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Published as doi: 10.1096/fj.07-8237com.
 (The FASEB Journal. 2007;21:3584-3591.)
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Do mosquitoes acquire organophosphate resistance by functional changes in carboxylesterases?

Feng Cui*, Hong Qu{dagger}, Jian Cong{ddagger}, Xiao-Li Liu§ and Chuan-Ling Qiao*,1

* State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China;

{dagger} College of Life Sciences, Peking University, Beijing, China;

{ddagger} College of Food Science and Technology, Shanghai Fisheries University, Shanghai, China; and

§ Shijiazhuang Pharmaceutical Group, Shijiazhuang, China

1Correspondence: State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China. E-mail: qiaocl{at}ioz.ac.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Carboxylesterase-based metabolic resistance to organophosphates (OPs) in insects has been shown to originate either from mutations in esterase-encoding sequences or from amplification of esterase genes. This study aimed to test the hypothesis that mosquitoes can acquire OP resistance by functional changes in carboxylesterases. Mutations were introduced into the esterase B1 of mosquito Culex pipiens by site-directed mutagenesis at positions 110 and 224. Three single mutants (G110D, W224L, and W224S) and two double mutants (G110D/W224L and G110D/W224S) were expressed and purified. All five mutants lost native carboxylesterase activity. Mutation W224L converted esterase B1 to an OP hydrolase and increased its malathion carboxylesterase activity. No obvious OP hydrolysis was observed by G110D or W224S. Our data strongly support our hypothesis and suggest that mutation W224L might occur in natural populations of mosquitoes. Sequence comparison shows that the site 224 is especially highly conserved among various insect carboxylesterases. This leads to another hypothesis: that the position 224 plays a key role in insect carboxylesterases’ switching from their native physiological functions to other functional niches under selection pressure exerted by insecticides.—Cui, F., Qu, H., Cong, J., Liu, X.-L., Qiao, C. L. Do mosquitoes acquire organophosphate resistance by functional changes in carboxylesterases?


Key Words: organophosphorus hydrolase • site-directed mutagenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RESISTANCE TO INSECTICIDES IN MANY INSECT species has been ascribed to changes either in the sensitivity of the target protein acetylcholinesterase (AChE) or in the rate of metabolism of the insecticides by carboxylesterases (1 , 2) . Both AChE and carboxylesterases belong to the carboxyl/cholinesterase multigene family, a branch of the {alpha}/ß-hydrolase fold superfamily. Members of this superfamily have a highly conserved catalytic triad of noncontiguous residues in the primary sequence. The triad is generally Ser, Glu, and His, but can be Ser, Asp, and His or Cys, Asp, and His. Apart from this catalytic triad, other characteristic structural features are an "oxyanion hole" and an "acyl-binding pocket". Notwithstanding considerable sequence divergence among the carboxyl/cholinesterases, the conservation of their tertiary structure appears to be high (3) .

Carboxylesterase-based metabolic resistance to OPs in insects generally has two origins. One is a qualitative change in enzymatic properties due to mutations in esterase-encoding sequences, resulting in an acquired OP hydrolase activity at the cost of decreased carboxylesterase activity. This is a "mutant ali-esterase model" of resistance and results from a qualitative change in the enzymatic properties of the enzyme. The other is a quantitative change achieved by tandem amplification of carboxylesterase genes, resulting in overexpression of the proteins (4) .

Qualitative changes in esterases have been documented in some OP-resistant strains of higher dipterans, such as Lucilia cuprina, L. sericata, and Musca domestica (5 6 7) . Their OP resistance maps to orthologous genes (Lc{alpha}E7, Ls{alpha}E7, and Md{alpha}E7, respectively) in the {alpha}-cluster of carboxylesterases. Two single mutations have been detected in natural populations. One, G137D (in Lc{alpha}E7), is responsible for diazinon-type resistance and is located in the "oxyanion hole" in the three-dimensional structure of the enzyme. This mutant esterase catalyzes hydrolysis of diethyl OPs faster than dimethyl OPs (8) . Another mutation, W251L/S (in Lc{alpha}E7), is responsible for the malathion-type resistance and is located in the "acyl-binding pocket." It exhibits exceptionally high malathion carboxylesterase (MCE) activity (which is defined as the hydrolysis of the carboxylester linkages of malthion), intermediate hydrolase activity against dimethyl OPs, and low activity against diethyl OPs (8) .

