Contribution

Aerolysin - BBa_K4323000 & BBa_K4323001

Design

The ideal compound for controlling zebra mussels should be as specific as possible, readily bio-degradable, and able to be reliably produced by a large range of model organisms. Given that these compounds are intended for environmental deployment, specificity was identified as the primary design constraint. Proteins characteristically have high specificity interactions with many of their substrates. For this reason, protein toxins were chosen over small molecules as the bio-control effector.

In addition to the FitD toxin already in the registry, we wanted to add a novel molluscicide to the iGEM registry. While conducting a literature search for further toxins we came across the promising candidate Aerolysin from the organism Aeromonas hydrophila .¹ Like FitD this is a pore-forming toxin (PFT). It is small enough that it can be easily cloned and synthesized. The mode of action of this protein is complex since it can only function as a heptamer (7 copies working together). It was noted that membrane proteins need to be excreted to be active and tend to aggregate.²

Figure 1. Alphafold predicted structure (AF-P09167-F1) of aerolysin from Aeromonas hydrophila. This is the structure that was used as the starting point for the molecular dynamics simulation.

This was flagged as a potential concern, so we decided to run some simulations before proceeding to the build phase. We wanted to see if the produced protein would be stable. We opted for a molecular dynamics simulation originally using the GROMACS software³. This is included since it was part of the design and a useful tool, but ultimately we opted for the CARMM36 force field.

Figure 2. Molecular dynamics simulation of the aerolysin protein. Note the segment in blue has the most motion, and could just genuinely be a mobile part of the structure. The rest is fairly stable.

In a nutshell, molecular dynamics begin with a protein structure and then simulates the behaviour of the protein over a period of time. The aerolysin structure used for the simulation was an alpha fold predicted structure. There are crystal structures, but they are incomplete. The figure below summarizes the paramters we were most interested in from the model. This supports the idea that we could get a stable structure in solution.

Figure 3. Summary of the most important results from the dynamics simulation.

Build

In any type of design, there comes a point where you think something could work, but you cannot know until you test. After research and modelling, we proceeded to the build phase. The sequence was synthesized and then cloned using Gibson assembly into the pET28b vector, as outlined in the description. Ultimately we opted to keep the C-terminal pro-peptide so our sequence was very similar to Uniprot entry P09167. The coding region was 1497 base pairs in length coding for 499 amino acids. The construct was modified with a C-terminal His tag. This allows expression to be more easily tested as the protein could be purified by affinity chromatography.

Test

For proof of expression, the test is to purify the expressed protein by Immobilized metal affinity chromatography (IMAC).

Figure 4. His-Trap Ni-column purified ACT. L: ladder, C5: 10 % B wash, D1: fraction of gradient at ~30 % B, D4: fraction of gradient at ~50 % B, D8: elution at 100 % B. A buffer: 20 mM Tris-HCl, 200 mM NaCl, pH 8.0; B buffer: 20 mM Tris-HCl, 200 mM NaCl, 300 mM imidazole, pH 8.0. Arrow: purified ACT protein, MW: 55.2 kDa.

The Bioassay was conducted by our partners UNILausanne. The bioassay was conducted on quagga mussels (Dreissena bugensis). This is another invasive mussel species very similar to Zebra mussels (Dreissena polymorpha).

Figure 5. Notice that the non-induced sample shown in red is apparently more toxic than the induced sample shown in green.

Learn

The experiments conducted by UNILausanne demonstrate clearly that the aerolysin toxin when transformed into E.coli is clearly capable of eliminating quagga mussels. In this sense the part is functioning with its desired function, to be toxic to invasive mussels. In all cases the aerolysin exhibited better toxicity when cell lysate was added to the mussel tanks instead of whole cells. While mussels are filter feeders they can be very selective with the particle size they actually admit to their stomachs. Complete toxicity data provided by UNILausanne is provided in the Experiments page.

Through testing, we also uncovered some room for improvement. The greatest toxicity was observed in cells which had the plasmid but were never induced to produce the product. Toxin production was controlled by the common T7 system. Cells where no isopropyl β-D-1-thiogalactopyranoside (IPTG) was added showed the best performance. It appears that in our system more functional protein was being produced by “leaky expression” than by induction.

There are at least a couple of possible reasons for this

(1) Aerolysin is disrupting the membrane of E.coli itself. At high levels of production, the producer simply dies and cannot produce any more proteins. Membrane toxins are often kept safe to the host organisms by way of chaperones and or subcellular localization. Consider reducing temperature of cultures or adding weaker ribosom binding site to find the expression optimum.

(2) Overexpression causes the protein to aggregate and becomes non-functional. While still a possibility, this can usually be seen as a large SDS-PAGE band in the insoluble fraction. Resuspension in a denaturant should be able to detect such a scenario.

Conclusion and Set-Up of for DBTL(II)

By making an effort to follow engineering principles and the Design Build Test Lear cycle. We were able to demonstrate toxicity in invasive mussels for aerolysin produced by E.coli. Overall, we were very pleased with the aerolysin protein as a potential molluscicide. The initial data suggests that it may even be more potent than FitD.

If we had time for a second cycle we would focus on increasing the expression levels of this protein. One promising start would be to add localization signals or secretion tags. The Open E.coli Protein Expression Toolkit, provided in the iGEM distribution 2022, would be an excellent place to start. This could be a way to mitigate the toxicity of the product on the expression organism. UManitoba in partnership with UNILausanne has added a new option to the iGEM registry for treatment of invasive mussels, available as either the stand-alone sequence or as the combination part.

Main References

(1) Iacovache, I.; Degiacomi, M. T.; Pernot, L.; Ho, S.; Schiltz, M.; Dal Peraro, M.; van der Goot, F. G. Dual Chaperone Role of the C-Terminal Propeptide in Folding and Oligomerization of the Pore-Forming Toxin Aerolysin. , PLoS Pathogens 2011, 7 (7).

(2) Howard, S.P., Buckley, J.T. Molecular cloning and expression in Escherichia coli of the structural gene for the hemolytic toxin aerolysin from Aeromonas hydrophila . Molec. Gen. Genet. 204, 289–295 (1986). https://doi.org/10.1007/BF00425512

(3) J.A. Lemkul (2018) "From Proteins to Perturbed Hamiltonians: A Suite of Tutorials for the GROMACS-2018 Molecular Simulation Package, v1.0" Living J. Comp. Mol. Sci. 1 (1): 5068.