Identification of a novel class of insect glutathione S-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae

Abstract

The sequence and cytological location of five Anopheles gambiae glutathione S-transferase (GST) genes are described. Three of these genes, aggst1-8, aggst1-9 and aggst1-10, belong to the insect class I family and are located on chromosome 2R, in close proximity to previously described members of this gene family. The remaining two genes, aggst3-1 and aggst3-2, have a low sequence similarity to either of the two previously recognized classes of insect GSTs and this prompted a re-evaluation of the classification of insect GST enzymes. We provide evidence for seven possible classes of insect protein with GST-like subunits. Four of these contain sequences with significant similarities to mammalian GSTs. The largest novel insect GST class, class III, contains functional GST enzymes including two of the A. gambiae GSTs described in this report and GSTs from Drosophila melanogaster, Musca domestica, Manduca sexta and Plutella xylostella. The genes encoding the class III GST of A. gambiae map to a region of the genome on chromosome 3R that contains a major DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] resistance gene, suggesting that this gene family is involved in GST-based resistance in this important malaria vector. In further support of their role in resistance, we show that the mRNA levels of aggst3-2 are approx. 5-fold higher in a DDT resistant strain than in the susceptible strain and demonstrate that recombinant AgGST3-2 has very high DDT dehydrochlorinase activity.

Full text

Biochem. J. (2001) 359, 295–304 (Printed in Great Britain) 295
Identification of a novel class of insect glutathione S-transferases involved
in resistance to DDT in the malaria vector Anopheles gambiae
Hilary RANSON*†
1
, Louise ROSSITER*, Federica ORTELLI*, Betty JENSEN†, Xuelan WANG†, Charles W. ROTH‡,
Frank H. COLLINS† and Janet HEMINGWAY*
*School of Biosciences, Main College, Cardiff University, PO Box 915, Cardiff CF10 3TL, Wales, U.K., †Department of Biological Sciences, University of Notre Dame,
PO Box 369, Notre Dame, IN 46556, U.S.A., and ‡Unite
!
de Biochimie et Biologie Mole
!
culaire des Insectes, Institut Pasteur, 75015 Paris, France
The sequence and cytological location of five Anopheles gambiae
glutathione S-transferase (GST) genes are described. Three of
these genes, aggst1-8, aggst1-9 and aggst1-10, belong to the
insect class I family and are located on chromosome 2R, in close
proximity to previously described members of this gene family.
The remaining two genes, aggst3-1 and aggst3-2, have a low
sequence similarity to either of the two previously recognized
classes of insect GSTs and this prompted a re-evaluation of the
classification of insect GST enzymes. We provide evidence for
seven possible classes of insect protein with GST-like subunits.
Four of these contain sequences with significant similarities to
mammalian GSTs. The largest novel insect GST class, class III,
contains functional GST enzymes including two of the A. gambiae
GSTs described in this report and GSTs from Drosophila
INTRODUCTION
Glutathione S-transferases (GSTs) are a major family of detoxifi-
cation enzymes found in most organisms. They help to protect
cells from oxidative stress and chemical toxicants by aiding the
excretion of electrophilic and lipophilic compounds from the cell
(reviewed in [1]). Eukaryotes contain multiple GSTs with differing
catalytic activities to accommodate the wide range of functions
of this enzyme family. Mammalian GSTs have been classified
into eight cytosolic classes (Alpha, Mu, Pi, Theta, Sigma, Zeta,
Kappa and Omega) and a microsomal class [2–7], whereas only
two classes of insect GSTs (classes I and II) have so far been
described [8]. (An alternative nomenclature in which the insect
classes are assigned Greek letters in line with the mammalian
GST classification system has been proposed [9] and is discussed
in the present paper). The insect class I GSTs are encoded by a
large complex gene family. Additional heterogeneity within this
class is introduced by alternative splicing in Anopheles gambiae
and the presence of fusion genes in Musca domestica [10,11].
In Drosophila melanogaster and A. gambiae this gene family
is tightly clustered [10,12], in contrast with the family in
M. domestica, in which the class I GSTs are dispersed throughout
the genome [13]. The class II insect GST family consists of a
single gene in all three species [14,15].
