Functional Analyses of House Fly Carboxylesterases Involved in Insecticide Resistance

Article abstract of DOI:10.3389/fphys.2020.595009

Carboxylesterase-mediated metabolism is one of major mechanisms involved in insecticide resistance. Our previous study has identified multiple carboxylesterase genes with their expression levels were significantly upregulated in pyrethroid resistant house flies. To further explore their metabolic functions, we used insect Spodoptera frugiperda (Sf9) cells to express these carboxylesterases in vitro and measure their hydrolytic activities toward esterase substrates. Our results indicated that these carboxylesterases can efficiently hydrolyze α-naphthyl acetate rather than β- naphthyl acetate. A cell based MTT cytotoxicity assay indicated that carboxylesterase-expressing cells show enhanced tolerance to permethrin, suggesting important roles of these carboxylesterases in metabolizing permethrin and thereby protecting cells from permethrin treatments. The metabolic functions of carboxylesterases were further verified by conducting in vitro metabolism studies toward permethrin and its potential metabolites 3-phenoxybenzyl alcohol and 3-phenoxybenzaldehyde, which not only suggested the potential metabolic pathway of permethrin in insects, but also important roles of these candidate carboxylesterases in metabolizing permethrin and conferring resistance in house flies. Homology modeling and docking were finally conducted to reflect interactions between permethrin ligand and carboxylesterase proteins, visually confirming the metabolic functions of carboxylesterases to insecticides in house flies.

Full text of DOI:10.3389/fphys.2020.595009

ORIGINAL RESEARCH
published: 16 October 2020
doi: 10.3389/fphys.2020.595009
Functional Analyses of House Fly
Carboxylesterases Involved in
Insecticide Resistance
1
,2
1
Xuechun Feng and Nannan Liu *
1
2
Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, United States, Division of Biological
Sciences, University of California, San Diego, San Diego, CA, United States
Carboxylesterase-mediated metabolism is one of major mechanisms involved in
insecticide resistance. Our previous study has identified multiple carboxylesterase genes
with their expression levels were significantly upregulated in pyrethroid resistant house
flies. To further explore their metabolic functions, we used insect Spodoptera frugiperda
(
Sf9) cells to express these carboxylesterases in vitro and measure their hydrolytic
Edited by:
Peng He,
activities toward esterase substrates. Our results indicated that these carboxylesterases
can efficiently hydrolyze α-naphthyl acetate rather than β- naphthyl acetate. A cell based
MTT cytotoxicity assay indicated that carboxylesterase-expressing cells show enhanced
tolerance to permethrin, suggesting important roles of these carboxylesterases in
metabolizing permethrin and thereby protecting cells from permethrin treatments. The
metabolic functions of carboxylesterases were further verified by conducting in vitro
metabolism studies toward permethrin and its potential metabolites 3-phenoxybenzyl
alcohol and 3-phenoxybenzaldehyde, which not only suggested the potential metabolic
pathway of permethrin in insects, but also important roles of these candidate
carboxylesterases in metabolizing permethrin and conferring resistance in house flies.
Homology modeling and docking were finally conducted to reflect interactions between
permethrin ligand and carboxylesterase proteins, visually confirming the metabolic
functions of carboxylesterases to insecticides in house flies.
Guizhou University, China
Reviewed by:
Kai Lu,
Fujian Agriculture and Forestry
University, China
Shaoli Wang,
Institute of Vegetables and Flowers
(
CAAS), China
Youssef Dewer,
Agricultural Research Center, Egypt
*
Correspondence:
Nannan Liu
liunann@auburn.edu
Specialty section:
This article was submitted to
Invertebrate Physiology,
a section of the journal
Keywords: carboxylesterases, insecticide resistance, Musca domestica, in vitro metabolism, MTT assay,
homology modeling and docking analysis
Frontiers in Physiology
Received: 14 August 2020
Accepted: 25 September 2020
Published: 16 October 2020
INTRODUCTION
House flies, Musca domestica, are ubiquitous agricultural and sanitary pests that can mechanically
transmit more than 100 human and animal disease pathogens, including bacterial, protozoan,
helminthic, and viral pathogens (Sasaki et al., 2000; Barin et al., 2010; Gordon Hewitt, 2011; Abbas
et al., 2014; Scott et al., 2014). Pyrethroids are currently the most widely used insecticides for the
management of many different insects including house flies due to their high insecticidal potency,
low mammal toxicity and environmental friendliness (Casida et al., 1983; Soderlund and Knipple,
2003). However, house flies can rapidly develop resistance and cross-resistance to insecticides,
Citation:
Feng X and Liu N (2020)
Functional Analyses of House Fly
Carboxylesterases Involved
in Insecticide Resistance.
Front. Physiol. 11:595009.
doi: 10.3389/fphys.2020.595009
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October 2020 | Volume 11 | Article 595009

Feng and Liu
Carboxylesterases in Insecticide Resistance
which is a major concern for house fly control strategies
worldwide (Liu and Yue, 2000; Kaufman et al., 2010; Scott et al.,
MATERIALS AND METHODS
2
013; Abbas et al., 2014).