Quantitative amplification of specific esterase genes is only documented in culicine mosquitoes (C. pipiens complex and C. tarsalis) and aphids (Myzus persicae). OP resistance in the C. pipiens complex is mainly due to coamplification (e.g., of allele pairs A2-B2, A4-B4, A8-B8, and A9-B9) but sometimes to single amplification (B1) or up-regulation (A1 only) of two esterase loci on chromosome II (Est-3 encoding esterase A and Est-2 encoding esterase B; refs. 9 , 10 ). Overproduced esterase proteins act as high-affinity sinks that sequester and very slowly hydrolyze OPs (11) . Although their OP hydrolytic activity is negligible, their abundance (e.g., up to 12% of the mosquito soluble proteins for esterase B1) enables them to sequester sufficient OP for the organism to survive (12) .

Is it possible for mosquitoes to evolve to be resistant to OPs by qualitative changes of carboxylesterases as higher dipterans do? This study aimed to test the hypothesis that mosquitoes can acquire OP resistance by functional changes in carboxylesterases, based on the results of site-directed mutagenesis of esterase B1 of C. pipiens.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Localization of mutagenesis sites by protein structure comparisons
With the use of the SwissModel server (http://swissmodel.expasy.org; refs. 13 14 15 ), the tertiary structures of esterase E3 (amino acid residues 1–294) from L. cuprina, esterase B1 (residues 1–309) from C. pipiens, and esterase Mda-E7 (residues 4–295) from M. domestica were predicted based on the dimensional structures of known homologs (1q84A, 1ku6A, 1q83A, 1n5mA and 1p0mA for E3; 1ku6A, 1c2oD, 1c2oA, 1maaD, and 1maaA for B1; and 1c7jA, 1qe3A, 1c7iA, 1q84A, and 1q83A for Mda-E7). The predicted structures and the structure of AChE (lacj) were compared and used as a basis of selecting positions for site-directed mutagenesis of esterase B1. Two positions were chosen: G110 and W224, corresponding to G137 and W251 in esterase E3 and to G119 and W233 in AChE.

Mutagenesis and expression of fusion proteins
Mutations were introduced into the cloned ORF of B1 gene from C. pipiens (16) by site-directed mutagenesis with the QuickChange kit using the manufacturer’s (Stratagene, La Jolla, CA, USA) protocols. Only one base was changed at each site (GGC->GAC for G110D, TGG->TTG for W224L, and TGG->TCG for W224S). Details of primers and PCR systems are available on request. All mutant genes were validated by sequencing, and the validated sequences were then transferred into the pET28a plasmid vector. Three single mutants (G110D, W224L, and W224S), two double mutants (G110D/W224L, and G110D/W224S), and the wild-type sequence, plus a vector-only construct, were expressed in Escherichia coli strain BL21 (DE3) to produce fusion proteins with a sequence of histidine residues (His-Tag) for purification of target proteins. Transformation was performed according to the pET System Manual (10th edn. TB055, Novagen 2002). One colony from freshly transformed cells was used to inoculate 2 ml Luria-Bertani (LB) medium containing 50 µg·ml–1 kanamycin and incubated overnight at 37°C. Then, a 1 ml cell suspension was used to inoculate 500 ml LB medium containing 50 µg/ml kanamycin. Optimal production of fusion proteins was obtained when midexponential-phase cells (OD600=0.5) were induced with 1 mM IPTG for 24 h at 18°C with 200 rpm agitation.