Abbreviations used: BAC, bacterial artificial chromosome ; CDNB, 1-chloro-2,4-dinitrobenzene; DCNB, 1,2-dichloro-4-nitrobenzene; DDE, 1,1-
dichloro-2,2-bis-(p-chlorophenyl)ethane; DDT, 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane; GST, glutathione S-transferase; RACE, rapid amplifi-
cation of cDNA ends.
1
Present address and address for correspondence : Parasite and Vector Biology Division, Liverpool School of Tropical Medicine, Pembroke Place,
Liverpool L3 5QA, U.K.) (e-mail HRanson!liverpool.ac.uk).
The nucleotide sequence data reported will appear in DDBJ, EMBL and GenBank2 Nucleotide Sequence Databases under the accession numbers
AF316635 to AF316638.
melanogaster, Musca domestica, Manduca sexta and Plutella
xylostella. The genes encoding the class III GST of A. gambiae
map to a region of the genome on chromosome 3R that contains
a major DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane]
resistance gene, suggesting that this gene family is involved in
GST-based resistance in this important malaria vector. In further
support of their role in resistance, we show that the mRNA levels
of aggst3-2 are approx. 5-fold higher in a DDT resistant strain
than in the susceptible strain and demonstrate that recombinant
AgGST3-2 has very high DDT dehydrochlorinase activity.
Key words: classification of insect GST enzymes, Drosophila
genome, insecticide resistance.
Interest in insect GSTs is focused on the role of these enzymes
in insecticide resistance. Elevated GST activity has been de-
tected in strains of insects resistant to organophosphates [8] and
organochlorines [16] and this enzyme family has recently been
implicated in resistance to pyrethroid insecticides [17,18].
A. gambiae GSTs are of particular interest because of their
involvement in resistance to DDT [1,1,1-trichloro-2,2-bis-
(p-chlorophenyl)ethane] in this important malaria vector. In the
1950s and 1960s house spraying with DDT was the primary line
of defence against malaria and, although the advent of DDT-
resistant strains of mosquitoes has decreased the effectiveness of
this control measure, this insecticide is still used today for
malaria control in many parts of the world [19]. In A. gambiae,
an increased rate of DDT dehydrochlorination in the resistant
strain is associated with quantitative increases in multiple GST
enzymes [20].
We have studied the A. gambiae class I and class II GST genes
to ascertain their role in conferring DDT resistance. The single
class II GST, aggst2-1 [15] is highly expressed in A. gambiae
larvae but is barely detectable in adult insects. Because DDT
resistance in this species is life-stage specific and the insecticides
are used as adulticides both in the field and for selection of the
resistant strain in the laboratory, the developmental expression
profile discounted a prominent role for aggst2-1 in conferring
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296 H. Ranson and others
DDT resistance. The class I GSTs are expressed at high levels in
both larvae and adults [10]. Several recombinant A. gambiae
GST enzymes are able to metabolize DDT but, by using
antibodies raised against these class I GSTs, we have demon-
strated that these enzymes are not the most important family in
DDT resistance [21]. Furthermore, we have no evidence to
suggest that any of these genes are overexpressed in resistant
mosquitoes (N. Roberts and J. Hemingway, unpublished work).
Hence either A. gambiae contains additional genes encoding
class I GSTs, or further classes of insect GSTs exist. We now
present evidence to support both these hypotheses. We report the
cloning of five novel A. gambiae genes encoding GSTs. Three of
these have been classified as class I GSTs, whereas the remaining
two genes belong to a previously undescribed class, which we
have named class III. We propose that the class III GSTs
represent the major enzyme family conferring resistance to DDT
in the malaria mosquito, A. gambiae.
EXPERIMENTAL
Mosquito strains
The ZAN\U strain of A. gambiae was colonized from a DDT-
resistant field population from Zanzibar, Tanzania, in 1982. This
strain has been maintained under regular adult selection pressure
with DDT. Kisumu is a laboratory insecticide-susceptible strain
originally colonized from Kisumu, western Kenya. The PEST
strain is fixed for the standard chromosome arrangement [22]
and was used to construct the A. gambiae bacterial artificial
chromosome (BAC) library (X. Wang, Z. Ke, A. J. Cornel, D.
Smoller and F. H. Collins, unpublished work).
DNA extraction and sequencing
BAC DNA was isolated with Qiagen Plasmid maxi kits. BAC
sequencing reactions were performed with 1 µg of BAC DNA as
a template and ABI BigDye Terminator chemistry. After electro-
phoresis on an ABI 377 automatic sequencer, contigs were
assembled and the sequences annotated with the LASERGENE
software package (DNAstar, Madison, WI, U.S.A.).
