Proof Of Concept

Introduction

Our project aims to kill and prevent the attachment of Dreissena bugensis (also known as quagga mussels). Our first strategy relied on killing quagga mussels by applying the FitD toxin which will be overproduced from the Pseudomonas protegens CHA0 strain. To prevent the attachment of quagga mussels, our second strategy was based on engineering the metabolic pathway to produce zosteric acid in Escherichia coli strain BL21 (DE3).

Overproduction of the FitD toxin

One of our strategies is to kill quagga mussels using FitD, a toxin naturally produced by P. protegens. The fitD gene is part of a virulence operon regulated by three regulatory proteins, including an activator encoded by the fitG gene and a type I secretion system. The FitD toxin has been shown to have insecticidal activity, by inducing the apoptosis of the midgut epithelium as well as paralyzing and destroying insect phagocytes (Péchy-Tarr et al., 2013). Interestingly for our project, FitD is also known for its potential as a molluscicidal agent.

Hence, we tried to overexpress the FitD protein in P. protegens using plasmids bearing either the fitG or fitD genes. To achieve this, we designed and successfully built two constructs to regulate each gene using either the inducible lacIQ promoter or the constitutive EM7 promoter (Figure 1).

FitD plasmids
Figure 1. Plasmid maps of the constructed plasmids for the overexpression of the FitD toxin in P. protegens.

We tested the efficacy of our constructs by applying lysed cells of the P. protegens strains transformed with our different plasmids on live quagga mussels. We reasoned that if the efficiency of the FitD toxin was the same between live and dead cells, it was advantageous to use lysed cells so we theoretically could apply them in a real-life context without any restriction concerning the release of genetically modified organisms. We also used different concentrations of our engineered cells to find the optimal amount of the FitD toxin to kill quagga mussels. In summary, we applied P. protegens CHA0 strains bearing different constructs to live mussels:

  • A plasmid expressing an inducible fitD gene that was induced with IPTG (FitD (+))
  • A plasmid expressing an inducible fitD gene that was not induced with IPTG (FitD (-))
  • A plasmid expressing a constitutively expressed fitG gene (FitG (const))
  • The wild-type P. protegens strain (i.e., containing a native copy of the fitD gene in its genome).

We also applied lake water only to the mussels as a control. We tested each treatment on 45 mussels with two concentrations of P. protegens: OD 0.5 or OD 0.25. More details about our measurements can be found here.

lysed cells
Figure 2. Lysed P. protegens cells engineered to overexpress the fitG gene kill quagga mussels. The Kaplan-Meier graph shows mussel survival's probability over time when subjected to the different lysed bacterial treatments applied to an OD of 0.25. Gehan-Breslow-Wilcoxon test, * significant for p-value < 5%, n = 45 mussels.

We were pleased to observe that our engineered and lyzed P. protegens cells overexpressing the fitG gene were able to significantly reduce the probability of survival of the tested mussels (p-value 0.0117; Figure 2). Indeed, our data show that the probability of survival of the quagga mussels decreases remarkably to 86 % after 80 hours when exposed to our engineered strain, in comparison to the wild-type P. protegens that did not impact the mussel’s fitness at all. Although we could not measure a statistically significant difference in the probability of survival of the mussels between the wild-type P. protegens treatment and our engineered strain overexpressing the fitD gene, we noted that the latter, however, seems to modestly reduce the mussel survival as well. We would therefore argue that both our modified P. protegens strains, expressing fitD or fitG, are able to kill the invasive quagga mussels.

