Método de identificação da mutação Gly137/Asp

MÉTODO DE IDENTIFICAÇÃO DA MUTAÇÃO Gly137/Asp

Como já visto, a mutação Gly137/Asp encontrada em C. hominivorax é responsável pela aquisição da resistência à maioria dos inseticidas OP, incluindo os inseticidas usados para controle dessa espécie no Brasil. Sendo assim, foi desenvolvido um método mais rápido e barato do que o seqüenciamento para a identificação dessa mutação.

METODOLOGIA DETALHADA

Amostras de C. hominivorax

As amostras de C. hominivorax utilizadas nesta etapa foram as mesmas utilizadas no Capítulo 1, e também amostras do Uruguai provenientes de coletas já realizadas anteriormente para estudos conduzidos no Laboratório de Genética Animal sobre a estrutura genética de populações desta praga ao longo da atual distribuição geográfica.

DNA genômico

A extração do DNA total dos indivíduos de C. hominivorax foi realizada como descrito por Azeredo-Espin (1993), já detalhada no Capítulo 1.

Amplificação

Para a realização da amplificação do fragmento contendo a mutação Gly137/Asp, foram utilizados os ‘”primers” 7F1a e 7R3a (Tabela 1), que juntos amplificam um fragmento de 536pb. As condições de amplificação apresentaram as seguintes concentrações: 20mM Tris-HCl (pH 8.4), 50mM KCl, 1U de enzima (‘Taq DNA Polimerase’), 200µM de cada dNTP, 1,8mM de MgCl2,1µM de

cada “primer” e 1µL de amostra de DNA preparadas como descrito anteriormente.

As reações de PCR foram realizadas para um volume final de 15µl utilizando o termociclador “Perkin Elmer GenAmp PCR System 9.600”. Estas reações foram submetidas a uma etapa inicial de desnaturação a 96° C por 3 minutos (1 ciclo), seguida de 35 ciclos de 95° C por 1minuto, 52° C por 1 minuto e 72° C por 2 minutos. Um passo prolongado de extensão de 72° C por

eletroforese em gel de agarose (1%) corado com brometo de etídeo e, em seguida, visualizadas em transiluminador UV.

Identificação dos indivíduos mutantes por PCR-RFLP

A mutação em um dos códons do gene da esterase responsável pela resistência à maioria dos OP ocasionou o surgimento de um sítio de restrição no alelo mutante. Através do aplicativo Nebcutter (http://tools.neb.com/NEBcutter2/index.php), a enzima Tsp45I (New England BioLabs), cujo sítio de reconhecimento é a seqüência GTSAC ('S' representa as bases nitrogenadas 'G' ou 'C'), foi escolhida para realizar a digestão dos fragmentos obtidos pela reação de PCR. Esta endonuclease cliva o fragmento proveniente do alelo mutante, mas não do selvagem, possibilitando a identificação dos alelos (Figura 10). Dessa maneira, a reação de digestão dos produtos de PCR a uma temperatura de 65° C por 60 minutos cliva fragmento proveniente do alelo mutado em duas partes, sendo a visualização feita em gel de agarose (2%). Como forma de avaliar o método PCR- RFLP na identificação de indivíduos de C. hominivorax mutantes, foram analisados alguns indivíduos provenientes de diferentes regiões do Brasil e do Uruguai. O posterior seqüenciamento de alguns destes alelos confirmaram os resultados.

