Detection of anatoxin-a(s) in environmental samples of cyanobacteria by using a biosensor with engineered acetylcholinesterases

Abstract

Bioassays are little used to detect individual toxins in the environment because, compared to analytical methods, these assays are still limited by several problems, such as the sensitivity and specificity of detection. We tentatively solved these two drawbacks for detection of anatoxin-a(s) by engineering an acetylcholinesterase to increase its sensitivity and by using a combination of mutants to obtain increased analyte specificity. Anatoxin-a(s), a neurotoxin produced by some freshwater cyanobacteria, was detected by measuring the inhibition of acetylcholinesterase activity. By using mutated enzyme, the sensitivity of detection was brought to below the nanomole-per-liter level. However, anatoxin-a(s) is an organophosphorous compound, as are several synthetic molecules which are widely used as insecticides. The mode of action of these compounds is via inhibition of acetylcholinesterase, which makes the biotest nonspecific. The use of a four-mutant set of acetylcholinesterase variants, two mutants that are sensitive to anatoxin-a(s) and two mutants that are sensitive to the insecticides, allows specific detection of the cyanobacterial neurotoxin.

FIG. 1.

Inhibition rates of Drosophila AchE mutants. The highest k_i values indicate the mutants most sensitive to inhibition by purified anatoxin-a(s). The arrow indicates the position of wild-type AChE.

FIG. 2.

Twenty-angstrom slab of Drosophila AChE, showing the positions of mutations that provided the best sensitivities to anatoxin-a(s) (Tyr71 and Tyr73) in white at the entrance of the active site gorge. Amino acids important for catalysis (Ser238 and Trp83) at the bottom of the active site are shown in yellow.

FIG. 3.

Determination of the amount of anatoxin-a(s) by progressive inhibition. In this experiment, 10, 7, 5, 3, 2, 1, and 0.5 pmol of AChE were mixed with aliquots of an anatoxin-a(s)-containing sample. Inhibition was monitored over time. The plateaus correspond to the free enzyme remaining when all of the neurotoxin had reacted with the AChE.

FIG. 4.

Identification of organophosphorous compounds inhibiting AChE by determining the relative inhibition rates for two mutants. The inhibition rate constants of four enzymes (Y71D Y73Q, Y71A, E69W, and E69Y) were estimated for anatoxin-a(s) and for some insecticides. Four inhibition ratios were then tested to obtain a signature for inhibition by anatoxin-a(s) versus inhibition by an insecticide. Y71D Y73Q and Y71A were more rapidly inhibited than E69W and E69Y by anatoxin-a(s) but not by the insecticides tested.

FIG. 5.

Detection of anatoxin-a(s) in cyanobacterial bloom samples from three freshwater lakes and analysis of purified anatoxin-a(s) reference toxin by using four AChE mutants.