Purification, identification, and quantification of expressed carboxylesterases
The expressed wild-type and mutant B1 esterases, fused with His-Tag at the N terminus, were purified using His Bind Kits (Novagen). The collection and disruption of cells by sonication and precipitation steps were carried out as described in the Novagen user’s manual. Before the crude cell extract was loaded onto an ion exchange chromatography column, the binding buffer was allowed to drain to the top of the column bed. A flow rate of ~10 column vol.·h–1 was optimal for efficient purification. The column was washed with 10 vol. of 1x binding buffer, followed by 6 vol. of 1x wash buffer. The bound protein was eluted with 6 vol. of 1x elute buffer and collected in a fraction collector; 12% SDS-PAGE gel was performed using the Bio-Rad Mini-Protein II apparatus to identify and check the purified target proteins (17) , which were visualized by staining with Coomassie blue, and quantified using the Bradford method (18) with bovine serum albumin as standard.

Assays of enzymatic activity
Carboxylesterase activities of the wild-type and mutants of B1 were assayed with ß-naphthyl acetate as substrate in 0.2 M phosphate buffer (pH 7.0). An appropriate amount of purified enzyme solution was added into a range of concentrations of substrate solutions spanning the Km (based on preliminary tests), to a final volume of 3.0 ml. After the mixture was incubated for 5 min at 37°C, 0.5 ml freshly prepared diazo blue SDS reagent (0.3% fast blue B salt in 3.5% aqueous SDS) was added. The color developed as a result of ß-naphthol formation was measured at 555 nm on a Beckman DU-800 spectrophotometer (Becton-Dickinson, Fullerton, CA, USA) and quantified using a ß-naphthol standard curve. Three replicates were carried out for each substrate concentration.

Organophosphorus hydrolase and MCE activities were determined using gas chromatography (19) and the following substrates: monocrotophos (dimethyl OP), chlorfenvinphos (diethyl OP), methyl parathion (reductive OP), and malathion. The structures of these OPs are given in Fig. 1 . An appropriate amount of purified enzyme was added into a range of concentrations of substrate solutions in potassium-phosphate buffer (pH 7.0) spanning the Km (based on preliminary tests) to a final volume of 15 ml and incubated at 37°C for 45 min; 0.5 ml of sample was taken out and mixed with 0.5 ml of petroleum ether and then dried with anhydrous sodium sulfate. The product was extracted with redistilled hexane and analyzed on a Hewlett-Packard 5890 series II GC with a nitrogen phosphorous detector (NPD), using N2 as the carrier gas at 1 ml·min–1. A fused silica capillary column (0.53 mm idx30 mx0.5 µm film thickness, Supelco Corp., Bellefonte, PA, USA) was used in each assay. Injector, column, and detector temperatures were set at 250°C, 200°C, and 300°C, respectively. Concentration of each OP insecticide was determined by comparing the peak area to a standard curve. Three replicates were carried out for each assay.


Figure 1
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  		Figure 1. Chemical structures of substrates used.

The kinetic constants, Vmax and Km, of wild-type B1 enzyme and its mutants for ß-naphtyl acetate and the OPs were computed with HYPER software according to Lineweaver-Burk method.

Phylogenetic analysis
The deduced amino acid sequence of esterase B1 from C. pipiens was aligned with E3 (Lc{alpha}E7) from L. cuprina, E7 (Md{alpha}E7) from M. domestica, and AChE from T. californica using the CLUSTALX (1.83) program, which also gave the pairwise sequence identities. A phylogenetic tree was constructed with the distance neighbor-joining method (pairwise deletion and JTT model) of the MEGA 3.1 software to elucidate the evolutionary relationship of various carboxylesterases. Bootstrap analysis (1000 replicates) was applied to evaluate the internal support of the tree topology. Accession numbers in NCBI database for the sequences used in phylogenetic analysis are summarized in Table 1 .