The House Fly Strain
Previous work to characterize the underlying molecular basis
ALHF, a multi-insecticide resistant house fly strain, was used
in this study. ALHF was collected from a poultry farm in
Alabama in 1998 (Liu and Yue, 2000). This strain was further
selected with permethrin in the laboratory for six generations
after collection and maintained under biannual selection with
permethrin, reaching to a high resistance level (∼2000 fold)
to permethrin (Liu and Yue, 2000; Tian et al., 2011; Feng
for the development of insecticide resistance has already laid
the foundation for a better understanding of this important
issue and facilitated efforts to design novel strategies to
efficiently prevent or minimize the spread and evolution of
resistance development in many insect pests (Hemingway and
Ranson, 2000; Roush and Tabashnik, 2012). The interactions
of multiple mechanisms (i.e., increased detoxification and
decreased target site sensitivity) or genes (i.e., cytochrome
P450s and carboxylesterases) responsible for insecticide
resistance have been extensively studied in recent years
◦
et al., 2018). The house flies were reared at 25 ± 2 C under a
photoperiod of 12:12 (L: D) hours and fed with sugar and water.
Construction of pENTRTM Expression
Plasmids of Carboxylesterase Genes
(
Corbel et al., 2007; Bass et al., 2014; Liu, 2015). In particular,
carboxylesterases, as one of the major detoxifying enzymes
in insects, have attracted attentions for their potential role in
sequestering and metabolizing insecticides (Wheelock et al.,
The total RNA was extracted from 20 3-day old adult female
ALHF house flies using the acidic guanidine thiocyanate-phenol-
chloroform method (Chomczynski and Sacchi, 2006). The DNA
was removed from the total RNA using DNase (TURBO
DNA-free, Ambion). The first-strand cDNA was synthesized
with DNA-free total RNA using a Transcriptor First Strand
2
005; Grigoraki et al., 2015; Grigoraki et al., 2017). Multiple
carboxylesterase genes have shown to be transcriptionally
up-regulated in various resistant insects, including house
flies (Cao et al., 2008; Bao et al., 2010; Zhang et al., 2010;
Adelman et al., 2011; Bass and Field, 2011; Fuentes-Contreras
et al., 2013; Demkovich et al., 2015). These overexpressed
carboxylesterases are thought to sequester the insecticides and
hydrolyze them into less harmful substances, thus facilitating
excretion outside the insect bodies (Field and Blackman, 2003;
Wheelock et al., 2005). In both Aedes aegypti and Anopheles
gambiae mosquitoes, pyrethroids can be metabolized by
carboxylesterases to form PBOH (phenoxybenzoic alcohol)
and PBCHO (phenoxybenzaldehyde), which can be further
metabolized by cytochrome P450 monooxygenases to
PBCOOH (phenoxybenzoic acid) (Somwang et al., 2011;
Chandor-Proust et al., 2013).
Our previous studies explored expression profiles of all
carboxylesterases in different house fly strains and revealed
four carboxylesterase genes, MdαE7, MdβE2, MdαE17, and
MdIntE7, with their expressions of 3- to 15-fold higher
in pyrethroid resistant house fly strain compared to those
in susceptible house fly strains, indicating the potential
involvements of these carboxylesterases in insecticide
resistance of house flies (Feng et al., 2018). To further
characterize the metabolic roles of carboxylesterase against
insecticides, we investigated the functions of MdαE7 in
cDNA Synthesis Kit (Roche) and oligo-dT primer following
TM
the manufacturer’s instructions. The pENTR
expression
plasmids of carboxylesterases were constructed with gene-specific
1
primers designed based on their full-length nucleotide sequences
0
with four nucleotide bases CACC added to the 5 end of
forward primer (immediately upstream of the ATG transcription
start codon) (Supplementary Table 1), which enables the
TM
carboxylesterase genes to be directly cloned into the pENTR
ꢀR
TOPO vector (Invitrogen) by annealing the CACC sequence in
the PCR products with the overhang tag GTGG in the vector.
The GFP gene was amplified from a plasmid kindly provided as
TM
a gift, with CACC added to allow the construction of pENTR
expression plasmid (Supplementary Table 1). The recombinant
ꢀR
vector was then transformed into One Shot competent E. coli.
TM
pENTR
plasmids with target carboxylesterase genes were
purified using the PureLink HQ Mini plasmid purification Kit
Invitrogen). The orientation of the inserted genes was detected
(
by using the forward primer of each of the specific genes and
the reverse primer of M13. Expression plasmids were further
verified by sequencing.
Recombinant Baculovirus Expression of
metabolizing permethrin via MTT cytotoxicity assay and Carboxylesterases in Sf9 Cells
in vitro metabolism assay, revealing high efficiency of The detailed methods follow our previous protocol (Feng
TM
MdαE7 against permethrin in different ways (Feng and Liu, and Liu, 2018). Briefly, the pENTR
plasmid of each
2
018). We hypothesized that the development of insecticide carboxylesterase gene and GFP gene was ligated with
TM
resistance was conferred by the combination of multiple BaculoDirect Linear DNA using the LR clonase II enzyme mix
TM
overexpressed metabolic genes in house flies. To test our through the BaculoDirect
Baculovirus Expression System.
hypothesis, in the current study, we functional characterized The constructed recombinant baculovirus was then transfected
the up-regulated carboxylesterase genes through in vitro into Spodoptera frugiperda (Sf 9) cells using Cellfectin II Reagent
metabolism studies, insect cell viability measurements, (Invitrogen) to produce recombinant baculovirus stock solutions.
and homology modeling and docking analysis. Our study The large-scale expression of carboxylesterase proteins in the Sf 9
provided direct evidence for the involvement of multiple cells was performed according to the manufacturer’s instructions
carboxylesterases in metabolizing permethrin, resulting in
pyrethroid resistance in house flies.
