Toxin Engineering

As up to now, no toxin specific to Varroa mites is known, we tried to engineer one. As basis for this, the Cry3Aa toxin – toxic to Coleoptera as the mealworm Tenebrionis molitor – has been chosen. It is also toxic to 4 different kinds of mites and has been well characterized in structure and mechanism of function, leaving us to believe to have a good basis. The engineering towards specificity has been performed in two ways:
• Binding site mutagenesis
• Phage display

Binding site mutagenesis

Design

As it has been shown in previous studies, mutations in the binding sites of Cry toxins can both decrease and enhance the specificity of a toxin towards its target organism (citation here). Knowing this, the 3D structure of the Cry3Aa toxin has been analysed. Three putative binding sites have been identified. (picture of structure with red loops) These can be seen in the following table.

To mutate these, the plasmid pSB1A3_Cry3Aa_TEV_HIS has been used as a template. This plasmid contains the toxin Cry3Aa as it can be found in Bacillus thuringiensis var. tenebrionis. Its expression is regulated by the arabinose inducible promotor pBAD/araC (Bb_I0500). From this, three different plasmids have been created: three plasmids with the same properties as the template, but with a series of Ns at the place of the binding sites. Thus, having cells containing one of these variants grow, a library of Cry3Aa proteins with randomly modified binding sites has been obtained.

Screening

The mutants have been screened as described in the protocols. Per binding site, 48 mutants have been screened. From these, 30 mutants have been chosen for more in depth characterisation of in vitro toxicity. As a reference, the vesicle breaking reaction kinetics of a sample prepared from expression strain Lemo21 has been chosen. In this way, both the effect of proteins possibly expressed by the strain and the effect of lipids in the samples are minimized. Thus, we can assume that a strain expressing a toxin that revoces a stronger response than the negative control Lemo21, indicates the expression of a toxin being able to break down Varroa vesicles. This then indicates toxicity to Varroa destructor.

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. Thus, it can be concluded that a Cry3Aa mutant has been created that shows implications to be toxic to Varroa destructor.

Sequencing of mutants

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.  In work, will be done by Thursday, I hope. I sequenced two mutants with a high value and one with a low value

Phage display

Due to a lack of mites from the beginning on, the phage display experiments have also been performed on mealworms, Tenebrio molitor. The aim of this was to provide a proof of principle for the phage display, to proof its applicability for protein engineering towards Varroa specificity. The advantages of using mealworms are that there is already a known Cry toxin targeting them. Furthermore, mealworms can be purchased in a lot of places and they are easy to maintain. To determine what binding sites there are to find in the midguts of T. molitor and V. destructor, phage displays with several libraries can be used. The aim of these displays is to find a consensus sequence of a receptor in the gut. This sequence can then be targeted specifically and the Cry3Aa protein can be engineered in a way that it contains this consensus sequence in one of its binding sites. In this way, it should be able to bind to a receptor in the gut of the attacked species and thus it might even be specifically toxic to it.

Display of M13KE-PIII libraries

For phage display, the phage M13KE has been chosen. The two libraries used were from NEB and included the expression of a C7C and a 12-mer peptide library at the pIII position of M13KE.

in vivo

In vivo, the phage libraries have been fed to T. molitor and V. destructor and the phages in the gut of T. molitor and inside of V. destructor have been recovered for further use(protocol). 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. 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 lot of plaques with morphologies different from the ones expected occurred (see plate). 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, the plates. The source of the unexpected plaques turned out to be the mealworm gut itself. 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, 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. 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 benn obtained.  Sequencing data

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.

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.

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.

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.

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 toxin ¹⁸. 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.

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.

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.

References

 

References for Varroa isolates

1. Lacey, L. A., Grzywacz, D., Shapiro-Ilan, D. I., Frutos, R., Brownbridge, M., & Goettel, M. S. (2015). Insect pathogens as biological control agents: back to the future. Journal of invertebrate pathology, 132, 1-41. ↩

2. de Maagd, R. A., Bravo, A., & Crickmore, N. (2001). How Bacillus thuringiensis has evolved specific toxins to colonize the insect world.TRENDS in Genetics, 17(4), 193-199. ↩

3. Marvier, M., McCreedy, C., Regetz, J., & Kareiva, P. (2007). A meta-analysis of effects of Bt cotton and maize on nontarget invertebrates.science, 316(5830), 1475-1477. ↩

