Skip to main content
ARS Home » Research » Publications at this Location » Publication #309960
Title: Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): construction, expression and biochemical properties of the G119S orthologous mutant

Author
item Temeyer, Kevin
item TONG, FAN - University Of Florida
item TOTROV, MAXIM - Molsoft, Llc
item Tuckow, Alexander
item CHEN, QIAO-HONG - Virginia Tech
item CARLIER, PAUL - Virginia Tech
item Perez De Leon, Adalberto - Beto
item BLOOMQUIST, JEFFREY - University Of Florida

Submitted to: Parasites & Vectors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/27/2014
Publication Date: 12/10/2014
Publication URL: http://handle.nal.usda.gov/10113/62222
Citation: Temeyer, K.B., Tong, F., Totrov, M.M., Tuckow, A.P., Chen, Q., Carlier, P.R., Perez De Leon, A.A., Bloomquist, J.R. 2014. Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): construction, expression and biochemical properties of the G119S orthologous mutant. Parasites & Vectors. 7:577.

Interpretive Summary: Sand fly transmission of leishmaniasis is a serious concern for military operations, as well as for several hundred million people residing in intertropical and temperate regions of the world. There is currently no vaccine available for leishmaniasis and chemical control of the sand fly vector is important to prevent disease transmission. Acetylcholinesterase (AChE) is a protein that functions as an enzyme because it accelerates the degradation of the neurotransmitter acetylcholine. In sand flies AChE is a key target for organophosphates and carbamates, which are chemical pesticides used for sand fly control. The gene coding for a sand fly AChE was cloned and the recombinant form of the protein encoded by that gene was produced in the laboratory to determine the biochemical properties of the recombinant protein. The AChEs of sand flies and many mosquitoes were found to be very similar. A mutation in mosquito AChE that was known to result in high level resistance to pesticides has now been produced in the recombinant AChE from sand flies. As expected, the mutation made the sand fly AChE resistant to available pesticides, but also enabled researchers to test and identify new synthetic pesticides that were effective against the mutant sand fly AChE, and to develop an improved molecular model of the AChE enzyme. Research is continuing to test the newly identified synthetic pesticides against live sand flies and other pests in the laboratory, and to test for the presence of the mutation in wild sand fly populations. It is expected that this research will aid in continuing efforts to control sand fly populations and reduce transmission of leishmaniasis and other diseases.

Technical Abstract: Phlebotomus papatasi vectors zoonotic cutaneous leishmaniasis, widespread in intertropical and temperate regions of the world. Previous cloning, expression, and biochemical characterization of recombinant P. papatasi acetylcholinesterase 1 (PpAChE1) revealed 85% amino acid sequence identity to mosquito AChE and identified synthetic carbamates that were effective inhibitors of PpAChE1 with improved specificity for arthropod AChEs compared to mammalian AChEs. We hypothesized that the G119S mutation responsible for high level resistance to organophosphate insecticides in mosquitoes might also occur in PpAChE1 and may result in reduced sensitivity to inhibition. This report describes construction, expression, and biochemical properties of rPpAChE1 containing the G119S orthologous mutation. Targeted mutagenesis introduced the G119S orthologous codon substitution in PpAChE1 cDNA. Recombinant PpAChE1 enzymes containing or lacking the G119S orthologous mutation were expressed in the baculoviral system. Biochemical assays were conducted to determine changes in catalytic properties and inhibitor sensitivity resulting from the G119S orthologous substitution in rPpAChE1. A molecular homology model was constructed to examine the modeled structural interference with docking of representative inhibitors of different classes. Genetic tests were devised and conducted to determine if the G119S orthologous codon existed in polymorphic form in a laboratory colony of P. papatasi. Recombinant PpAChE1 containing the G119 orthologous substitution exhibited significantly altered biochemical properties, and reduced inhibition by a variety of compounds that bind to the acyl pocket on the enzyme (except eserine). Less resistance was directed against bivalent or peripheral site inhibitors, in good general agreement with modeled docking of representative inhibitors. Eserine appeared to be a special case capable of inhibition in the absence of covalent binding at the acylation site. Genetic tests did not detect presence of the G119S orthologous mutation in a laboratory colony of P. papatasi but did reveal that the G119S orthologous codon existed in polymorphic form (GGA + GGC). The finding of G119S orthologous codon polymorphism in a laboratory colony of P. papatasi strongly suggests that a single nucleotide transversion (GGC'AGC) might readily occur, causing relatively rapid development of resistance to organophosphate and phenyl-substituted carbamate insecticides if subjected to strong selection. Careful management of pesticide use in IPM programs is important to prevent or mitigate development and fixation of the G119S mutation in susceptible pest populations. Availability of the recombinant AChEs may enable identification of novel inhibitory ligands with improved efficacy and specificity for AChEs of arthropod pests.