Total RNA was extracted from individual mosquitoes with the
TRI reagent (Sigma), in accordance with the manufacturer’s
instructions. The RNA was treated with DNase to remove any
contaminating genomic DNA and the mRNA was reverse-
transcribed into cDNA by using Superscript II (Gibco BRL) and
an oligo(dT) adapter primer (5h-GACTCGAGTCGACATCGA-
(dT)
"(
-3h).
Genomic DNA was extracted from individual adult mos-
quitoes as described previously [23].
In situ hybridization
BAC clones were physically mapped to polytene chromosomes
prepared from half-gravid ovaries of the PEST strain of
A. gambiae as described previously [24].
Quantification of aggst3-2 mRNA levels
Incorporation of the fluorescent dye SYBR GreenI (Molecular
Probes) into double-stranded PCR products was used to de-
termine the mRNA copy number of aggst3-2 in individual
mosquitoes. An aggst3-2 standard plasmid was constructed by
inserting a 353 bp fragment from the coding region of the
aggst3-2 gene, amplified from ZAN\U cDNA with the primers
3-2f (5h-GTACGATCATCACCGAGAGC-3h) and 3-2r (5h-
CTTCGACTGCTCCAACGGC-3h), into pGEM T-easy vector
(Promega). A control plasmid was constructed by inserting a
partial fragment from the gene encoding ribosomal S7 protein
[25], amplified with primers SPC (5h-GTGCCGGTGCCGA-
AACAGAA-3h) and SPD (5h-AGCACAAACACTCCAATA-
ATCAAG-3h), into the pGEM T-easy vector. These plasmids
were used as template DNA at concentrations ranging from 1 ng
to 10 fg to produce standard curves using a Roche Lightcycler
in accordance with the manufacturer’s recommended protocols.
For quantification of the copy number, approx. 2% of the
cDNA from an individual mosquito was used as a template for
the control (S7) primers and 6 % was used for quantifying
aggst3-2 expression; 40 rounds of amplification were performed
in glass capillaries containing 5 pmol of each primer, 1iSYBR
GreenI mix and a final concentration of 3 mM MgCl
#
. The
amplification cycle was as follows : 95 mC for 1 s, 62 mC for 3 s
and 72 mC for 15 s, with incorporation of fluorescence measured
at 87 mC for aggst3-2 quantification, and 95 mC for 1 s, 60 mC for
3 s and 72 mC for 10 s, with incorporation of fluorescence
measured at 86 mC for S7 quantification. Each sample was
analysed in duplicate in each experiment and the results are
means for two separate experiments. The data were quantified
with LightCycler Software V3 (Roche) and converted into copy
number as described in [26].
Expression of aggst3-2 in vitro
The coding region of aggst3-2 was amplified in a PCR reaction
with ZAN\U cDNA as a template, Pfu polymerase (Stratagene)
and primers that contained the initiation and termination codons
of the gene preceded by BamH1 sites. The single product of
approx. 680 bp was subcloned into T-easy (Promega) and
sequenced to ensure that no errors had been introduced during
amplification. The insert was then isolated by digestion with
BamH1, ligated into the BamH1 site of the pET3a vector
(Novagen) and the resultant expression construct was used to
transform Escherichia coli Origami (DE3)pLysS cells. The orient-
ations of the inserts were determined by restriction digestion ;
colonies containing the insert in both the forward and reverse
orientations were grown at 37 mC to an attenuance (D
'!!
) of 0.6.
Expression of the recombinant protein was induced by the
addition of isopropyl β--thiogalactoside to 0.4 mM and, after
incubation for a further 3 h, the cells were harvested by centri-
fugation for 10 min at 5000 g. After a single round of freeze–
thawing, the cells were resuspended in 50 mM Tris\HCl (pH 8.0)\
2 mM EDTA\0.1 M NaCl. Protein concentration was deter-
mined with Bio-Rad protein reagent [27] ; GST activity was
assayed spectrophotometrically by measuring the conjugation of
GSH to the standard GST substrates 1-chloro-2,4-dinitrobenzene
(CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) [28].