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  		Table 1. Amino acid polymorphism at two sites homologous to 110 and 224 of Culex pipiens esterase B1 among various insect carboxylesterases


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  		Table 1A. (continued)


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Modeled tertiary structure of esterase B1 and conservation of the mutated sites among insect carboxylesterases
Although the sequence similarities between the esterase B1 of C. pipiens and E3 of L. cuprina or Md{alpha}E7 of M. domestica were as low as 38% identity, and even lower (at 25%) between B1 and AChE of Torpedo californica (Fig. 2 ), their predicted tertiary structures were very similar. In the case of B1 and AChE, 309 of the 540 {alpha} carbons (57%) overlapped with a root mean square (r.m.s.) deviation of 0.75 Å (Fig. 3 ). The catalytic triad (S200, E327, and H440, numbered according to TcAChE) and the two positions of mutagenesis G119 (G110 in B1) and W233 (W224 in B1) were all conserved in the primary and tertiary structures among the four enzymes. Residues G110 and W224 were located in the "oxyanion hole" and "acyl-binding pocket" within the active-site gorge of the enzymes. Sequence comparisons showed that the position 224 was highly conserved and even more so than the site 110 among various insect carboxylesterases. Thus, 86% of 88 insect carboxylesterases (spanning 31 species from 7 orders) have tryptophan at position 224, whereas at position 110, 51% have glycine and 47% have alanine (Table 1) . Phylogenetic analysis showed that although Culex esterases were not orthologous to any of D. melanogaster {alpha}-esterases, they clustered more closely to them than to aphid M. persicae esterases or to D. melanogaster ß-esterases (Fig. 4 ).


Figure 2
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  		Figure 2. Comparison of amino acid sequences of esterase B1 (CpB1) from resistant strain (Tem-R) of C. pipiens (16) ; Md{alpha}E7 from OP resistant strain (Rutgers) of M. domestica (7) ; Lc{alpha}E7 from OP susceptible strain (LS2) of L. cuprina (5) ; and AChE of T. californica (TcAChE). Identical residues in the 4 sequences are shaded black. *Indicates catalytic triad residues and regions surrounding them; specifically the "nucleophilic elbow," "acid turn," and "histidine loop" are boxed. Mutation positions G137D and W251L/S (numbered in Md{alpha}E7) are shown with an arrow ({downarrow}).


Figure 3
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  		Figure 3. Comparison of tertiary structure between AChE of T. californica and esterase B1 of C. pipiens. Only residues 1–309 of B1 were included (in blue) and are compared with TcAChE (in yellow). RMSD = 0.75 Å. Catalytic triad and mutation amino acids are shown with rods.


Figure 4
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  		Figure 4. An unrooted neighbor-joining tree of the {alpha}-esterase cluster (Dm{alpha}E1-Dm{alpha}E10) and ß-esterase cluster (DmEstP and DmEst6) from D. melanogaster, {alpha}E7 (Md{alpha}E7) from M. domestica, E3 (Lc{alpha}E7) from L. cuprina, {alpha}E7 (Ls{alpha}E7) from L. sericata, esterase A2 (CpA2) and B1 (CpB1) from C. pipiens, esterase B1 (CtriB1) from C. tritaeniorhynchus, esterase B3 (CtarB3) from C. tarsalis, esterase E4 (MpE4), and FE4 (MpFE4) from M. persicae. Numbers at nodes are bootstrap percentage values, which indicate percentage of bootstrapped replicates that group taxa together when data set is randomly sampled 1000 times. Only values >70% are shown. Branch lengths are proportional to distance.