Alelo Selvagem

Alelo Mutante

CAT GGT GGT GGT TTT GTA CCA CCA CCA AAA

CAT GGT GGT GAC TTC GTA CCA CCA CTG AAG





→ _←

← 50bp

Alelo Selvagem

Alelo Mutante

CAT GGT GGT GGT TTT GTA CCA CCA CCA AAA

CAT GGT GGT GAC TTC GTA CCA CCA CT

CAT GGT GGT GAC TTC GTA CCA CCA CT





→ _←

←

Figura 10. Esquema da reação de digestão com a enzima Tsp45I dos produtos de PCR amplificados com os “primers” 7F1a/7R3a

RESULTADOS E DISCUSSÃO

Identificação dos mutantes por PCR-RFLP

A técnica de PCR-RFLP foi usada pra identificar, em nível molecular, indivíduos mutantes de C. hominivorax possivelmente resistentes. Usando o aplicativo Nebcutter, as endonucleases Tsp45I, MaeIII e HphI foram preditas de reconhecer o sítio no qual está inserida a mutação Gly137/Asp. Para validar este método, a enzima Tsp45I foi escolhida para analisar indivíduos de C. hominivorax de populações naturais. O fragmento proveniente do alelo mutado amplificado pelo par de “primers” 7F1a/7R3a foi clivado em dois fragmentos (52pb e 484pb), enquanto que o fragmento proveniente do alelo selvagem não foi clivado. A eletroforese em gel de agarose permitiu a identificação dos indivíduos (Figura 11). Um posterior seqüenciamento de alguns alelos confirmaram os resultados obtidos por PCR-RFLP.

Figura 11. Análise eletroforética em gel de agarose da reação de digestão com a endonuclease Tsp45I dos produtos amplificados em C. hominivorax com o par de ”primers” 7F1a/7R3a. ‘L’ - 1 Kb DNA Ladder (Invitrogen); 2 – indivíduo heterozigoto (Asp/Gly); 3 – indivíduo homozigoto selvagem (Gly/Gly); 4 – indivíduo homozigoto mutante (Asp/Asp).

Uma vez que a digestão com a endonuclease Tsp45I resultou em padrões específicos que diferenciaram os alelos selvagens (Gly137) e mutados (Asp137) de C. hominivorax, a técnica de PCR-RFLP representa, então, um método rápido, fácil e confiável para detectar resistência nessa

1 2 3 4

517 pb

506 pb

detecção. Bioensaios no campo são largamente usados para testar a suscetibilidade de C. hominivorax aos inseticidas, mas eles consomem muito tempo e estão sujeitos a diversas variáveis que podem reduzir sua confiabilidade. Além disso, esses bioensaios tradicionais provêem pouca informação sobre a presença e distribuição populacional de alelos associados à resistência (Cassanelli et al., 2005).

Dessa forma, a técnica de PCR-RFLP permite que a diversidade alélica do ChαE7, gene codificador da E3 em C. hominivorax, seja estimada em populações naturais. Uma evidência de que tal mecanismo de resistência possa ter evoluído em C. hominivorax é o fato de que as mesmas mutações de aminoácidos foram independentemente selecionadas nos genes ortólogos LcαE7 e MdαE7, ambos envolvidos em resistência a OP através da aquisição da atividade OP hidrolase em L. cuprina e M. domestica, respectivamente (Newcomb et al., 1997b; Claudianos et al., 1999).

Esse procedimento de rápida identificação de indivíduos/linhagens contendo alelos mutantes resultou em um pedido de patente de invenção no Instituto Nacional da Propriedade Industrial (INPI), descrevendo o método e kit de detecção de indivíduos de C. hominivorax mutantes para o gene da esterase relacionado com resistência a inseticidas organofosforados (PI0503997-5). Uma vez que essa associação seja comprovada, a técnica PCR-RFLP pode ser uma importante ferramenta no estudo de resistência a inseticidas permitindo uma estimativa de mudanças nas freqüências gênicas em estudos populacionais desta praga ao longo de sua distribuição geográfica. O contínuo monitoramento de populações naturais de C. hominivorax proverá importantes informações para a escolha do inseticida apropriado e para a implementação de programas mais efetivos de controle.