DDT dehydrochlorinase activity was assayed by incubating
the crude cell extract with 0.1 mM DDT and 10 mM GSH in
0.1 M sodium phosphate buffer, pH 6.5, for 2 h at 30 mC. The
samples were extracted twice with chloroform, air-dried and then
resuspended in propan-2-ol. HPLC analysis of DDT metabolites
was performed as described by Prapanthadara et al. [20], with a
flow rate of 0.6 ml\min. Constructs containing the insert in the
negative orientation were assayed to control for non-enzymic
DDT metabolism. Controls omitting GSH in the incubation
mixture were also included to verify the dependence of the
reaction on GSH.
Phylogenetic analysis of insect GSTs
A search of the GenBank2 database located 21 non-Drosophila
insect GST sequences including seven A. gambiae GSTs. These
were retrieved and the putative amino acid sequences were aligned
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297Glutathione S-transferases and DDT resistance in Anopheles gambiae
against the A. gambiae GST sequences described here, with the
CLUSTAL W program [29] A total of 42 Drosophila sequences
predicted to contain GST protein domains are present in the D.
melanogaster genome [30]. These sequences were retrieved from
FlyBase (http:\\flybase.bio.indiana.edu) to enable us to in-
corporate them into our phylogenetic analysis. Ten of these
sequences are identical to previously submitted Drosophila
GST sequences and were included in our analysis as they appear
in GenBank2 (with the exception of DmGST26 and DmGST22,
which are reported to be pseudogenes [12] and were therefore
excluded). Of the remaining 32 putative Drosophila genes for
GST, four sequences (CG15100, CG4623, CG12304 and
CG11901) were considered unlikely to encode functional GST
enzymes on the basis of their transcript length and\or low degree
of similarity to genes encoding GST; they were therefore
discarded. The annotations of the remaining genes for GST were
studied and most of the amino acid translations were accepted as
published with the following exceptions. (1) Two putative full-
length transcripts were derived from the annotations for
CG12930 and CG6673. The derived amino acid sequences of
both of these were included in the study (denoted by A and B).
(2) The translation of CG1681 seems to be lacking 37 residues
at the N-terminus. A search of the nucleotide sequence of this
annotation identified a putative 5h exon, approx. 3 kb upstream
from the 3h end of the gene, which was joined to the amino acid
translation to produce a putative full-length gene. (3) Anno-
tations CG1702, CG10065 and CG17639 predicted very large
translation products of which only approx. 220 residues showed
significant similarity to GSTs. These translations were therefore
trimmed manually.
After updating the alignment to contain these Drosophila
sequences, evolutionary distances were calculated by using the
Jukes–Cantor algorithm [31]; phylogenetic trees were determined
by the neighbour-joining method [32] with TREECON for
Windows [33]. The amino acid translation of a Rat Kappa GST
(rGSTTK1-1 [5]) was used as an outgroup to root the tree.
RESULTS
Cloning of GSTs
As part of the A. gambiae genome initiative, the insert ends of
each clone from a BAC library have been determined by single-
pass sequencing at Genoscope and the Institut Pasteur
(www.genoscope.cns.fr\externe\English\Projets\ProjetIAK\
AK.html). The resultant sequences were queried against the
GenBank2 database and two BAC clones were identified in which
end sequences had significant similarity to GSTs (04H09 and
28I09). The end sequence of clone 04H09 (sequenced with primer
SP6) was predicted to encode the carboxy region of a GST.
Primers were designed to amplify this partial gene encoding a
GST and used to screen the BAC library for overlapping clones.
Four positive clones were identified (05C11, 06I12, 12H09 and
28A19) and primers designed against the 3h GST sequence in
04H09 were used for partial sequencing of clone 06I12. The
results of this sequencing not only completed the genomic
sequence of the GST gene present in the end sequence of clone
04H09, but also identified an additional gene encoding a GST,
approx. 350 bp downstream from this gene. These A. gambiae
GST genes have low levels of similarity to the two insect GST
classes previously recognized (class I and class II) and were
therefore tentatively assigned to a third class of insect GSTs and
named aggst3-1 and aggst3-2 (see the Discussion).
The end sequence of BAC clone 28I09 (sequenced with primer
SP6) contained the 3h end of one gene for GST (later named
aggst1-9) and the 5h end of a second gene encoding a GST
Figure 1 Alignment of deduced amino acid sequences of the five A.
gambiae genes encoding GST described in this report
Gaps introduced to maximize sequence identity are shown by a horizontal dash. Residues shown
in bold are shared by all class I and class III A. gambiae GSTs. The residues denoted by an
asterisk are shared by all known GSTs [37]. The arrows indicate intron positions (see the text
for further details).
