What is Anatoxin-a?
Anatoxin-a is a neurotoxin produced by multiple cyanobacteria species living in freshwater. It is a secondary bicyclic amine alkaloid with a molecular weight of 165 Da, and structurally mimics the neurotransmitter acetylcholine.
Figure 1: Structure of Anatoxin-a
As the latter, it binds nicotinic acetylcholine receptors (nAChR) at the neuromuscular junction. However, unlike acetylcholine, Anatoxin-a binds irreversibly to nAChR and is not degraded by acetylcholinesterases. This leads to overstimulation of muscles and fatigue which can cause respiratory failure and death.
Anatoxin-a poisonings occur mostly in livestock and dogs through drinking or swimming in contaminated waters. A 2015 report by the Centers for Disease Control including data from 15 US states revealed over 200 confirmed events of poisoning over a four-year period. Climate change can strongly influence the occurrence of cyanobacteria and, hence, Anatoxin-a – however its exact impacts are still unclear. It is estimated that climate change can promote mass proliferation of cyanobacteria in some water bodies and and prohibit it in others (1).As there is no antidote to Anatoxin-a, prevention remains the most effective measure against this cyanotoxin.
Why is a biosensor for Anatoxin-a useful?
The presence of Anatoxin-a in potentially contaminated waters is usually detected via high-performance liquid chromatography analysis, which is laborious, expensive and not widely available (2). A functional biosensor could be used to exclude the presence of Anatoxin-a in waters. This would limit the use of LC-MS testing to those occasions when Anatoxin-a is detected and its levels need to be accurately determined to establish whether they exceed the limit values. Furthermore, the biosensor would allow testing to be de-centralized, thus speeding up the time between sample collection and data acquisition.
What is the idea for our biosensor?
We aimed to establish an E. coli-based biosensor for Anatoxin-a, which emits light in presence of the toxin. The general idea was to engineer the strain to express a hybrid chemoreceptor system that upon sensing, i.e., binding, to Anatoxin-a, induces the expression of the reporter GFP.
Figure 2: Working principle of the biosensor
The hybrid receptor consists of two parts. The extracellular and transmembrane domains belong to the chemoreceptor PctD (also known as PA4633) which has been shown to bind acetylcholine(3). Those parts are fused to the intracellular domains of the EnvZ histidine kinase, which is naturally located in the bacterial cytoplasmic membrane and senses changes in osmolarity. Activation of EnvZ activates the transcriptional activator OmpR to induce the transcription of genes including ompC from its promotor (4).
The engineered E. coli biosensor was thus envisioned to carry 2 plasmids:
- A plasmid carrying the gene that encodes for the hybrid receptor we designed.
- A reporter plasmid carrying the GFP-encoding under the control of the ompC promoter.
- Example 3
We first aimed to test this system with the naturally occurring ligands of PctD, acetylcholine and choline. Simultaneously, we aimed at determining whether Anatoxin-a binds to the extracellular domain of PctD, first in silico and then in vitro. In the last stage of our project, the goal was to modify the PctD binding site to improve binding of Anatoxin-a.
What have we achieved?
We successfully created and episomally expressed the gene encoding the hybrid chemoreceptor. We also demonstrated that the natural ligands of PctD acetylcholine and choline increase the GFP expression from the reporter plasmid in the E. coli biosensor strain. In silico analysis suggested that Anatoxin-a binds only weakly to the extracellular domain of PctD . Unfortunately, we were unable to obtain Anatoxin-a since the supplier we contacted had problems with the synthesis of the compound. Therefore, we could neither test the binding of Anatoxin-a to the sensor domain of PctD in vitro nor its effect on GFP expression in our biosensor strain.
In the end, we managed to create a hybrid chemoreceptor which activates the Pompc promotor when no ligand is bound, but without the function to alter this activation in presence of ligands. Improvements will need to be made to create a functional hybrid chemoreceptor. This could then be tested for signal transduction in response to Anatoxin-a and improved further to optimally detect the toxin.