Characterization of the promoters of Epsilon glutathione transferases in the mosquito Anopheles gambiae and their response to oxidative stress

Abstract

Epsilon class GSTs (glutathione transferases) are expressed at higher levels in Anopheles gambiae mosquitoes that are resistant to DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] than in insecticide-susceptible individuals. At least one of the eight Epsilon GSTs in this species, GSTe2, efficiently metabolizes DDT to DDE [1,1-dichloro-2,2-bis-(p-chlorophenyl)ethane]. In the present study, we investigated the factors regulating expression of this class of GSTs. The activity of the promoter regions of GSTe2 and GSTe3 were compared between resistant and susceptible strains by transfecting recombinant reporter constructs into an A. gambiae cell line. The GSTe2 promoter from the resistant strain exhibited 2.8-fold higher activity than that of the susceptible strain. Six polymorphic sites were identified in the 352 bp sequence immediately upstream of GSTe2. Among these, a 2 bp adenosine indel (insertion/deletion) was found to have the greatest effect on determining promoter activity. The activity of the GSTe3 promoter was elevated to a lesser degree in the DDT-resistant strain (1.3-fold). The role of putative transcription-factor-binding sites in controlling promoter activity was investigated by sequentially deleting the promoter constructs. Several putative transcription-factor-binding sites that are responsive to oxidative stress were present within the core promoters of these GSTs, hence the effect of H2O2 exposure on the transcription of the Epsilon GSTs was investigated. In the DDT-resistant strain, expression of GSTe1, GSTe2 and GSTe3 was significantly increased by a 1-h exposure to H2O2, whereas, in the susceptible strain, only GSTe3 expression responded to this treatment.

Figure 1. Organization of the Epsilon GST gene cluster in A. gambiae and details of the promoter constructs used in the present study

The direction of transcription is indicated by an arrow. The size of the intergenic space is from the stop codon of the preceding gene to the AUG initiation codon of the following gene (or between AUG initiation codons for genes arranged in head to head orientation) and thus includes UTRs of flanking genes. Experiments not attempted are indicated by a dash, those which gave no activity are indicated by X. The ratio of transcript levels between the ZAN/U and Kisumu strains are the results of previous experiments carried out on adult mosquitoes [12].

Figure 2. Scheme of putative core promoter regions of members of the Epsilon GST family

Exons are shown by boxes, non-coding regions are shown by a horizontal line. Vertical lines denote the boundaries of the UTRs of the genes, where known. Polyadenylation (Poly A) signals were determined from the tentative consensus sequences for A. gambiae ESTs retrieved from the TIGR. Core promoter elements were identified by the MatInspector program. DPE, downstream promotor element.

Figure 3. Sequence of the GSTe3 promoter

Alignment of the sequence upstream from GSTe3 in the ZAN/U and Kisumu strains of A. gambiae obtained using the EMBOSS pairwise alignment programme (http://www.ebi.ac.uk/emboss/align/). Gaps inserted to maintain sequence alignment are shown by a horizontal dash. Potential transcription factors and the consensus sequences of high homology (100% core similarity and 95% matrix similarity) identified by MatInspector are underlined. Allelic variations between the two strains are boxed and shaded grey. The putative TSS of GSTe3 is indicated by a triangle. The start of the coding sequence of GSTe3 is boxed, and the amino acid translation is shown below the sequence alignment. Arrows indicate the boundaries of the constructs used for promoter analysis. AREB, Atp1a1-regulatory-element-binding factor; C/EBP, CCAAT/enhancer-binding protein; Sox-5, Sry-type high-mobility group box protein; HNF-6, hepatocyte nuclear factor 6.

Figure 4. Sequence of GSTe2 promoter

Details are as for GSTe3 in Figure 3. NMP4, nuclear matrix protein 4.

Figure 5. Normalized firefly luciferase expression following transfection into A. gambiae Sua 4.0 cells of a panel of progressive deletion constructs from the upstream sequence of GSTe3

The arrows represent the putative TSS of GSTe3. Distances are from the A of the AUG initiation codon of GSTe3. Values shown on the panel on the right are the mean (±S.D.) luciferase activities (normalized to control Renilla activity) for two separate experiments performed in triplicate. The promoterless pGL3-Basic vector is designated basic. The promoter activity of the various constructs was analysed by pairwise Student's t tests. Statistically significant differences are shown by square brackets whose ends designate the sequences being compared (*P<0.05, **P<0.01).

Figure 6. Normalized firefly luciferase expression following transfection into A. gambiae Sua 4.0 cells of a panel of progressive deletion constructs from the intergenic spacer region between GSTe1 and GSTe2

The arrows represent the putative TSS of GSTe2. Distances are from the A of the AUG initiation codon of GSTe2. Values shown on the panel on the right are the mean (±S.D.) luciferase activities (normalized to control Renilla activity) for two separate experiments performed in triplicate. Potential transcription-factor-binding sites are shown above the constructs. Cross stars denote allelic variations in the promoter region between the two strains. The promoterless pGL3-Basic vector is designated basic. Promoter activity of constructs was analysed by pairwise Student's t test. Statistically significant differences are shown by square brackets whose ends designate the sequences being compared (**P<0.01). AREB, Atp1a1-regulatory-element-binding factor; NMP4, nuclear matrix protein 4.

Figure 7. Effects of altering the number of consecutive adenosine residues in the GSTe2 promoter on luciferase activity

The number of adenosine residues at −265 in the GSTe2 promoter was altered by site-directed mutagenesis. The resultant constructs were transfected into A. gambiae Sua 4.0 cells and the luciferase activity was measured. The mean (±S.D.) luciferase activities (normalized to control Renilla activity) for two separate experiments performed in triplicate are shown. Pairwise Student's t tests were used for statistical analysis (**P<0.01).

Figure 8. Induction of GST expression by H₂O₂ exposure

To test whether GSTe3, GSTe1 and GSTe2 in A. gambiae are inducible by oxidative stress, larvae of Kisumu and ZAN/U strains were exposed to 3 mM H₂O₂ for 1 h. mRNA was extracted and reversed-transcribed into cDNA. The copy number of the Epsilon GST, GSTe3, GSTe1 and GSTe2 (inset) was determined by real-time PCR. The data are from duplicates of three biological replicates. The transcript copy number in control (no H₂O₂ exposure) was compared with the H₂O₂-exposed samples using the pairwise Student's t test (**P<0.01; N.S., not significant).