1https://www.ncbi.nlm.nih.gov/genome/?term=Musca+domestica
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Carboxylesterases in Insecticide Resistance
(
Invitrogen). The titer of the baculovirus was measured by plaque
MTT Cytotoxicity Assay
8
forming assay and a titer of ∼2 × 10 pfu/mL P2 virus was used
to infect Sf 9 cells for large-scale expression of carboxylesterase
proteins, largely because the cells own the highest infection
ratio at the same time with lowest cell death ratio under P2
The cytotoxicity assay of the carboxylesterases was conducted
according to Gong et al. (2017) with modifications. The cell
infection procedures follow methods described previously (Feng
and Liu, 2018). The cells at P2 infected stage (with a titer of
infection stage compared with at other infection stages (Feng
and Liu, 2018). The parental Sf 9 cells and pENTR
8
2
◦
∼
2 × 10 pfu/mL) were cultured in 25 cm flasks at 27 C.
TM
CAT
TM
The pENTR
CAT expressing cells cultured under the same
(
plasmid producing baculovirus expressing chloramphenicol
conditions was served as controls. After 48 h cultivation, cells
expressing either GFP or different carboxylesterases were seeded
acetyltransferase (CAT) protein [Invitrogen]) infected cells
served as controls. The cell lysate protein was harvested after
5
onto 24 well plates with a density of 2 × 10 cells/well, and
7
4
2 h infection and centrifuged at 1000 rmp for 10 min at
later treated with permethrin standard solutions (the mixture
of cis- and trans-isomers dissolved in acetonitrile) (analytical
standard, Sigma-Aldrich), with final concentrations ranging from
◦
C. The cell pellets were washed twice using ice-cold PBS
TM
buffer (pH 7.4) and then re-suspended in insect cell PE LB
buffer. Subsequently, the dissolved cell lysate was centrifuged at
9
5
0 to 400 µM. The cytotoxic effects of the permethrin standards
800 rmp for 15 min, and then the supernatant was collected
were evaluated with a MTT cell viability assay kit (Sigma).
After 48 h treatment, the cell culture medium was removed
and the cells were washed with PBS buffer (0.1 M, pH 7.4).
Later, 200 µL of thiazolyl blue tetrazolium bromide solution
◦
and stored at −80 C. The protein concentration was measured
with Bradford method (Bradford, 1976). For each protein, three
replications for individual infection were performed to produce
different batch of carboxylesterase proteins.
(
Sigma-Aldrich) (5 mg/mL) was added to each well and the
◦
plate was incubated at 37 C for 4 h, after which the absorbance
values were measured at 540 nm using the Cytation 3 imagine
reader (BioTek, United States). Four replications were conducted
with independent protein preparations. The cell viability was
calculated in comparison with acetonitrile-treated cells. For the
inhibition assay, the inhibitor S, S, S-tributyl phosphorotrithioate
Carboxylesterase Activity Assays
The activities of the carboxylesterases were determined by
measuring their hydrolysis toward α-naphthyl acetate (α-NA)
and β-naphthyl acetate (β-NA), both of which are ester substrates
commonly used for esterase activities. The method follows Zhang
et al. (2010) with modifications based on our pretests, in which
we found that the protein amount around 50 µg is adequate for
measuring the hydrolytic activities and meet the detection limit
of the spectrophotometer. Briefly, a 25 µL of carboxylesterase
protein solution (with a final concentration of 2.0 µg/µL protein
(DEF) (Sigma-Aldrich) (with final concentrations of 0.1, 1,
and 10 µM) was added, together with 200 µM permethrin in
each well, and cell viability was calculated in comparison with
treatments with no DEF added.
−
4
in 0.1 M PBS buffer, pH 7.5) and 90 µL of 3 × 10 M substrate
In vitro Metabolism of Permethrin and Its
solution (either α-NA or β-NA dissolved in 0.1 M PBS buffer)
◦
were added to a 96-well microplate and incubated at 30 C for Metabolites by Carboxylesterases
3
0 min. The reaction was stopped by the addition of 45 µL Each substrate standard (Permethrin, PBOH or PBCHO) was
of freshly prepared diazo blue-sodium lauryl sulphate solution initially dissolved in acetonitrile to make 1 mM stock solution.
containing 2 parts of 1% fast blue B salt and 5 parts of 5% sodium Serial dilutions of stock solution were then prepared in
(
dodecyl sulfate solution) in each well. After 15 min incubation at acetonitrile to create the standard curve for each. A total
room temperature, the absorbance value of hydrolysis product of 700 µL metabolism reaction contained 20 µM substrate
α-naphthol or β-naphthol was measured at 600 or 550 nm, standard and 1 mg carboxylesterase protein (MdαE17, MdβE2
respectively, with a 96-well microplate reader (Cytation 3 imagine or MdIntE7) dissolved in 0.2 M Tris–HCl buffer. After 2 h
◦
reader, BioTek, United States) and then converted to product incubation at 30 C with orbital shaking, the reaction was
formation rate (pmol/min/mg protein) based on the standard quenched by adding 700 µL ice-cold acetonitrile and incubated
curves for α-naphthol or β-naphthol. For the kinetic assay, we with shaking for an additional 15 min. After that, the mixture
conducted pretest for the range of substrate concentration (0.01 was centrifuged at 10000 rmp for 2 min and the supernatant
to 2 mM) before the real experiments and showed that the was collected by filtering through 0.45 µm membranes and
substrate concentration at a range of 0.1 to 1.2 mM had the transferred to ultraclean glass vials for HPLC analysis. The
better degradation curve by enzymes and met the detection HPLC analysis was monitored by a reverse-phase HPLC system
limits of the spectrophotometer reader. Accordingly, the kinetic (Alliance Waters 2695) equipped with a Nova-Pak C18 column
parameters, including Michaelis constant (Km) and maximal (60 Å, 4 µm, 3.9 mm × 150 mm, 1/pkg [WAT086344]) and a
velocities (Vmax) for each carboxylesterase were measured using Waters 2487 Dual λ absorbance detector. Two mobile phases
a series of substrate (α-NA or β-NA) concentrations ranging (mobile phase A: 90% acetonitrile and 10% water; mobile phase
from 0.1 to 1.2 mM (add 3.8, 7.7, 11.5, 15.3, 19.2, 23.0, 26.8, B: 5% acetonitrile adjusted to pH 2.3 with 85% phosphoric acid)
−
3
3
0.7, 34.5, 38.3, 42.2, and 46.0 µL of 3 × 10
M substrate were used for the gradient elution with a flow rate of 1 ml/min
solution, respectively, into each well). The reactions with proteins and measured at a wavelength of 232 nm. The gradient system
extracted from the parental Sf9 cells or CAT-recombinant was initiated with 50% of solvent A and 50% of solvent B rising to
baculovirus infected cells served as controls. Three replications 75% of mobile phase A at 6 min and finishing at 100% of solvent
were performed with independent prepared proteins.