ARTIGO

A verificação em C. hominivorax da presença das mutações relacionadas com resistência a inseticidas OP e o desenvolvimento do método de identificação da principal mutação relacionada com a resistência resultou em um artigo publicado no periódico “Veterinary Parasitology”, intitulado: A survey of mutations in the Cochliomyia hominivorax (Diptera: Calliphoridae) esterase E3 gene associated with organophosphate resistance and the molecular identification of mutant alleles

Carvalho, R. A., Torres, T. T., Azeredo-Espin, A. M. L. Veterinary Parasitology, 140: 344-351 (2006)

A survey of mutations in the Cochliomyia hominivorax

(Diptera: Calliphoridae) esterase E3 gene associated

with organophosphate resistance and the molecular

identification of mutant alleles

Renato Assis de Carvalho*, Tatiana Teixeira Torres, Ana Maria Lima de Azeredo-Espin

Laborato´rio de Gene´tica Animal, Centro de Biologia Molecular e Engenharia Gene´tica (CBMEG), Universidade Estadual de Campinas (UNICAMP), P.O. Box 6010,

13083-875 Campinas, SP, Brazil

Received 9 November 2005; received in revised form 4 April 2006; accepted 4 April 2006

Abstract

Cochliomyia hominivorax (Calliphoridae) is one of the most important myiasis-causing flies and is responsible for severe economic losses to the livestock industry throughout the Neotropical region. In Brazil, C. hominivorax has been controlled mainly with organophosphate (OP) insecticides, although the inappropriate use of these chemicals can result in the selection of resistant flies. Changes in carboxylesterase activity have been associated with OP insecticides in some arthopodan species. In this work, we isolated and characterized part of the E3 gene in C. hominivorax (ChaE7), which contained the same substitutions responsible for the acquisition of OP hydrolase activity in Lucilia cuprina (Calliphoridae). Digestion of the polymerase chain reaction products with a restriction enzyme that specifically recognized the mutation site unambiguously differentiated wild and mutated esterase alleles. The PCR-RFLP assay therefore provided a fast, reliable DNA-based method for identifying C. hominivorax individuals with a mutation in the esterase gene. Further bioassays to determine the association of this mutation with OP resistance in C. hominivorax should allow the development of more effective strategies for managing this species. # 2006 Elsevier B.V. All rights reserved.

Keywords: Carboxylesterase; Insecticide resistance; Myiasis; New World screw-worm; Pest management; PCR-RFLP

1. Introduction

The infestation of live vertebrate hosts by dipteran larvae, a condition known as myiasis (Zumpt, 1965), remains an unsolved problem for animal production in Neotropical regions. Cochliomyia hominivorax

www.elsevier.com/locate/vetpar Veterinary Parasitology 140 (2006) 344–351

* Corresponding author. Tel.: +55 19 3788 1141; fax: +55 19 3788 1089.

E-mail address:rassis@unicamp.br(R.A. de Carvalho).

0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.04.010

(Coquerel) is one of the most important myiasis- causing flies in the Neotropics (Hall and Wall, 1995) and is responsible for severe economic losses to the livestock industry, mainly by reducing the quality of leather and the production of milk and meat. In South American countries, C. hominivorax has been controlled by applying insecticides, particularly organophosphate (OP)-based compounds. However, the improper and continuous use of these chemicals can lead to the selection of OP-resistant strains.

The major mechanisms of insecticide resistance described so far involve either metabolic detoxification of the insecticide before it reaches its target site, or changes in the sensitivity of the target site that abolish its susceptibility to the insecticide (Hemingway, 2000). The carboxylesterase-mediated detoxification of insec- ticides has been reported for more than 30 medical, veterinary and agricultural insect pests (reviewed in

Hemingway and Karunaratne, 1998). A decrease in carboxylesterase activity has been observed in OP-resistant strains of Lucilia cuprina (Hughes and Raftos, 1985), Musca domestica (Van Asperen and Oppenoorth, 1969) and Chrysomya putoria (Townsend and Busvine, 1969). These findings have been hypothesized to involve a mutant ali-esterase in which a structural mutation in a carboxylesterase results in a reduced ability to hydrolyze aliphatic ester substrates, such as methyl butyrate and naphthyl acetate, but an acquired ability to hydrolyze OP substrates (Claudia- nos et al., 1999).

