Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Summary

Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies.

Abstract

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were found up-regulated in the resistant house fly strain, whereas their roles in conferring insecticide resistance remained to be explored. Here, we designed a protocol for the functional characterization of carboxylesterases. Three example experiments are presented: (1) expression and isolation of carboxylesterase proteins through a baculovirus-mediated insect Spodoptera frugiperda (Sf9) cell expression system; (2) a cell-based MTT (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) cytotoxicity assay to measure the tolerance of insect cells to different permethrin treatments; and (3) in vitro metabolic studies to explore the metabolic capabilities of carboxylesterases toward permethrin. The carboxylesterase gene MdαE7 was cloned from a resistant house fly strain ALHF and used to construct a recombinant baculovirus for Sf9 cells infection. The cell viabilities against different permethrin treatments were measured with the MTT assay. The enhanced cell tolerance of the experimental group (MdαE7-recombinant baculovirus infected cells) compared with those of the control groups (CAT-recombinant baculovirus infected cells and GFP-recombinant baculovirus infected cells) to permethrin treatments suggested the capabilities of MdαE7 in metabolizing insecticides, thereby protecting cells from chemical damages. Besides that, carboxylesterase proteins were expressed in insect Sf9 cells and isolated to conduct an in vitro metabolic study. Our results indicated a significant in vitro metabolic efficiency of MdαE7 toward permethrin, directly indicating the involvement of carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.

Introduction

Insecticide resistance is currently a major issue of house fly control worldwide^1,2. Efforts to determine the mechanism of insecticide resistance facilitates better understanding of this issue and thus provide novel strategies to effectively prevent or minimize the spread of resistance development³. Carboxylesterases, as one of the major detoxification enzymes, have attracted a lot of attention for their roles in sequestering and metabolizing insecticides in various insects^4,5,6. Our previous s....

Protocol

1. Expression and Isolation of Target Proteins with a Baculovirus-mediated Insect Cell Expression System

1. Directionally clone blunt-ended PCR products of target proteins from house flies.
   1. Design PCR primers of green fluorescent protein (GFP) and the house fly MdαE7 gene based on their sequences and the special requirements of the chosen vector (Table 1).
   2. Use a thermostable, proofreading DNA polymerase and primers from step 1.1.1 to perform a 150 µL PCR reaction (30 µL of reaction buffer, 3 µL of 10 mM dNTPs, 1.5 µL of DNA polymerase, 6 µL of house fly template DNA, 7.5 µL of forward primer, 7.5 µL of reverse primer, w....

Results

The cell viability toward different permethrin treatments (MTT assay)

The cytotoxicity of permethrin was examined in MdαE7-recombinant baculovirus infected Sf9 cells (experimental group) and CAT-recombinant baculovirus (provided by baculovirus infected kit) infected cells (control groups). The enhanced cell tolerances to permethrin in MdαE7 expressing cells strongly support the metabolic roles of this carboxylesterase against insec.......

Discussion

In recent decades, heterologous expression systems have been widely used to express and isolate large amounts of proteins, thus allowing biochemical and functional determination and characterization of enzymes in vitro. To date, several different model systems including Escherichia coli, Pichia pastoris, Sacccharomyces cerevisiae, and Spodoptera frugiperda have been adapted for recombinant protein expression, and the choice of the in vitro system is crucial for large .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Q5 High-Fidelity DNA Polymerase	New England Biolabs inc.	M0491L
QIAquick Gel Extraction Kit	QIAGEN	28704
pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli	Invitrogen by life technology	K240020	S.O.C medium and universal M13 sequence primers were included in this kit.
PureLink HiPure Plasmid Miniprep Kit	Invitrogen by life technology	K210002
Gateway LR Clonase II Enzyme mix for BaculoDirectTM Kits	Invitrogen by life technology	11791-023
BaculoDirect C-Term Linear DNA Transfection Kit	Invitrogen by life technology	12562-019	Cellfectin transfection reagent and ganciclovir were included in this kit
pENTR-CAT plasmid	Invitrogen by life technology		Included in BaculoDirect C-Term Linear DNA Transfection Kit, concentration: 0.5 ug/uL
Heat inactivated Fetal Bovine Serum, Certified	Gibco by Life Technologies	10082-139
Sf9 cells in Sf-900 III SFM	Gibco by Life Technologies	12659017
Insect Cell-PE LB Insect Cell Protein Extraction & Lysis Buffer	G Biosciences by A Geno Technology Inc	786-411
Sf-900 III SFM (1×) Serum Free Medium Complete	Gibco by Life Technologies	12658-019
Grace's Insect Medium, unsupplemented	Gibco by Life Technologies	11595030
Permethrin (isomers) analytical standard	SUPELCO by Solutions WithinTM	442748
Methanol (analytical graded)	Sigma-Aldrich	67-56-1
Acetonitrile (analytical graded)	Sigma-Aldrich	75-05-8
GHP Acrodisc 25 mm Syringe Filters with 0.45 μm GHP Membrane (HPLC Certified)	Pall Life Sciences	21890388
Alliance Waters 2695 HPLC System	Waters
T100 Thermal Cycle	Bio-Rad Laboratories Inc.	1861096
Nanodrop 2000/2000c Spectrophotometers	ThermoFisher Scientific	ND2000CLAPTOP
Cytation 5 Cell Imaging Multi-Mode Reader	BioTek