Carboxylesterase activity of the wild-type and mutant B1 esterases
Three single mutants (G110D, W224L, and W224S), two double mutants (G110D/W224L and G110D/W224S), as well as the wild-type carboxylesterase B1, cloned from mosquito C. pipiens, were expressed and purified. The kinetic constants measured with ß-naphthyl acetate as substrate showed strong carboxylesterase activity by the wild type enzyme (Vmax=9.9±1.2 µmol·min–1·mg–1, Km=26.3±2.5 µM, kcat=717±18 min–1, and kcat/Km=26.8±1.9 µM–1·min–1), while all five mutants showed no appreciable activity with ß-naphthyl acetate and thus had no carboxylesterase activity.

OP hydrolase and MCE activities of the wild-type and mutant B1 esterases
No obvious hydrolysis of the four OP insecticides was observed for the wild-type enzyme or for the mutant enzymes G110D, W224S, and G110D/W224S, which means G110D and W224S did not gain OP hydrolase or MCE activity at the cost of losing the carboxylesterase activity of esterase B1. On the other hand, both the W224L and the G110D/W224L enzymes displayed moderate hydrolysis of monocrotophos, chlorfenvinphos, and malathion, with similar kinetic characteristics (Table 2 ). This means that changing tryptophan at position 224 to leucine converts carboxylesterase B1 to an OP hydrolase and increases its MCE activity. The hydrolysis of methyl parathion, a reductive OP insecticide, was only detected by the double mutant G110D/W224L.


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  		Table 2. Kinetic constants of esterase B1 mutants as hydrolyzing insecticides


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Carboxylesterase B1 from the mosquito C. pipiens was mutated in vitro at two positions, namely 110 and 224, based on our inspection of a predicted three-dimensional structure for the enzyme. Specifically, we created mutants G110D and W224L/S, corresponding to the resistant {alpha}-esterase E3 of L. cuprina, which exists in nature. The G110D mutant did not display the change from carboxylesterase activity to OP hydrolase activity that is observed in its paralogues Lc{alpha}E7, Ls{alpha}E7, and Md{alpha}E7. On the other hand, the W224L enzyme (although not W224S) did acquire OP hydrolase and MCE activities at the expense of carboxylesterase activity. This is a change in activity that has not been detected in natural OP-resistant populations of C. pipiens. However, one cannot entirely rule out the possibility that the B1 mutants G110D or W224S can hydrolyze other OP insecticides or analogs such as diethyl 7-hydroxycoumaryl phosphate (dECP) and dimethyl 4-methylumbelliferyl phosphate (dMUP), which could possibly be detected using more sensitive assay methods than the GC assay we have used here (8 , 20) . Increased OP hydrolyase and MCE activities in esterase B1 were in fact observed in this study in the mutant enzyme W224L. This observation strongly supports the hypothesis that it is feasible for mosquitoes to acquire OP resistance by functional changes in carboxylesterases (notwithstanding its apparent absence in nature), in addition to quantitative amplification of esterase genes.

It has been proposed that hydrolysis of carboxylester substrates proceeds via a two-step reaction mechanism involving covalent binding of the carbonyl carbon of the substrate to the catalytic serine with loss of the alcohol side chain to generate an acyl-enzyme intermediate, followed by nucleophilic attack by a water molecule on the carbonyl-serine bond to liberate the acid and regenerate free enzyme (21) . Molecular modeling of Lc{alpha}E7 (esterase E3) suggests that the G137D mutation (G110D in B1, which is located in "oxyanion hole") alters the orientation of the Ser-200-attacking water molecule. This new orientation facilitates its attack on a tetrahedral-phosphorylated serine, but on a planar acylated serine, and then leads to an increase in the rates of OP hydrolysis and loss of esterase activity (5 , 22) . Another mutation, W251L/S (W224L/S in B1), located in the "acyl-binding pocket," creates more space to accommodate substrates with bulky acid groups (e.g., malathion) and reduces the steric obstruction to the inversion that must occur around phosphorus during hydrolysis of OPs (8) .