The LcaE7 gene (Newcomb et al., 1997b) encodes the major ali-esterase of L. cuprina, also known as esterase isozyme E3. L. cuprina aE7 alleles from three strains (an OP-susceptible, a diazinon and a malathion- resistant) have been isolated and expressed in vitro (Campbell et al., 1998). Biochemical assays using these expressed proteins showed that a single amino acid substitution at position 137 (Gly137Asp) in the diazinon-resistant strain simultaneously abolished the ali-esterase activity and increased the rate of depho- sphorylation to yield OP insecticide hydrolase activity, especially of diethyl OPs. Diethyl OP insecticides contain two ethoxy groups attached to their central phosphorus atom and are commonly used to control C. hominivorax. In contrast, malathion-resistant strain contained a different amino acid substitution (Trp251Leu) that conferred specificity towards dimethyl OPs (Campbell et al., 1998), which are not

commonly used to control C. hominivorax. In the putative E3 model based on the solved tertiary structure of the related acetylcholinesterase from Torpedo californica (Sussman et al., 1991), the Gly137Asp mutation is located in the motif known as the oxyanion hole and Trp251Leu is part of the acyl binding site within the active site of the insect esterases (Newcomb et al., 1997b; Campbell et al., 1998).

Campbell et al. (1997) showed that there is substantial functional equivalence in the mutant enzymes associated with esterase-mediated resistance across the species M. domestica (MdaE7), Drosophila melanogaster (DmaE7) and L. cuprina (LcaE7). There are many similarities among these systems, including amino acid sequence, electrophoretic mobilities and inhibitor sensitivities, which strongly suggests that they are orthologous (Bigley and Plapp, 1961; Hughes and Raftos, 1985; Spackman et al., 1994; Parker et al., 1996). Based on the foregoing studies, we have isolated and sequenced a region of the E3 gene in C. hominivorax, which is orthologous to that previously described for L. cuprina and contains the Gly137Asp and Trp251Leu mutations. The characterization of this sequence allowed the development of a rapid and efficient method for identifying Gly137Asp mutants of C. hominivorax.

2. Material and methods

2.1. Amplification and sequencing

Larvae of C. hominivorax were collected from the wounds of infested animals from several locations and

stored at
70 8C. Genomic DNA from eight

individuals of C. hominivorax was extracted according to Infante and Azeredo-Espin (1995). Four primers previously described for L. cuprina, 7F1, 7F1a, 7R2 and 7R4 (Newcomb et al., 1997a,b), were used to amplify the orthologous region in C. hominivorax. A novel primer, 7R3a (50-ATCCTTATCATTATTTT- CACCC-30), was designed to specifically amplify C. hominivorax sequences.

The initial conditions were the same as described in

Newcomb et al. (1997b) and, once the amplification conditions had been standardized, the PCR was done in a Perkin-Elmer 9600 thermal cycler. The 15 mL PCR mix contained 20 mM Tris–HCl (pH 8.4), 50 mM KCl,

1 unit of Taq polymerase (Invitrogen), 200 mM of each dNTP, 1.8 mM MgCl2, 1 mM of each primer and

10–15 ng of DNA. After an initial denaturing step of 3 min at 96 8C, 35 cycles were performed, each one consisting of 1 min at 95 8C, 1 min at 50 8C and 2 min at 72 8C, with a final step of 10 min at 72 8C to fully extend all amplicons. Direct sequencing of the PCR products was done using the primer 7R3a, an ABI Prism Terminator Cycle sequencing kit and an ABI 377 automatic sequencer (Applied Biosystems).

