The influence of Cry3Aa on T. molitor vesicles can be used as a positive control. The process of optimization of the positive control can be found in the notebook. It should be noted, however, that the relationship between the effect of the Cry toxin on vesicles and the Cry toxin on the whole organism might be different for each organism. To know with certainty whether a certain Cry toxin is lethal for V. destructor, an in vivo test should be performed.

Toxin Engineering

As mentioned above, no specific toxin to Varroa mites is known. To overcome such limitation, we tried to engineer one. As a basic part, we used the Cry3Aa toxin – toxic to a number of Coleoptera as the Colorado potato beetle Leptinotarsa decemlineata and the mealworm Tenebrio molitor –, previously shown to be active against 4 different types of mites (Acarus siro L., Tyrophagus putrescentiae, Dermatophagoides farinae Hughes, and Lepidoglyphus destructor)¹. This engineering focused two different strategies:
1) finding specific binding motifs to the receptors within the mite gut – Phage display and
2) modification of the previously described motifs within the Cry3Aa structure –Random mutagenesis of the binding sites.

Binding site mutagenesis

Design

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. Knowing this, the 3D structure of the Cry3Aa toxin has been analysed. Three putative binding sites have been identified. These can be seen in the following table.

Region Name	Amino Acid Region	Amino Acid Sequence
3.1	312-317	NNLRGY
3.2	349-355	GYYGND
3.3	410-416	VWPSAVY

To mutate these, the plasmid pSB1A3_Cry3Aa_TEV_HIS has been used as a template. This plasmid contains the wildtype Cry3Aa toxin (Bacillus thuringiensis var. tenebrionis, Taxonomy ID: 1444). Its expression is regulated by the arabinose inducible promotor pBAD/araC (Bb_I0500). From this, three different plasmids have been created with randomized sequences within the binding sites (see protocols). By this we expect that each individual colony harbouring this plasmid containing an individual variants, in other words, a library of Cry3Aa proteins with randomly binding sites to be screened.

Screening

The mutants have been screened following the hightroughput procedure developed by Jacco (see above). In short, random colonies were picked from each of the binding mutants (n=144) for toxin expression and from these (n=24) were selected for activity testing (see protocols).As a negative control, cell free extract from empty expression strain, BL21(DE3) has been used, see Figure X... .

Figure x. relative k-values of 24 different mutants compared to Cry3Aa and the empty expression strain.

Figure x. Heatmap of the relative k values.

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. This one has both a higher mean reaction velocity than the reference value and a relatively narrow range of measured relative k-values obtained for the 9 tested samples. Furthermore, the negative control shows that other proteins present in the cell free extract or cell debris (e.g membrane residue) interfere with our reading. Nevertheless, our preliminary results show that the mutant revokes a stronger response than the negative control, suggesting a specific binding to the Varroa vesicles.

Library sequencing

To find an indication what future engineering steps could be of use, the sequences of the altered binding sites have been obtained and looked at.

Phage display

Due to difficulty that is to obtain mites, the phage display experiments were set up in a model organism where a Cry toxin as already described, Tenebrio molitor (Cry3Aa). Furthermore, mealworms can be purchased in a lot of places and they are easy to maintain. In here, we expected to proof the applicability of this technique for specific protein engineering towards Varroa destructor. M13 was used and two different libraries were used; 7-mer flanked by a pair of cysteine residues (The Ph.D.™-C7C Phage Display Peptide Library) and the 12-mer (The Ph.D.™-12 Phage Display Peptide Library).

Display of M13KE-PIII libraries

Two different strategies for phage display were used, in vivo (conventional phage display) and in vitro (panning). For the in vitro display, a modified biopanning protocol, the Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL) method, was used.

in vivo

First as a proof of concept feeding experiments were performed with fluorescine on mealworms and mites (see protocol). In short, the insects were fed with either a piece of carrot soaked with fluorescine or a bee larvae soaked with fluorescine. After washing with PBS buffer, the insect were ground up and fluorescence was measured.

