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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR BIOLOGY AND PHYSIOLOGY OF BITING FLIES AFFECTING LIVESTOCK

Location: Tick and Biting Fly Research

Title: Expression and Biochemical Properties of a Recombinant Acetylcholinesterase 1 of the Sand Fly, Phlebotomus papatasi (Scopoli) Insensitive to Organophosphate Inhibition

Authors
item Temeyer, Kevin
item Tuckow, Alexander
item Li, Andrew
item Perez De Leon, Adalberto

Submitted to: Entomological Society of America Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: May 31, 2013
Publication Date: N/A

Interpretive Summary: Sand flies are fragile blood-feeding flies that carry infectious agents causing diseases to humans, including leishmaniasis, and are a significant hindrance to military operations and a serious threat to local populations throughout the Middle East and much of the tropical and subtropical areas of the world. Organophosphate and carbamate pesticides work by inhibiting an enzyme called acetylcholinesterase that is essential to proper function of the insect nervous system, resulting in death of the insect. We demonstrated that replacement of a single specific amino acid within the structure of the sand fly acetylcholinesterase enzyme results in production of an altered form of the enzyme that is now 500-1000 times less sensitive to organophosphate pesticides. This observation suggests that sand flies may be able to readily mutate to become highly resistant to organophosphate and carbamate insecticides. Studies are underway to determine if this mutation occurs in the field. The recombinant form of the mutant enzyme has been produced in a test tube that will allow testing of new chemical compounds to identify and develop novel pesticides that can inhibit the sand fly acetylcholinesterases specifically and efficiently.

Technical Abstract: Phlebotomine sand flies are small hematophagous flies present throughout tropical and subtropical areas of the world and are vectors of human and zoonotic leishmaniases. Human cutaneous leishmaniasis is a debilitating disease presenting major problems for U.S. military operations in the Middle East, and people living in areas endemic to phlebotomine sand flies. We recently identified and expressed recombinant acetylcholinesterase 1 (rPpAChE1) of Phlebotomus papatasi, one of the principal vectors of human cutaneous leishmaniasis in the Eastern Hemisphere. The enzyme shared considerable sequence identity with AChE of Lutzomyia species, generally known as New World sand flies, that exists from Argentina to the southern United States. It was hypothesized that an altered PpAChE1 containing a single amino acid substitution corresponding to that responsible for high level organophosphate insensitivity in AchE1 of mosquitoes may generate a rPpAChE1 insensitive to organophosphate inhibition. We conducted targeted mutagenesis to generate a cDNA containing the desired codon substitution and expressed the recombinant altered enzyme in a baculovirus expression system. Biochemical properties of the altered rPpAChE1 containing a substitution of serine for glycine at amino acid position 256 (GenBank accession AFP20868) were quite different from the wild type enzyme, including a significant increase in the Michaelis–Menten constant (Km) reflecting increased concentration of substrate required to achieve maximal velocity, and 500-1000 fold decreased sensitivity to inhibition by eserine or oxon forms of organophosphate pesticides.

Last Modified: 12/19/2014
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