2.2. Sequence analysis

The nucleotide sequences of C. hominivorax were submitted to BLASTN (Altschul et al., 1997) to search for similarities, and sequence alignments were done using ClustalX (Thompson et al., 1997). The predicted amino acid sequences were obtained using the ‘Biological Sequence Alignment Editor’ program (Hall, 1999).

2.3. PCR-RFLP assay

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to develop a fast and reliable diagnostic method for identifying the Gly137Asp mutation. The selection of enzymes that recognized sites that included the point mutation was done using the Nebcutter program (http:// tools.neb.com/NEBcutter2/index.php). The endonu- clease Tsp45 I (New England BioLabs) was chosen and used to digest the PCR products obtained with primers 7F1a and 7R3a, according to the manufac- turer’s instructions. To assess the applicability of this method, 50 individuals collected from several C. hominivorax populations across South America were analyzed. The digested fragments were separated by electrophoresis in 2.5% agarose gel and stained with ethidium bromide. The PCR-RFLP results were confirmed by sequencing some of the products.

3. Results

3.1. Amplification and sequencing

The primer pairs 7F1/7R2 and 7F1/7R4 did not amplify the expected fragments, probably because

of the low specificity between these fragments and the C. hominivorax sequences. The 7F1a/7R4 primer pair amplified a fragment of approximately 700 bp, as expected from L. cuprina sequences (Newcomb et al., 1997b), and this fragment was used to design a C. hominivorax-specific reverse primer (7R3a). The 7F1a/7R3a primer pair amplified a fragment of 536 bp and was used for all subsequent amplifications, which yielded fragments of the expected size. The amplified exon region corre- sponded to positions 359–831 of the L. cuprina aE7 nucleotide sequence (Newcomb et al., 1997b). The region of the intron comprised 63 bp and its position (nt 511) corresponded exactly to the position of intron III reported for the orthologous L. cuprina and M. domestica a7 genes, which have 56 and 62 bp, respectively (Newcomb et al., 1997b; Claudianos et al., 1999).

The putative coding region of the C. hominivorax wild type alleles showed high nucleotide sequence similarity (84.5%) with L. cuprina (U56636). Since the substitutions were not located in the putative active site of the enzyme, they probably did not interfere with the protein’s activity. The predicted amino acid sequence showed 88.5% identity with the equivalent residues of L. cuprina E3. Three possible genotypes (wild type homozygotes, mutated homozygotes and heterozygotes) were found among the C. hominivorax sequences analyzed (Fig. 1). The exon sequences showed 97.9% similarity to each other.

The codon GGG at positions 409–411 of the wild alleles of the C. hominivorax a7 nucleotide sequence (Fig. 1) translated to glycine. Mutation of this Gly137 to an aspartate residue (Asp137) was seen in the mutated alleles of the C. hominivorax a7 nucleotide sequences, which contained the codon GAC in the corresponding region. This amino acid substitution, which is associated with OP resistance in L. cuprina and M. domestica (Newcomb et al., 1997b; Claudianos et al., 1999), occurred in one of the conserved residues that constitute the putative oxyanion hole motif (G136, G137, A219) and is predicted to be located in the catalytic center of carboxyl/cholinesterase enzymes (Claudianos et al., 1999).

The alleles that contained Gly137 showed another mutation, namely, substitution of the codon TGG, which translates to tryptophan at position 251 of the L. cuprina E3 amino acid sequence, by the

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R.A. de Carvalho et al. / Veterinary Parasitology 140 (2006) 344–351 347

Fig. 1. Alignment of the nucleotide sequences of the three alleles of ChaE7 from C. hominivorax and of a heterozygote individual (GenBank accession numbers fromDQ266357toDQ266360) with the Lc743 cDNA clone of LcaE7 from L. cuprina (GenBank accession number U56636). ‘wild’ represents the C. hominivorax wild genotype, while ‘mut1’ and ‘mut2’ represent the mutant genotypes and ‘het’ represents the heterozygote. The position of intron 3 was determined by comparison with the cDNA sequence of LcaE7 from L. cuprina and is indicated with abutting arrowheads above the nucleotide sequence. The nucleotide sites responsible for the acquisition of the OP hydrolase activity are shaded. Dots indicate identity with the wild allele ChaE7. R = A/G, S = G/C, Y = C/T and W = A/T.

codon TCG, which translates to serine (Fig. 2). In addition to these two substitutions, a further seven nucleotide substitutions and one amino acid sub- stitution were seen among the C. hominivorax sequences.

