Elevated activity of an Epsilon class glutathione transferase confers DDT resistance in the dengue vector, Aedes aegypti

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Abstract

Glutathione transferases (GSTs) play a central role in the detoxification of xenobiotics such as insecticides and elevated GST expression is an important mechanism of insecticide resistance. In the mosquito, Anopheles gambiae, increased expression of an Epsilon class GST, GSTE2, confers resistance to DDT. We have identified eight GST genes in the dengue vector, Aedes aegypti. Four of these belong to the insect specific GST classes Delta and Epsilon and three are from the more ubiquitously distributed Theta and Sigma classes. The expression levels of the two Epsilon genes, a Theta GST and a previously identified Ae. aegypti GST [Grant and Hammock, 1992. Molecular and General Genetics 234, 169–176] were established for an insecticide susceptible and a resistant strain. We show that the putative ortholog of GSTe2 in Ae. aegypti (AaGSTe2) is over expressed in mosquitoes that are resistant to the insecticides DDT and permethrin. Characterisation of recombinant AaGSTE2-2 confirmed the role of this enzyme in DDT metabolism. In addition, unlike its Anopheles ortholog, AaGSTE2-2 also exhibited glutathione peroxidase activity.

Introduction

The mosquito Aedes aegypti is the major vector of yellow fever, dengue and dengue haemorrhagic fever (DHF). Globally there are an estimated 50 million cases of dengue infection each year with 500,000 cases of DHF and over 24,000 deaths (WHO, 2002). Thailand suffers one of the highest rates of dengue and DHF infection in the world. In addition to the public health impact, DHF is leading to social and economic problems (Gubler, 1998). Effective vector control is essential to reduce the transmission of DHF, but this is hindered by the development of insecticide resistance in Ae. aegypti. In northern Thailand, populations of Ae. aegypti resistant to the organochlorine insecticide DDT and to pyrethroid insecticides have been isolated (Somboon et al., 2003). A colony of Ae. aegypti was established and a resistant line PMD-R selected. The molecular basis of insecticide resistance in this strain has now been investigated, with the goal of improving monitoring and management of insecticide resistance in this disease vector.

DDT and pyrethroid insecticides share the same target site, the neuronal voltage-gated sodium channel (Soderlund and Knipple, 2003). Amino acid substitutions in the insecticide-binding domain of this protein can lead to resistance to both of these insecticide classes. One amino acid substitution occurs in subunit 6 of domain II of the sodium channel protein in Ae. aegypti populations from Thailand (Brengues et al., 2003; Prapanthadara et al., 2002) but the correlation between this allele and the resistance phenotype is, as yet, unknown. A second, highly prevalent, mechanism of insecticide resistance is an increase in the rate of insecticide metabolism. There are three major groups of enzymes involved in insecticide detoxification, carboxylesterases, cytochrome P450s and glutathione transferases (GSTs) (Hemingway et al., 2004). Elevated activity of these enzymes has been associated with resistance to DDT and/or pyrethroids in different mosquito species (Hemingway et al., 2004; Kasai et al., 2000; Prapanthadara et al., 2002; Vaughan and Hemingway, 1995). Preliminary biochemical characterisation of the DDT/pyrethroid resistant PMD-R strain from Thailand, found 10-fold higher levels of DDT metabolism compared to the susceptible strains, associated with elevated levels of total GST activity (Prapanthadara et al., 2002).

Multiple GSTs are found in all insect species. A subset of these enzymes is able to catalyse the glutathione dependent dehydrochlorination of DDT to the non-insecticidal product, DDE (Clark and Shamaan, 1984; Prapanthadara et al., 1995; Tang and Tu, 1994). GSTs are also implicated in resistance to other insecticide classes, through metabolism of the insecticide or its metabolites (Chiang and Sun, 1993), sequestration (Kostaropoulos et al., 2001) or by protecting against secondary toxic effects, such as increases in lipid peroxidation, induced by insecticide exposure (Vontas et al., 2001). Elevated GST activity has previously been associated with DDT resistance in the GG strain of Ae. aegypti (Grant et al., 1991) and expression of the GST subunit, GST-2, was correlated with the resistance phenotype (Grant and Hammock, 1992).

The majority of GSTs are cytosolic, dimeric enzymes with subunit sizes ranging from 17 to 28 kDa. There are at least six classes of cytosolic GSTs in insects: Delta, Epsilon, Omega, Sigma, Theta and Zeta (Ranson et al., 2002). Additional GSTs exist in insects that cannot be readily assigned to any of these classes (Ding et al., 2003). GST-2, previously isolated from Ae. aegypti, belongs to this ‘unclassified’ group of insect GSTs. The two largest insect GST classes, Delta and Epsilon, are both insect specific whereas the remaining classes are also found in other organisms. Elevated expression of Epsilon GSTs occurs in DDT resistant populations of the malaria vector, Anopheles gambiae. There are eight Epsilon GSTs arranged sequentially on chromosome 3R of An. gambiae in close association with the DDT resistance locus rtd1 (Ding et al., 2003; Ranson et al., 2000). One of these, AgGSTe2, encodes an enzyme that possesses high DDT dehydrochlorinase activity (Ranson et al., 2001). We have identified several GST genes in Ae. aegypti by using a combination of homology based PCR and data mining of the incipient Ae. aegypti genome database (Severson et al., 2004). The expression of these newly identified genes and that of GST-2 in insecticide susceptible and resistant Ae. aegypti were compared and those with elevated expression were characterised further.

Section snippets

Mosquito strains

The PMD strain of Ae. aegypti was colonized from field caught material originating from Ban Pang Mai Dang, Mae Tang district, Chiang Mai Province in northern Thailand. A highly resistant line (PMD-R) was generated from the parental PMD colony by selection of one-day adults with either 4% DDT or 0.75% permethrin using standard WHO susceptibility kits (WHO, 1981). The resistance ratios of the parental PMD colony and the selected line, PMD-R were determined by comparison to New Orleans strain, a

Identification of genes encoding GSTs in Ae. Aegypti

The database of expressed sequence tags (ESTs) maintained by The Institute for Genomic Research (TIGR) (www.tigr.org) was searched firstly using the keyword ‘GST’ and secondly by local BLAST searches using An. gambiae GSTs as the query sequences. Nine putative GST sequences were retrieved and each of these was then searched against the non-redundant database at NCBI, using BLASTX, to confirm their identity as putative GST genes. Of the nine Ae. aegypti GSTs, two were classified as Delta, two as

Discussion

We have identified eight GST genes from the mosquito Ae. aegypti and conducted a preliminary phylogenetic analysis to establish their relationship with other insect GSTs. A complete examination will only be possible once the Ae. aegypti genome sequence is determined and the full extent of the GST gene family in this species is known. Nevertheless, several interesting points arise from this initial analysis. The topology of the tree in Fig. 1 suggests that the insect GST family had diverged

Acknowledgements

We thank D. W. Severson for providing the Ae. aegypti BAC library. The use of EST sequence database from The Institute Genome Research (TIGR) (http://www.tigr.org/) is gratefully acknowledged. We thank Dr. John Vontas and Dr. Nicola Hawkes for their advice, Ms. Amanda Ball for DNA sequencing and BAC DNA preparation and Ms. Alison Helm for assistance with the HPLC analysis. This work was supported by the Wellcome Trust and the World Health Organization. The Aedes aegypti resistant strain was

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    The sequences described in this manuscript have been deposited to Genbank and have the following accession numbers: AY819709, AY819710, AY819711 and AY819712.

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