A at 8 min. The flow of 100% mobile phase A was maintained
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Carboxylesterases in Insecticide Resistance
for 4 min and then reduced to 50% at 13 min and continued were isolated from Sf 9 cells and further used for biochemical
for a further 4 min to return the column to the initial conditions characterization. Our results indicated that carboxylesterase
for the next run. Reactions containing no enzymes were used to proteins obtained from insect Sf 9 cells were capable of
calculate the substrate depletion percentage. Three replications hydrolyzing α-naphthyl acetate (α-NA) to produce α-naphthol
were performed, and a paired t-test was used to analyze the at different efficiencies, with a hydrolytic activity ranging from
−
1
−1
results. Reactions with proteins extracted from CAT expressing 6083.5 to 13810.1 pmol · min · mg , which were 1.8–4.0 fold
cells served as controls.
higher than that measured in either the parental Sf 9 cells or
TM
the pENTR
CAT (plasmid producing baculovirus expressing
In silico Modeling and Docking Analysis
chloramphenicol acetyltransferase (CAT) protein [Invitrogen])
In silico 3D structure modeling of each carboxylesterase infected cells served as controls (Table 1), indicating the
protein was performed by the I-TASSER server utilizing the strong hydrolytic capabilities of carboxylesterases in metabolizing
2
combined methods of threading and ab initio modeling (Roy esterase substrate α-NA. The kinetic parameter Km and Vmax for
et al., 2010; Zhang et al., 2011). Five models were predicted each hydrolytic reaction were also measured by using substrate
for each carboxylesterase gene and the top scoring model α-NA with final concentrations from 0.1 to 1.2 mM and listed in
submitted to the FG-MD server for fragment guided molecular Table 1. These data indicated that all these reactions followed the
dynamics structure refinement (Zhang et al., 2011). Model Michaelis-Menten equation; The maximum velocities (Vmax)
quality was controlled by Ramachandran plots generated with of carboxylesterase reactions were ranging from 41048.9 to
3
4
−1
−1
Procheck (Laskowski et al., 1993) and ProSA-web (Sippl, 1993; 71586.7 pmol · min · mg , significantly higher than the
−
1
−1
for parental
Wiederstein and Sippl, 2007). Proteins and ligands were prepared values in controls (9503.9 pmol · min · mg
5
−1
−1
TM
for docking with Autodock Tools V1.5.6 . Molecular docking Sf9 cells and 10343.6 pmol · min · mg for pENTR
CAT
was performed by Autodock 4.2 (Morris et al., 2009). Ligand infected cells, respectively), suggesting the strong hydrolytic
6
permethrin structures were retrieved from the ZINC database
capabilities of these carboxylesterases toward substrate α-NA.
(
Irwin and Shoichet, 2005). For all dockings, a search space Simultaneously, we examined the hydrolytic activities and
with a grid box of 60 × 60 × 60 Å centered on the serine of kinetic parameters of different carboxylesterases to another
the catalytic triad of the carboxylesterase was used. All protein substrate, β-naphthyl acetate (β-NA), and the hydrolytic values
7
−1
−1
structure images were produced by Pymol (DeLano, 2002). The are low (less than 20.0 pmol · min · mg ) in all these
binding cavity and its constitutive amino acids were predicted by genes (Table 1).
LigPlot (Wallace et al., 1995). Protein structure diagrams were
produced using TopDraw (Bond, 2003).
Cytotoxicity of Permethrin in
Carboxylesterase-Expressing Sf9 Cells
RESULTS
(
MTT Assays)
To confirm that Sf 9 cells can be applied to study in vivo
metabolic functions of carboxylesterases, and to prove that
MTT assay is a feasible method to measure cell viabilities
against different permethrin stimuli, we firstly build a GFP-
recombinant baculovirus and use it to infect insect Sf 9 cells.
The GFP expression make it visible to directly observe cell
viabilities under different conditions. The 48 h-post infection
cells at P2 stage were then chosen to perform the permethrin-
treated experiments due to the relative higher cell viability
and protein expression levels at this stage (Feng and Liu,
2018). By treating the GFP-expressing cells with permethrin
Carboxylesterase Activity
The expression of carboxylesterase protein was accomplished
by infecting insect Sf 9 cells with constructed carboxylesterase-
recombinant baculovirus. The carboxylesterase proteins obtained
2
http://zhanglab.ccmb.med.umich.edu/I-TASSER/
http://services.mbi.ucla.edu/SAVES/
3
4
5
6
7
http://prosa.services.came.sbg.ac.at/prosa.php
http://mgltools.scripps.edu/downloads
http://zinc.docking.org/
6
http://www.pymol.org/
TABLE 1 | Hydrolytic activities and kinetic parameters of M. domestica carboxylesterases.
