Expression And Characterization Of Acetylcholinesterases From Drosophila Melanogaster And Musca Domestica

Acetylcholinesterase (AChE, EC 3.1.1.7) catalyzes the hydrolysis of the neurotransmitter acetylcholine (ACh) at cholinergic synapses to keep the normal transmission of nerve impulses. It is the target of the organophosphate and carbamate compounds, which phosphorylate or carbamoylate the active site serine to block the hydrolysis of ACh and so lead to the death of insects. Insecticide resistance is now common due to the frequent and intensive use of chemicals to control insects, which provides a useful tool to study the adaptation of eukaryotic genome to various environments. In this study, we made use of molecular enzymology principles to elucidate the mechanisms involving in a propoxur-resistant strain of Musca domestica resistant to the organophosphate and carbamate compounds. In order to discover the most frequent point mutations of Drosophila melanogaster AChE associated with insecticide resistance and the different patterns of mutation combinations, we investigated mutation distribution of AChE in natural populations. At the molecular level, we revealed the fitness cost of possible resistant alleles of Drosophila melanogaster AChE. Finally, we attempted to identify one Drosophila melanogaster AChE displaying more sensitive to aldicarb detection via screening a library of genetically engineering mutant emzymes. The major results are summarized as following:1) A propoxur-resistant strain of Musca domestica exhibited 185-, 47-, 41-, and 51-fold greater resistance to propoxur, methomyl, paraoxon, and dichlorvos, respectively, than a susceptible strain. The greater maximal velocity (Vmax) of AChE from the resistant strain appeared to be due to both the increased turnover number and the higher concentration of AChE active site. AChE from the resistant strain was 21-, 21-, 33-, and 19-fold less sensitive to methomyl, propoxur, dichlorvos, and paraoxon, respectively. The decreased affinity of the insecticides for AChE from the resistant strain was the main reason that conferred the insensitivity of AChE in the resistant strain to these inhibitors. The decreased sensitivity of AChE was associated with modified catalytic efficiency of the enzyme in the resistant strain, suggesting that AChE from the resistant strain might be qualitatively altered.2) The cDNA encoding AChE from susceptible and propoxur-resistant strains of Musca domestica had been cloned using RT-PCR. A novel mutation D421V was identified in AChE from the resistant strain. D421 was positioned at the outer surface of the protein, but this residue could not interact with amino acid residues lining the active site gorge. The homologous modelling of three dimensional structure of houselfy AChE implicated that D421 might be involved in a ionic bond with H341. The distance between D421 and H341 is 3.42 ?. Modification of charge on the side chain of D422V mutation would be expected to affect the profile of acyl pocket via Y339 losing anchorage. By in vitro site-directed mutagenesis, we expressed and purified the wild type and three mutant enzymes(D421V,V260L/G342A/F407Y,V260L/G342A/F407Y/ D421V) to tested their catalytic activity and enzymatic stability. The results showed that D422V mutation increased catalytic efficiency and diminished the stability of the mutated enzyme. 3) The Ace gene encoding AChE was cloned and sequenced in 30 strains of Drosophila melanogaster harvested from different parts of the world. Comparison with the wild type sequence allowed evidencing four widespread mutations, I161V, G265A, F330Y and G368A. Their combinations in the same protein were also detected in most of the strains. We expressed 15 mutated proteins in vitro with different patterns of mutation combinations to measure the resistance levels relative to the wild type protein. The results showed that four single mutations provided specific insecticide resistance. The higher level and wider spectrum of resistance was achieved by combining three or four mutations in the same protein. Most natural populations of Drosophila melanogaster were heteroge...