No OP hydrolase activity was observed by the G110D mutant at B1 in this study, probably because of unknown effects of the linear alcohol side chains of the two tested substrates, monocrotophos and chlorfenvinphos (Fig. 1) , as compared to the naphthyl alcohol side chains common in ß-naphtyl acetate, dECP and dMUP. As for the mutant W224S, it is difficult to explain its failure to have increased MCE activity or OP hydrolysis rate considering the more space it provides, just as does the Leu substitution. Perhaps the hydrophilic character of the Ser destroys the hydrophobic environment of "acyl-binding pocket." Although the mutation W224L in B1 can increase MCE activity and OP hydrolysis, its first-order rate constant (kcat) for malathion is only 0.2 min–1, much lower than that (220 min–1 or 43 min–1) of mutant W251L of Lc{alpha}E7, while its kcat (0.2 min–1) for chlorfenvinphos is much higher than that (0.86 h–1) of mutant W251L of Lc{alpha}E7 (20 , 22) . The increases in OP hydrolase and MCE activities caused by the W224L mutation in B1 are difficult to quantify because the basal activity in the wild-type enzyme is so low that accurate rate measurements are not possible with our GC assay. Furthermore, it is difficult to explain the unique hydrolysis of one reductive OP, methyl parathion, by the G110D/W224L double mutant. The functional differences between the resistant esterases encoded by the {alpha}E7 gene in higher dipterans and the mutant esterases from C. pipiens could be related to some subtle differences in their tertiary structures. These subtle differences are the prerequisites for the same mutations to produce different OP hydrolase or MCE activities among members of the carboxyl/cholinesterase multigene family.

According to the current study and to previous studies on OP-resistant L. cuprina and M. domestica, it should be possible for C. pipens to acquire OP resistance if the mutant W224L carboxylesterase appeared in natural populations. The question then arises as to why this mutant has not been detected in resistant C. pipiens field populations. Two factors may account for this. One is that selection of mutants with amplified esterase genes may predominate because of gene-amplification’s ability to sequester a wider range of insecticides than the mutant ali-esterase mechanism, thus providing greater protection. Another is that the mutant ali-esterases’ loss of their wild-type function leads to biologically significant loss of fitness. It is noteworthy that in L. cuprina resistance did not rise to high frequency until a well-characterized Modifier locus was observed (23) .

In contrast to the situation in C. pipiens, a possible mutant ali-esterase mechanism has been reported in some malathion-resistant strains of Anopheles arabiensis, An. culicifacies, An. stephensi, and C. tarsalis mosquitoes (24) . These strains have enzymes that metabolize malathion not malaoxon do not show elevated expression in native PAGE gels and appear to be qualitatively different from the susceptible enzymes. The molecular basis for the resistance phenotype in these mosquitoes is probably the same as in L. cuprina and M. domestica. On the other hand, there is no report of diazinon- or paraoxon-type resistance in any mosquito population, which is consistent with our observation that OP hydrolysis was not detectable for the C. pipiens mutant G110D. Thus, mutation at this position may not be able to provide protection to mosquitoes as it does to higher dipterans.

Finally, the current study points out that postion 224 (and likely position 110) could play a key role in insect carboxylesterases’ switching from their native physiological functions to other functional niches, especially under the selection pressure of insecticides. Data from insect carboxylesterases need to be accumulated to verify this hypothesis.


   ACKNOWLEDGMENTS
 
We thank G. Reeck of Kansas State University, Manhattan, KS, USA, A. Fallon of University of Minnesota, St. Paul, MN, USA, and S. Zhu of Institute of Zoology, and Chinese Academy of Sciences, Beijing, China, for helpful comments on the manuscript. This work was supported by the National Natural Science Foundation (no. 30470322), by the Innovation Program of Chinese Academy of Sciences (KSCX2-YW-G-008), and by the State Key Laboratory of Integrated Management of Pest Insects and Rodents (grant no. ChineseIPM0701).

Received for publication March 6, 2007. Accepted for publication May 3, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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