Enzyme
α-naphthyl acetate
β-naphthyl acetate
Activitya
Kmb
Vmaxc
Activitya
Kmb
Vmaxc
Sf9 cells
CAT gene
MdαE7
3458.2 ± 168.2
3645.0 ± 173.5
11396.6 ± 484.8
13810.1 ± 635.6
8610.8 ± 335.2
6083.5 ± 326.6
186.4 ± 7.6
213.2 ± 10.7
568.0 ± 22.3
651.9 ± 33.8
523.3 ± 19.5
521.7 ± 15.4
9503.9 ± 151.0
10343.6 ± 186.8
53555.5 ± 1649.7
71586.7 ± 2585.8
46414.3 ± 1401.9
41048.9 ± 1748.2
8.4 ± 0.5
8.8 ± 0.6
12.9 ± 0.6
18.4 ± 1.7
13.8 ± 1.0
11.5 ± 0.6
56.4 ± 3.0
58.1 ± 3.0
146.8 ± 4.1
190.1 ± 4.3
188.3 ± 3.5
146.1 ± 4.8
9.9 ± 0.2
9.8 ± 0.1
15.7 ± 0.3
21.9 ± 0.7
16.0 ± 0.3
13.9 ± 0.1
MdαE17
MdβE2
MdIntE7
All data were listed as mean ± STE a: COE activity = pmol · min−1 · mg−1. b: Km (Michaelis constant) = µM. c: Vmax (Maximum velocity) = pmol · min−1 · mg−1
.
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Carboxylesterases in Insecticide Resistance
at different concentrations of 50, 100, 200, and 400 µM, the controls, ranging from 73.3 to 85.5% (Figure 1B-1). The
we observed that cell viabilities were gradually decreased as results show that carboxylesterase-expressing cells can greatly
the increment of permethrin concentrations (Supplementary enhance their tolerance to permethrin treatments compared
Figure 1) and no significant differences were observed in two with control cells.
controls (one is cells without permethrin treatments and the
To deeper explore roles of these carboxylesterases in
other with only acetonitrile added) (Supplementary Figure 1), detoxifying permethrin in insect cells, we then tested the cell
suggesting that the permethrin rather than the virus itself viabilities against permethrin in presence of the carboxylesterase
can greatly influence the cell viability. Also, no significant inhibitor, S, S, S-tributyl phosphorotrithioate (DEF). The cell
differences were observed in two controls (one is cells without viabilities in MdαE17 expressing cells were significantly dropped
permethrin treatments and the other with only acetonitrile from 84.7% (200 µM permethrin treatment in absence of
added) (Supplementary Figure 1), indicating that acetonitrile DEF) to 77.8, 64.4, and 53.8% when co-treated with 0.1,
used to dissolve permethrin will not affect cell viabilities. We 1, or 10 µM DEF, respectively (Figure 1A-2). For MdβE2,
then use the same method to test the cytotoxicity of permethrin a slight decrease in cell viability was detected between the
among all carboxylesterases and cell viabilities against different control (79.1%) and the cells subjected to co-treatment with
permethrin concentrations (50, 100, 200, and 400 µM) was 200 µM permethrin and 0.1, 1 or 10 µM DEF (70.5, 70.2, and
calculated in comparison with cells treated with acetonitrile 67.4%, respectively) (Figure 1B-2). For MdIntE7, although no
TM
alone. The pENTR
CAT expression Sf9 cells was used as significant difference was detected between the control (89.4%)
the controls. Our results showed that the viabilities of MdαE17 and cells co-treated with 200 µM permethrin and 0.1 µM DEF
expressing cells were significantly higher (78.8–94.1%) than (91.0%), significant decreases to 76.8 and 78.9% were observed
those of controls, which were 57.7–88.9% (Figure1A-1) when when co-treated with 200 µM permethrin and 1 or 10 µM
exposed to permethrin at different concentrations. The cell DEF, respectively (Figure 1C-2). No significant differences in
viabilities of MdIntE7 expressing cells ranged from 89.4 to 92.4%, the cell viabilities were detected in pENTR CAT infected cells
again more tolerant than the controls (Figure 1C-1); the cell when co-treated with 200 µM permethrin and DEF at different
viabilities of MdβE2 expressing cells, though weaker than those concentrations. The significant decrease in cell viability against
of MdαE17 and MdIntE7, were still significantly higher than permethrin cytotoxicity in carboxylesterase expressing cells when
FIGURE 1 | Roles of carboxylesterases in detoxification of permethrin in insect Sf9 cells. (A-1), (B-1), and (C-1) The comparisons of Sf9 cell viabilities expressing
MdαE17, MdβE2, and MdIntE7 to Sf9 cell viabilities expressing CAT gene (control), respectively, treated with 50, 100, 200, and 400 µM of permethrin. (A-2), (B-2),
and (C-2). The comparisons of Sf9 cell viabilities expressing MdαE17, MdβE2, and MdIntE7 to Sf9 cell viabilities expressing CAT gene (control), co-treated with
200 µM permethrin and 0.1, 1, or 10 µM of DEF. Three replications with independent prepared proteins were performed. Student’s t-test was used for the statistical
significance analysis. Different letters above the bars indicate the significant differences of cell viabilities under different treatments (P ≤ 0.05). The blue color line and
shape indicated the control groups (CAT-infected cells).
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Carboxylesterases in Insecticide Resistance
co-treated with DEF at different concentrations strongly supports percentages for these carboxylesterases were significantly higher
the involvement of these carboxylesterases in metabolizing than that of pENTRTM CAT gene (7.3 ± 0.8%) used as the
permethrin in insect cells.
control (Figure 2A).