After seeing that the mites and mealworms could take up fluorescine, the phage libraries were fed to T. molitor and V. destructor and the bound phages recovered for further use(see protocols). Due to a lack of mites, only two rounds of mite in vivo display could be performed. For the mealworms, the titre decreased drastically per round, see Figure X. Thus, a second run with propagation of phages between the round has been performed. During this second round, an unexpected incident caused us to stop the in vivo phage display. While tittering a phage library obtained from a mealworm, no plaques with the expected morphology (blue, lysogenic) showed up on the plate. Instead, a M13 plaques, several lytic virus were recovered (see XXX for different morphologies). The experiments have thus been stopped due to a fear of contamination in the samples. To investigate the source of the contamination, all possible sources have been looked at: used buffer, used top-agar, and the plates. The source of the unexpected plaques turned out to be the mealworm gut. XXX et al. found that phages can be found in mealworms naturally. When using guts of mealworms that have not been fed any phages prior to the process, still a lot of plaques of different kinds could be seen on the titre plates. From this, we could conclude that there are naturally some bacteriophages that E. coli can be a host for in the gut of T. molitor. While those phages could be of interest for general bacteriophage studies, or further specific binding studies, they do interfere with our phage display experiments in two ways: 1) Knowing that phages are already inside of the gut (in relatively high amounts) they might block the receptors for our phage libraries 2) As the phages and their properties are not known, working with them might not be safe As there was a shortage of living mites for the in vivo display anyway, we decided to switch to an in vivo phage display method.  Sequencing data

in vitro

For an in vitro assay, vesicles of the midgut of T. molitor and V. destructor have been produced, according to the general protocol (see protocols). Briefly, cells were lysed, lipids were separated and then left for reassembly. These vesicles should have no phages attached on the outside, due to the way they have been prepared. The only chance of having phages that are naturally inhabitating mealworms or Varroa mites was thus that they have been trapped inside of the vesicle during the preparation steps and so relatively low. Unfortunately, the titre of phages bound to the vesicles prepared from Varroa destructor decreased dramatically after the first round of display and dropped to 0 after the second round. This indicates that the phages did not bind to the vesicles in a way that is strong enough to keep them attached during the separation step of vesicles with attached phages from phages. The reasons for this might be investigated and the protocol used for this phage display might still be optimized. However, the sequences of the phages left after one round have been obtained.

Varroa Isolates

Staining Isolates

To provide an alternative to currently available pesticides, we wanted to adopt Cry toxins to kill Varroa destructor. Cry toxins, also known as Bt toxins, have been used commercially for decades and can limit the environmental impact of pesticide use ¹. Cry toxins are produced by the insect pathogen Bacillus thuringiensis. They are activated by the alkaline conditions of the insect gut, after which the protoxin is cleaved off. The toxin can then recognize receptors on the gut membrane and bind to them. It will form oligomers with other toxin molecules and make a pore in the gut membrane. This is what causes the insect’s death ². Their mode of action can make them highly specific to certain species of insects.

While over 800 Cry toxins are known, research on the workings of these toxins mainly focuses on conventional pests such as the Colorado potato beetle, cabbage moths and mosquitoes. Field experiments on non-target invertebrates show that Acari (mites and ticks) are not significantly affected by Bt crops³ (transgenic crops which express Cry toxins, such as Bt cotton or Bt maize), but these use only a few known Cry toxins. Other studies have shown that there are strains of B. thuringiensis which are harmful to Acari. While most of these strains or toxins were tested against herbivorous species of mites^4,5,6,7, some are known to kill species of ticks^8,9,10,11. In fact, two strains have been discovered which show toxicity to V. destructor^12,13! Unfortunately, they did not identify a particular Cry toxin.

To find our own Cry toxin, we adapted a procedure from Rampersad et al.¹⁴ and Alquisira-Ramírez et al¹². We gathered over 800 dead mites from the Dutch foundation “De Duurzame Bij”¹⁵, Bennekom, The Netherlands.

Figure 1. Photo of the beehives from De Duurzame Bij. We gathered most of our dead mites from these beehives.

Our method of sample preparation selected for spore formation, but not the B. thuringiensis rod-shaped morphology. Therefore, 106 colonies from the mites were investigated with brightfield microscopy. They were stained with Coomassie Brilliant Blue to make Cry toxins more visible. Five isolates were identified as Bacillus-like species. Some of them, including Bacillus thuringiensis HD-350, are shown in Figure 2.

Figure 2. Microscopy images of Coomassie-stained isolates, 1000x magnification with a Zeiss Axio Scope.A1 brightfield microscope. (a) *Bacillus 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.

In Vivo Toxicity

We wanted to test the strains we identified for their toxicity to Varroa destructor. Protein extracts and SDS-PAGE were performed. The toxicity assay was based on the assay used by Alquisira-Ramírez et al.¹², but we faced one major problem: the assay, including the negative control, killed all of the Varroa mites. Therefore, it was not a reliable indicator of the isolates’ toxicity to Varroa. An alternative would be to use an assay where we dip honeybee larvae in protein extracts from the strains; a Varroa mite which feeds on the larvae should then ingest any toxins. Unfortunately, Varroa mites tend to share feeding sites and use them repeatedly¹⁶, which means that not all mites would consume the protein extract.

