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Title: Acetylcholinesterase of the Sand Fly Phlebotomus papatasi (Scopoli): cDNA Sequence, Baculovirus Expression and Biochemical Properties

item Temeyer, Kevin
item BRAKE, DANETTE - Former ARS Employee
item Tuckow, Alexander
item Li, Andrew
item Perez De Leon, Adalberto - Beto

Submitted to: Parasites & Vectors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/10/2013
Publication Date: 2/4/2013
Citation: Temeyer, K.B., Brake, D.K., Tuckow, A.P., Li, A.Y., Perez De Leon, A.A. 2013. Acetylcholinesterase of the sand fly Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression and biochemical properties. Parasites & Vectors. 6(1):article 31.

Interpretive Summary: The World Health Organization reports that human cutaneous leishmaniasis is an emerging and uncontrolled infectious disease affecting millions of people in parts of Central and South America, Europe, Africa, and Asia, causing flu-like symptoms and severe skin ulcerations that often result in permanent scarring and disfiguration. The disease is transmitted to humans by the bite of sand flies, tiny mosquito-like insects that feed on the blood of humans and other animals. Like malaria, leishmaniasis can be a severe problem for military operations in regions where the disease and its insect vector are present. In Iraq and Afghanistan, attempts to control sand flies at military installations have been largely ineffective, and military readiness and operations have been hampered by incidence of leishmaniasis among military personnel. Research reported in this study discovered the DNA sequence necessary to produce acetylcholinesterase, an enzyme essential for the proper function of the sand fly's central nervous system. Organophosphate and carbamate chemical insecticides kill flies by inactivating the enzyme, which causes failure of the fly’s central nervous system and death. Knowing the DNA sequence enabled scientists to produce the enzyme in the laboratory and determine its functional properties. This work will allow us to screen and identify new and more effective chemicals to control sand flies. In addition, the genetic information can be used to quickly identify fly populations that have mutated to become resistant to available insecticides, allowing for more rapid tests to guide selection and deployment of effective control strategies.

Technical Abstract: Millions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduce or interrupt Leishmania transmission. Zoonotic cutaneous leishmaniasis caused by L. major is vectored mainly by Phlebotomus papatasi (Scopoli) in Asia and Africa. Organophosphates comprise a class of insecticides used for sand fly control, which act through the inhibition of acetylcholinesterase (AChE) in the central nervous system. Point mutations producing an altered, insensitive AChE are a major mechanism of organophosphate resistance in insects and preliminary evidence for organophosphate-insensitive AChE has been reported in sand flies. This report describes the identification of complementary DNA for an AChE in P. papatasi and the biochemical characterization of recombinant P. papatasi AChE. A 2309 nucleotide sequence of PpAChE1 cDNA (GenBank accession JQ922267) of P. papatasi from a laboratory colony susceptible to insecticides is reported with 73-83% nucleotide identity to acetylcholinesterase mRNA sequences of Culex tritaeniorhynchus and Lutzomyia longipalpis, respectively. The P. papatasi cDNA ORF encoded a 710-amino acid protein (GenBank accession AFP20868) exhibiting 85% amino acid identity with acetylcholinesterases of Cx. pipiens, Aedes aegypti, and 92% amino acid identity for L. longipalpis. Recombinant P. papatasi AChE1 was expressed in the baculovirus system and characterized as an insect acetylcholinesterase with substrate preference for acetylthiocholine and inhibition at high substrate concentration. Enzyme activity was strongly inhibited by eserine, BW284c51, malaoxon, and paraoxon, and was insensitive to the butyrylcholinesterase inhibitors ethopropazine and iso-OMPA. Results presented here enable the screening and identification of PpAChE mutations resulting in the genotype for insensitive PpAChE. Use of the recombinant P. papatasi AChE1 will facilitate rapid in vitro screening to identify novel PpAChE inhibitors, and comparative studies on biochemical kinetics of inhibition.