The depletion percentages of two permethrin metabolites,
PBOH and PBCHO, were also measured by incubating 20 µM
substrate together with different carboxylesterase proteins
extracted from insect Sf 9 cells. The reaction of 20 µM
In vitro Metabolism of Permethrin, PBOH
and PBCHO by Carboxylesterases
TM
substrates incubated with proteins extracted from pENTR
Permethrin metabolism was assayed by incubating a 20 µM
permethrin standard together with different carboxylesterase
proteins extracted from infected Sf9 cells. The reactions of 20 µM
CAT infected cells again was served as the control. The
depletion percentage of the substrate was again calculated in
comparison with reactions in which only 20 µM substrates
were added. Reactions were monitored by reverse-phase HPLC
after a 120 min incubation period. The retention time of
PBOH was 3.3 min (Supplementary Figure 2B). The depletion
percentages of PBOH by MdαE17 and MdβE2 were 7.5 ± 0.5
TM
permethrin incubated with proteins isolated from pENTR
CAT infected cells was served as the control. The depletion
percentage for permethrin was calculated in comparison with
reactions where only 20 µM permethrin was added. Reactions
were monitored by reverse-phase HPLC after a 120 min
incubation period. Since the permethrin standard is actually a
mixture of cis- and trans- isomers, two peaks were observed in the
HPLC chromatographic profiles, with elution times of 10.7 and
and 7.2 ± 0.6%, respectively, none of which were significantly
TM
different from that of the pENTR
CAT infected cells
(
8.9 ± 0.5%) (Figure 2B). For MdIntE7, the depletion percentage
of PBOH was 2.2 ± 0.5%, lower than that achieved by
the control (Figure 2B). The retention time of the other
substrate, PBCHO, was 5.4 min (Supplementary Figure 2C).
Again, no significant differences were found in the depletion
percentages of PBCHO achieved by MdαE17 (12.1 ± 1.00%)
1
0.9 min for trans-permethrin and cis-permethrin, respectively
Supplementary Figure 2A). The highest depletion percentage
of permethrin was achieved by MdαE17, at 29.4 ± 2.3%
Figure 2A), followed by 16.4 ± 0.7% and 16.2 ± 0.7% for
MdIntE7 and MdβE2, respectively (Figure 2A). All depletion
(
(
FIGURE 2 | The depletion percentages of permethrin, PBOH and PBCHO by different carboxylesterases. (A) The depletion percentages of permethrin by
carboxylesterases. (B) The depletion percentages of PBOH by carboxylesterases. (C) The depletion percentages of PBCHO by carboxylesterases. Three
replications with independent prepared proteins were performed. Different letters above the bars indicate the significant differences for the depletion percentage of
substrates under different treatments (P ≤ 0.05).
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Carboxylesterases in Insecticide Resistance
and MdβE2 (13.0 ± 1.2%) compared with that of the control inserted after β1, two short α-helices inserted after β3 and four
(
9.9 ± 0.8%) (Figure 2C).
α-helices following β6 (boxed orange in Figure 3). Subdomain
II was composed of four α-helices inserted after β7 with the last
two α-helices near the C-terminals (boxed green in Figure 3).
A detailed comparison revealed that two small regions were
divergent in the four carboxylesterase proteins, which could have
Homology Modeling and Permethrin
Docking Analysis
To investigate the interactions between carboxylesterases a major impact on the rearrangement of the two subdomains
and permethrin, we conducted the homology modeling that form the binding cavity. The first is the antiparallel β-sheet
and permethrin docking analysis for four carboxylesterases. after β1 (shown as a red helix in Figures 3A-1,B-1), which
Several missing or inserted motifs were found in certain is present in MdαE7 and MdαE17, while absent in MdβE2
carboxylesterases, such as the missing of start antiparallel and MdIntE7. This structure can create a groove against the
β-sheets, β6 and αD in MdαE17 (Figures 3B-1,B-2); the missing N-terminal α-helices when packing, thereby preventing the
β1, β2, β5, and β6 and the insertion of a short helix following partial closure of the active site. The second is the short helix
αB in MdβE2 (Figure 3C-1,C-2); and a missing β4 and the before the αD (shown as red sheets in Figures 3A-1,B-1), which
insertion of a short sheet after the start antiparallel β-sheets in is present in MdαE7 and MdαE17 but absent in MdβE2 and
MdIntE7 (Figure 3D-1,D-2). The overall structures containing MdIntE7. This helix is thought to hold apart two subdomains
eight-stranded β-sheet (β1–β8) surrounded by six α-helices that comprise the binding cavity, leaving a much more open
(
αA-αF), together with two pairs of antiparallel β-strands at space for substrate binding.
the start and end of the protein structure, all of which were
Further docking analysis revealed the interactions between
comprised of the catalytic domain (shown as the magenta area carboxylesterase proteins and permethrin. Our preliminary tests
in Figure 3). Two bundles of α-helices at the top of the catalytic have individually docked four permethrin isomers into the
domain formed the αβ domain and the regulatory domain carboxylesterases and found that 1S-trans-permethrin isoform fit
(
shown as orange and green areas, respectively, in Figure 3). most snugly into the binding pockets with lowest binding energy.