4. Ahmed, N., Wang, M., & Shu, S. (2016). Effect of commercial Bacillus thuringiensis toxins on Tyrophagus putrescentiae (Schrank) fed on wolfberry (Lycium barbarum L.). International Journal of Acarology, 42(1), 1-6. ↩

5. Chapman, M. H., & Hoy, M. A. (1991). Relative toxicity of Bacillus thuringiensis var. tenebrionis to the two‐spotted spider mite (Tetranychus urticae Koch) and its predator Metaseiulus occidentalis (Nesbitt)(Acari, Tetranychidae and Phytoseiidae). Journal of Applied Entomology, 111(1‐5), 147-154. ↩

6. Dunstand-Guzmán, E., Peña-Chora, G., Hallal-Calleros, C., Pérez-Martínez, M., Hernández-Velazquez, V. M., Morales-Montor, J., & Flores-Pérez, F. I. (2015). Acaricidal effect and histological damage induced by Bacillus thuringiensis protein extracts on the mite Psoroptes cuniculi. Parasites & vectors, 8(1), 1. ↩

7. Erban, T., Nesvorna, M., Erbanova, M., & Hubert, J. (2009). Bacillus thuringiensis var. tenebrionis control of synanthropic mites (Acari: Acaridida) under laboratory conditions. Experimental and Applied Acarology, 49(4), 339-346. ↩

8. Fernández-Ruvalcaba, M., Peña-Chora, G., Romo-Martínez, A., Hernández-Velázquez, V., de la Parra, A. B., & De La Rosa, D. P. (2010). Evaluation of Bacillus thuringiensis pathogenicity for a strain of the tick, Rhipicephalus microplus, resistant to chemical pesticides. Journal of Insect Science, 10(1), 186. ↩

9. Hassanain, M. A., Garhy, M. E., Abdel-Ghaffar, F. A., El-Sharaby, A., & Megeed, K. N. A. (1997). Biological control studies of soft and hard ticks in Egypt. Parasitology research, 83(3), 209-213. ↩

10. Neethu, K. B., Priji, P., Unni, K. N., Sajith, S., Sreedevi, S., Ramani, N., ... & Benjamin, S. (2015). New Bacillus thuringiensis strain isolated from the gut of Malabari goat is effective against Tetranychus macfarlanei. Journal of Applied Entomology. ↩

11. Zhioua, E., Heyer, K., Browning, M., Ginsberg, H. S., & LeBrun, R. A. (1999). Pathogenicity of Bacillus thuringiensis variety kurstaki to Ixodes scapularis (Acari: Ixodidae). Journal of medical entomology, 36(6), 900-902. ↩

12. Neethu, K. Alquisira-Ramírez, E. V., Paredes-Gonzalez, J. R., Hernández-Velázquez, V. M., Ramírez-Trujillo, J. A., & Peña-Chora, G. (2014). In vitro susceptibility of varroa destructor and Apis mellifera to native strains of Bacillus thuringiensis. Apidologie, 45(6), 707-718. ↩

13. Tsagou, V., Lianou, A., Lazarakis, D., Emmanouel, N., & Aggelis, G. (2004). Newly isolated bacterial strains belonging to Bacillaceae (Bacillus sp.) and Micrococcaceae accelerate death of the honey bee mite, varroa destructor (V. jacobsoni), in laboratory assays. Biotechnology letters, 26(6), 529-532. ↩

14. Rampersad, J., & Ammons, D. (2005). A Bacillus thuringiensis isolation method utilizing a novel stain, low selection and high throughput produced atypical results. BMC microbiology, 5(1), 1. ↩

15. This is the homepage of “De Duurzame Bij”. They want to stop treating Varroa with pesticides. ↩

16. Kanbar, G., & Engels, W. (2005). Communal use of integumental wounds in honey bee (Apis mellifera) pupae multiply infested by the ectoparasitic mite Varroa destructor. Genetics and Molecular Research, 4(3), 465-472. ↩

17. Bavykin, S. G., Lysov, Y. P., Zakhariev, V., Kelly, J. J., Jackman, J., Stahl, D. A., & Cherni, A. (2004). Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms. Journal of clinical microbiology, 42(8), 3711-3730. ↩

18. Berry, C. (2012). The bacterium, Lysinibacillus sphaericus, as an insect pathogen. Journal of invertebrate pathology, 109(1), 1-10. ↩