3.2. PCR-RFLP

PCR-RFLP was used to identify C. hominivorax Gly137Asp mutants at the molecular level. Using the Nebcutter program, the enzymes Tsp45 I, Mae III and Hph I were predicted to recognize sites that included the mutation site. To validate this method, the enzyme Tsp45 I was chosen to analyze C. hominivorax individuals from natural populations. The amplified fragment from mutated alleles was digested in two fragments (52 and 484 bp), whereas the fragment from wild type alleles was not cut (Fig. 3). Electrophoresis on agarose gels allowed the identification of mutant individuals. Of 50 indivi- duals genotyped, 12 were homozygous for the wild type allele, 15 were heterozygous and 23 were homozygous for the mutant allele. The frequency of the mutated allele was very high (0.61). Additional sequencing of some alleles confirmed the PCR-RFLP results (data not shown).

4. Discussion

The primers 7F1a, previously described for L cuprina, and 7R3a, designed specifically for C.

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Fig. 2. Comparison of the inferred amino acid sequences (single letter code) of the alleles from C. hominivorax and part of the esterase from L. cuprina involved in organophosphate resistance (Newcomb et al., 1997b). Dots indicate identity with the wild allele ChaE7. ‘het’ represents the sequence from a heterozygote individual, with 1 = G/D, 2 = T/P and 3 = W/S. Of 24 conserved residues thatCygler et al. (1993)described for 29 catalytically active carboxyl/cholinesterases, 15 are present in this sequence and are shaded. One of the residues predicted to form the catalytic triad is indicated with an arrow and those contributing to the oxyanon hole are identified with a cross above the sequence.

Fig. 3. Electrophoresis on an agarose gel showing the PCR pro- ducts cleaved by the endonuclease Tsp45 I. L, Ladder 1 kb; WW, non-digested product from a wild homozygote; MM, digested product from a mutant homozygote; WM, digested product from a heterozygote. The small fragment (52 bp) derived from digestion is not shown.

hominivorax, amplified a 536 bp fragment that included intron III (63 bp) and the exon region coresponding to positions 359–831 in the L. cuprina aE7nucleotide sequence, as well as both amino acid positions (137 and 251) related to OP resistance. Like LcaE7, DmaE7 and MdaE7, the orthologous region in C. hominivorax (ChaE7) contained motifs that are highly conserved among carboxyl/cholinesterases and contribute to the catalytic mechanism of the active site. One of these motifs, the nucleophilic elbow (VFGESAG, residues 214–220 in ChaE7), which contains one of the residues of the catalytic triad (S218, E351, H471;Cygler et al., 1993), is conserved across all carboxyl/cholinesterases. The high similar- ity of the amino acid sequences of C. hominivorax to other carboxyl/cholinesterases was also confirmed by the presence of the conserved residues that Cygler et al. (1993) described for 29 catalytically active carboxyl/cholinesterases.

Site-directed mutagenesis of human butyrylcholi- nesterase (BuChE) has shown that a Gly/His mutation at the amino acid corresponding to Gly137 of the L. cuprina enzyme also confers OP hydrolase activity (Lockridge et al., 1997). As in the mutant human BuChE, the Gly/Asp substitution in E3 may also change the structure of the catalytic centre. In mutant enzymes, a water molecule can be appropriately oriented to hydrolyze the organophosphate-enzyme intermediate, which is tetrahedral with respect to the phosphate group (Newcomb et al., 1997a). This is thought to occur to the detriment of the appropriate