A catalytic triad made up of Ser, His, and Glu/Asp (labeled as We therefore chose 1S-trans-permethrin isomer for this analysis.
black dots and sticks in Figure 3) was highly conserved among From Figure 4, we observed the majority of constitutive amino
these four carboxylesterases. Two subdomains on either side acids forming binding cavities were hydrophobic, including Gly,
of the active site cleft on the upper face of the protein formed Phe, Ala, Val, Leu, Ile, Met, and Trp, providing a hydrophobic
the substrate binding cavity (white surface area in Figure 3). environment for permethrin binding. The conserved catalytic
Subdomain I consisted of two short antiparallel α-helices triad composed of Ser, His, and Glu/Asp (shown in Figure 3) is
FIGURE 3 | The structures of carboxylesterases in M. domestica. (A-1), (B-1), (C-1), and (D-1) Topology representations of secondary structures of MdαE7,
MdαE17, MdβE2, and MdIntE7 displaying the conserved α/β hydrolase fold (labeled as gray), conserved motifs among four carboxylesterase proteins (labeled as
blue) and unique motifs belong to certain carboxylesterase structure (labeled as red). Two subdomains made up of bundles of α-helices (framed by orange and green
box) formed the substrate binding cavity. Three conserved amino acids (Serine, Histidine, and Glutamine/Aspartic acid) consisted of a catalytic triad were also
indicated as black dots. (A-2), (B-2), (C-2), and (D-2) Cartoon representations of structures of MdαE7, MdαE17, MdβE2, and MdIntE7 highlighting the regulatory
domain (green area), αβ domain (orange area), catalytic domain (magenta area) and binding pocket (white surface area). The conserved catalytic residues were also
labeled as black sticks.
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Carboxylesterases in Insecticide Resistance
involved in the catalytic process. Briefly, Ser residue attacks the
carbonyl group of permethrin to form a tetrahedral intermediate,
DISCUSSION
which then collapses to release Ser, the alcohol portion of In insects, carboxylesterase is one of the major metabolic
permethrin and a acyl-enzyme complex, allowing for the His enzymes that detoxify insecticides in the first phase of
residue to further attack and release the acid portion of metabolism (Somwang et al., 2011; Chandor-Proust et al., 2013).
permethrin (Wheelock et al., 2005; Satoh and Hosokawa, 2006). Overexpressed carboxylesterases lead to increased activities,
Based on this, the distance between the oxygen atom in the which further results in the enhanced metabolism of xenobiotics
OH side chain of Ser residue and the carbon atom in the or endogenous compounds (Bass and Field, 2011; Zhang et al.,
carbonyl group of substrates is of great value to determine 2013; Grigoraki et al., 2015). In a previous study we identified
the distance between carboxylesterase and permethrin ligand. multiple carboxylesterase genes in house flies, whose expressions
Our results in Figure 4 show that MdαE7 has the shortest were not only constitutively up-regulated in the resistant house
distance (=2.95 Å) to permethrin with the lowest binding fly ALHF strain, but can also be induced to much higher levels
energy (=−8.74 Kcal/mol) (Figure 4A); The distance between in response to permethrin, indicating the important role they
MdαE17 and permethrin is 3.01 Å with a binding energy of play in metabolizing permethrin in house flies (Feng et al., 2018).
8
.02 Kcal/mol (Figure 4B); both MdβE2 and MdIntE7 were even However, a functional characterization of these carboxylesterases
further from permethrin, at 7.70 and 6.77 Å and with relatively in vitro is still lacking.
higher binding energies of −7.35 Kcal/mol and −6.26 Kcal/mol
Here, a baculovirus-mediated insect Sf 9 expression system
Figures 4C,D), respectively.
was used to investigate the heterozygous expression of these
(
FIGURE 4 | Stereo view of permethrin bound within the active site cavity. Permethrin bound within the active site cavity of: (A) MdαE7; (B) MdαE17; (C) MdβE2; and
D) MdIntE7. The Serine (Ser) and Histidine (His) residues are labeled as green sticks and the 1S-trans-permethrin isomer as cyan sticks. The distance between
(
permethrin and the Ser residue of the carboxylesterase is indicated by a red dashed line. The binding energy is also shown. All amino acid active site cavities are
labeled in each carboxylesterase structure.
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Carboxylesterases in Insecticide Resistance
carboxylesterases in vitro; their hydrolytic activities toward metabolism of permethrin metabolites (Somwang et al., 2011;
different esterase substrates and permethrin insecticides were also Chandor-Proust et al., 2013).
measured in this study.
The house fly MdαE7 is orthologous to blowfly LcαE7, an
Our results indicated that all carboxylesterases showed strong α-esterase isolated from the Australian sheep blowfly Lucilia
activities to α-NA rather than β-NA, indicating that β-NA cuprina with its roles in organophosphate (OP) resistance in
may not be the most favorable substrate for measuring the blow flies (Jackson et al., 2013). The metabolic functions of
activities of these carboxylesterases. The choice of a substrate house fly MdαE7 towered insecticides (either OP or permethrin)
with which to monitor carboxylesterase activity remains a have also been reported in several studies (Zhang et al., 2010;
major obstacle hampering efforts to accurately characterize Feng and Liu, 2018). However, few studies have explored the
carboxylesterase activities, especially for multiple isozymes interactions between carboxylesterases and insecticides through
(
Wheelock et al., 2005). Also, all carboxylesterases significantly homology modeling and docking analysis. In this study, we
differ in their hydrolytic activities toward esterase substrates, built homology models for four carboxylesterases based on the
which was partially attributed to the various protein structures crystal structure of LcαE7. We compared their modeling and
leading to different substrate specificities (Hosokawa, 2008; found that their overall structures are highly conserved with
Younus et al., 2017).