A sample-size calculation was performed in R with the following code to assess the feasibility of such an assay.
> p=0.66 > p0=0.5 > alpha=0.05 > beta=0.1 > (n=p*(1-p)*((qnorm(1-alpha)+qnorm(1-beta))/(p-p0))^2)

Here, p is the alternative hypothesis, p0 the null hypothesis, alpha the type I error and beta the type II error. The values of these variables were based on the following assumptions:

The null hypothesis is 50% mite mortality. Natural mite mortality is very high in the laboratory.
Linea Muhsal estimated during feeding experiments that approximately 16% of mites ingested the fluorophores she had dipped honeybee larvae in.
If an effective Cry toxin is ingested, we expect to see 100% mite mortality. This is an optimistic assumption.
16% of mites eat a toxin which causes 100% mite mortality, so if there is a toxin, we should see 66% mite mortality.

With these assumptions, a sample size of 75 was calculated. We were generally able to gather a maximum of 40 mites per week. Gathering enough mites to test all our samples was unfeasible. Therefore, pursuit of this type of toxicity assay was cancelled in favour of further development of an in vitro assay.

16s rRNA sequencing and SDS-PAGE results

All five isolates with Bacillus-like morphology had their 16s rRNA sequenced. A search of NCBI’s 16S ribosomal RNA sequences database resulted in the following alignments, each with approximately 99% identity of their best match. The isolates and their matches are displayed in Table 1.

Isolate	Best alignment
V46	Bacillus licheniformis
V47	Paenibacillus amylolyticus
V82	Lysinibacillus meyeri
V88	Paenibacillus chitinolyticus
V106	Bacillus sp.

Table 1. Best matches of a BLAST search of the isolates' 16s rRNA sequence against NCBI's 16 ribosomal RNA sequence database.

It can be difficult to tell Bacillus species apart with just a 16s rRNA sequence¹⁷, so an SDS-PAGE gel of the protein extracts was prepared to further analyse them. This gel is shown in Figure 3.

Figure 3. SDS-PAGE gel of the protein extracts from *Bacillus subtilis*, *Bacillus thuringiensis* HD350, *Bacillus thuringiensis* tenebrionis and isolates 46, 47, 81, 82 and 88. The ladder is a BioRad Precision Plus Protein Standard. The red arrow indicates a band that could be Cry1Aa, which is 133 kDa. The blue arrow indicates a band which could be Cry3Aa, a 73 kDa toxin. The green arrow indicates a band that was investigated with protein MS/MS – see Figure 3.

Proteomics analysis of isolate V82

Based on the previous results, our Lysinibacillus isolate V82 had the most potential as a putative mite pathogen. It is relatively closely related to a known insect pathogen, Lysinibacillus sphaericus, a species which is known to make a 100 kDa mosquiticidal toxin¹⁸. Our isolate also showed a strong band at the 100 kDa region (Figure 3). A new SDS-PAGE gel was prepared with independently grown V82 cultures and bands were cut out for peptide extraction, so the 100 kDa protein could be analysed with LC-MS/MS. Negative control bands were cut out approximately ~1 cM above the 100 kDa bands. MaxQuant was used to analyze the data.

Figure 4. LC-MS/MS spectra of 3 control samples (top) and 3 samples extracted from a ~100 kDa band on the SDS-PAGE gel with V82 protein. Click the figure for the full-resolution image.

Analysis of the LC-MS/MS results resulted in Figure 5. Figure 5 is a vulcano plot; the x-axis plots the protein abundance ratio between the 100 kDa band and its respective control band, while the y-axis plots the p-value of this difference in protein abundance. Therefore, a protein in the left half of the plot is more abundant in the control bands, while the opposite goes for proteins in the right half of the plot. The higher it is on the y-axis, the more significant this difference is.

Figure 5. Vulcano plot of LC-MS/MS results. The colours indicate whether the difference between the amount of protein in the 100 kDa bands and the control bands was significant, or whether the proteins were recognized as contaminants by the MaxQuant software.

Contaminations such as keratins and the trypsin used to digest the samples should not differ significantly between the samples. This is also shown in Figure 5 as most contaminants (the orange data points) are present in the bottom half of the vulcano plot. The relative label-free quantification (LFQ) intensity of the significant proteins shown in Figure 4 was very low, in all cases lower than the LFQ intensity of trypsin. From a biological perspective, this makes sense; there is no reason for an organism to produce mass amounts of alanine tRNA ligase. However, this and the fact that only 3% of peptides could be matched indicates that the 100 kDa protein was not present in the prepared Lysinibacillus sequence database. Therefore, the peptides were also matched against a Bacillus database, but this yielded similar results. Genomic DNA was extracted from V82 and sent for sequencing so the LC-MS/MS data could be run against the isolate's own DNA. This genomic DNA was also analyzed with the Toxin Scanner.