We proved that our engineered strains are able to effectively and remarkably kill quagga mussels and could therefore represent a tangible solution to the worldwide issue caused by this invasive species. In addition, the fact that lyzed cells appeared efficient in killing quagga mussels is of importance as it means our engineered P. protegens-based solution could be potentially applied outside confined laboratory conditions and provide real-world benefit by solving the quagga mussel crisis. Indeed, a product currently in use against quagga mussels called Zequanox, a solution with molluscicidal activity based on lysed wild-type P. protegens cells, was registered in 2014 by the U.S. Environmental Protection Agency (Murawski, 2016). While this product has been shown to work well against invasive mussels, our engineered cells have the potential to outcompete it in terms of efficiency and cost. We, indeed, demonstrated that our P. protegens strain overexpressing fitG notably kills more mussels than the wild-type P. protegens used in Zequanox and this, at extremely low cell concentrations where the wild-type cells do not exhibit any activity. A product based on our engineered strain could thus require less cell density to reach similar killing activity than the currently marketed solution, potentially being a novel and cost-effective product to fight invasive mussels.

Ultimately, these experiments allowed us to prove the efficiency of our engineered strain overproducing the FitD toxin under laboratory conditions. However, the ultimate goal of this project is to highlight a real word application. Since we could not test our proposed implementation on infested infrastructures within the framework of our experiments, we developed a mathematical model that allowed us to evaluate the benefits associated with periodical applications of our product into standard pipes.

Given a pipe with a cylindrical section, of known radius and length, we can easily calculate the amount of mussels required to clog such infrastructure and make it unusable. Our model recapitulates the growth of a mussel population under normal conditions and in presence of our toxic compound.

The evolution of the population over time, in absence of the FitD toxin, is captured by the following equation :

$$N(t+1)=N(t)+b\cdot N(t)-d\cdot N(t)$$

with N(0) and N(t) being the number of mussels at time zero and at time t, respectively, b and d being the birth and death rates, respectively.

We have proven experimentally that our toxin negatively affects mussels' probability of survival. Therefore we can consider that it increases the death rate of the animals. When the toxin is present, the previous equation becomes:

$$N(t+1)=N(t)+b\cdot N(t)-d_{FitD}\cdot N(t)$$

Where dfitD is the death rate parameter derived from our experimental data, the killing effect of FitD is not perpetual but vanishes due to the degradation of the protein approximately five days after the treatment application. Thereby, the population of treated mussels alternates growth regimes with standard and enhanced death rates, depending on the periodicity of FitD administration.

Building upon these assumptions and calculations, our model predicts the difference in time required to clog a pipe, considering an untreated population and a population systematically treated with our product. A graphical example of the output of our model is shown in figure 3.

lysed cells
Figure 3. Mussels population size in function of time without application of FitD toxin (black) and with application of FitD toxin (blue).

Without toxin application, the population size reaches the maximum number of mussels the given pipe can hold (pink). The modeled pipe has a radius of 20 cm and a length of 3m. The FitD toxin is applied every 60 days, and the initial population consists of 10 mussels.

Last but not least, we managed to represent the differences in plugging time for various pipe structural features with and without FitD administration.

lysed cells
Figure 4. Differences in plugging time for different combinations of pipe length and radius. The time saved by applying the FitD toxin becomes highly meaningful with increasing pipe volumes.

We can affirm that our product would significantly slow down the mussel invasion in this kind of infrastructure, resulting in a considerable reduction in maintenance time and cost.

More informations about our model can be found here

References

  1. Péchy-Tarr, M., Borel, N., Kupferschmied, P., Turner, V., Binggeli, O., Radovanovic, D., … Keel, C. (2013). Control and host-dependent activation of insect toxin expression in a root-associated biocontrol pseudomonad. Environmental Microbiology, 15(3), 736–750. https://doi.org/10.1111/1462-2920.12050
  2. Molloy, D. P., Mayer, D. A., Gaylo, M. J., Morse, J. T., Presti, K. T., Sawyko, P. M., … Griffin, B. H. (2013). Pseudomonas fluorescens strain CL145A - a biopesticide for the control of zebra and quagga mussels (Bivalvia: Dreissenidae). Journal of Invertebrate Pathology, 113(1), 104–114. https://doi.org/10.1016/j.jip.2012.12.012
  3. Murawski, B. (2016). Zequanox: A Potential Solution to Zebra Mussels. Aisthesis: Honors Student Journal, 7, 29–33. Retrieved from https://pubs.lib.umn.edu/index.php/aisthesis/article/view/783