majority of αβ hydrolase structures, especially for the catalytic
The data from GFP-expressing cells indicated that the triad. The detailed comparison among their structures revealed
MTT assay can be used to explore the cytotoxicity of several differences, and a small divergence in structures may
permethrin for all carboxylesterases. Although its accuracy significantly impact the overall topology of the substrate binding
was sometimes limited by numerous experimental parameters sites (Jackson et al., 2013). From our modeling, we can see MdαE7
correlated with cell metabolism and virus infections, but and MdαE17 have much more open spaces in their active sites
this can be diminished by performing more replicates compared to MdβE2 and MdIntE7, allowing much more favored
with independent cell and virus preparations, and it substrate binding and accommodations, which can be further
is no doubt that MTT assay is still a rapid and easily linked with their relatively higher metabolic efficiencies against
operated method to quickly detected cell cytotoxicity permethrin (Feng et al., 2018).
against permethrin treatments, giving a preliminary images
We further analyzed the binding modes of permethrin
in consideration of metabolic roles of carboxylesterases in within active cavities of different carboxylesterases. The catalytic
detoxifying permethrin in insect cells. Some other assays process of substrate within the carboxylesterases determines
can compensate to more precisely measure and compare the that the hydrogen bond distance as well as the binding energy
metabolic efficiencies.
are two important parameters to analyze the binding affinity.
Compared with MTT assay, the in vitro metabolism study is Usually, the lower binding energy together with a shorter
recognized as the most accurate strategy to directly reflect the distance between O residue of Ser and C in the carbonyl
metabolic efficiencies of detoxifying enzymes against xenobiotics. group of substrates indicate a stronger binding affinity between
In this study, the depletion percentages of permethrin and carboxylesterases and ligands. Among four carboxylesterase
its substrates have been successfully measured via in vitro binding modes, the MdαE7 showed lowest binding energy
metabolism assays and in turn reflecting the metabolic efficiencies as well as the shortest distance, which reflects its highest
of different carboxylesterases. By comparing the metabolic data binding affinity to permethrin, which also provide evidence
collected, we found that α-esterases, such as MdαE17 in this for its highest metabolic efficiencies against permethrin in this
study and MdαE7 reported in previous study (Feng and Liu, study. Also, through binding multiple permethrin isoforms in
2
018), have much higher metabolic abilities to permethrin protein cavities, we realized that the stereochemistry is another
compared with MdβE2 and MdIntE7, which is consistent with important factor in esterase-mediated metabolism, and our
the metabolism and detoxification roles of the α-esterase clade docking analysis found that 1S-trans-permethrin had the best
reported in classification studies (Campbell et al., 2003; Flores fit within binding cavities of four carboxylesterases, and the
et al., 2005; Wang et al., 2015; Feng and Liu, 2018). While similar findings have also been reported in carboxylesterase E4
the overall metabolic efficiencies of these carboxylesterases to of the aphid Myzus persicae, which exhibits absolute specificity
permethrin (∼16 to ∼30%) are less efficient than those achieved for hydrolyzed 1S-trans-permethrin rather than other isomers
by multiple CYP450s in mosquitoes (∼40−∼45%) (Gong et al., (Devonshire and Moores, 1982).
2
017), the carboxylesterases may serve as an “insecticide sink”
In conclusion, our study has firstly employed baculovirus-
that delays or prevents the interactions of insecticides and target mediated insect cell expression system to express
sites rather than directly metabolizing them (Oakeshott et al., carboxylesterases and characterized their functions in vitro
2
005; Li et al., 2007). Two main permethrin metabolites, PBOH via different assays, which provided multiple choices for
and PBCHO, were also measured for their depletion percentages exploring metabolic functions of detoxifying proteins in insects.
among these carboxylesterases, and no significant metabolic The results of these house fly carboxylesterases in metabolizing
effects were detected, which can be explained by the hypothesized permethrin have further provided directed scientific evidence
permethrin metabolic route proposed in mosquitoes, where for the esterase-mediated resistance in insects that sheds light on
carboxylesterases only play essential roles in the first phase of the mechanisms governing insecticide resistance development
permethrin metabolism, and other enzymes such as cytochrome and could lead to the development of innovative new pest
P450s or glutathione S-transferases are involved in following management strategies.
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October 2020 | Volume 11 | Article 595009

Feng and Liu
Carboxylesterases in Insecticide Resistance
DATA AVAILABILITY STATEMENT
SUPPLEMENTARY MATERIAL
The original contributions presented in the study are included The Supplementary Material for this article can be found
in the article/Supplementary Material, further inquiries can be online at: https://www.frontiersin.org/articles/10.3389/fphys.
directed to the corresponding author.
2020.595009/full#supplementary-material
Supplementary Figure 1 | The cell viabilities under different permethrin
treatments. The GFP-expressing cells at P2 infection stage were observed under
fluorescent microscope. As the increase of permethrin concentration, the cell
viabilities were gradually decreased. The GFP-expressing cells without permethrin
treatment and with only acetonitrile added were served as control groups.
AUTHOR CONTRIBUTIONS
NL and XF conceived and designed the study and wrote the
manuscript. XF performed the experiments. NL prepared the
materials. Both authors reviewed the manuscript.
Supplementary Figure 2 | HPLC profiles of permethrin, PBOH and PBCHO. (A)
The HPLC profile of permethrin standard (a mixture of trans-permethrin and
cis-permethrin isomers). The red arrows indicate the peaks for the
trans-permethrin and cis-permethrin isomers. (B) The HPLC profile of PBOH
standard. The red arrow indicates the peak for PBOH. (C) The HPLC profile of
PBCHO standard. The red arrow indicates the peak for PBCHO.
FUNDING
The project described was supported by Award AAES Hatch/
Multistate Grants ALA08-045 and ALA015-1-10026 to NL.
Supplementary Table 1 | The primer list.
twenty-something years on. Nat. Protoc. 1, 581–585. doi: 10.1038/nprot.
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