Figure 2 Schematic representation of the A. gambiae genome showing
the location of genes for GSTs and loci associated with resistance to DDT
(rtd1 and rtd2)
Cytological positions are shown to the right of the chromosome and the two major regions of
the genome associated with DDT resistance are shown as solid bars.
(aggst1-8). Primers designed against the end sequence of 28I09
were used to search the BAC library for overlapping clones and
a single positive, 10O16, was identified and used as a template for
obtaining the full length-genomic sequences of genes aggst1-8
and aggst1-9.
3h-Rapid amplification of cDNA ends (RACE) reactions with
primers incorporating the initiator methionine codon of the
putative genes encoding GST were used to verify that these genes
were expressed in A. gambiae. Transcripts of aggst3-1, aggst3-2
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298 H. Ranson and others
0
10
20
30
40
010203040
0
10
20
30
40
010203040
10 15 20 25 30
–2
0
2
4
10 15 20 25 30
–2
0
2
4
–4
0
0.002
0.004
0.006
Kisumu ZAN/U
Copy no. ratio (Aggst3-2 / SP7)
C
AB
Fluorescence
Fluorescence
Cycle number Cycle number
1 ng
100 pg 10 pg 1 pg 100 fg
1 ng
100 pg 10 fg
1 pg
1 fg
Log conc. (pg)
Log conc. (pg)
Crossing Point Crossing Point
1 ng
100 pg
10 pg
1 pg
100 fg
1 ng
100 pg
1 pg
10 fg
1 fg
Figure 3 Quantification of aggst3-2 mRNA expression levels in individual A. gambiae adults
(A, B) Upper panels: SYBR GreenI fluorescence acquisition by PCR products from serially diluted (1 ng to 1 fg) standard plasmids and individual adult mosquito cDNA against cycle number.
Lower panels: standard curves derived from plotting the crossing points against the logarithm of copy number for plasmids S7 and aggst3-2.(A) SP7 standardization control ; (B) aggst3-2. (C)
mRNA copy number of aggst3-2 transcript relative to the number of SP7 transcripts for each individual mosquito.
and aggst1-8 were detected in adult mosquitoes. 3h-RACE with
a primer complementary to aggst1-9 gave a product of the
expected size but with only 36.8 % sequence identity to aggst1-9.
This gene transcript has relatively high similarity to aggst1-5 and
aggst1-6 (42.9 % and 42.4% similarity at the nucleotide level)
and was therefore provisionally classified as a class I GST and
named aggst1-10. The BAC library was screened with primers
specific to aggst1-10 to enable the genomic organization and
physical location of aggst1-10 to be established. As attempts at
3h-RACE for aggst1-9 were unsuccessful we designed PCR
primer pairs specific to this gene and used these in PCR reactions
with fourth-instar larvae, pupae and adult mosquito cDNA as
templates; however, we were unable to detect a transcript in any
of these life stages. This gene might be expressed in earlier life
stages but the possibility that aggst1-9 is a pseudogene cannot be
discounted at this stage.
An amino acid alignment of the five A. gambiae genes for GSTs
identified in this study is shown in Figure 1. The four invariant
residues proposed to be crucial for the correct folding of GST
enzymes [34] are conserved in these, and in all previously
identified, A. gambiae GST sequences (indicated by an asterisk in
Figure 1). In addition to these, a further eight residues are
constant in all A. gambiae GSTs. aggst1-8 and aggst1-10 contain
a single intron within the 5h coding region at an identical position
to the intron in the alternately spliced A. gambiae gene for GST,
aggst1α [10]. aggst3-1 and aggst3-2 also contained an intron at
this position and an additional intron at position 119 (numbers
according to AgGST3-1 sequence) (Figure 1). aggst1-9 is unique
among the newly described A. gambiae GSTs in being intronless.
Physical mapping
The cytological location of the A. gambiae genes encoding GSTs
was determined by in situ hybridization. Figure 2 shows the
positions of all these genes on the polytene chromosomes. Six
genes for GSTs are located on chromosome 2R within divisions
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299Glutathione S-transferases and DDT resistance in Anopheles gambiae
Figure 4 Heterologous expression of AgGST3-2
E. coli cultures containing aggst3-2 expression constructs, prepared with the insert in either the
correct or the reverse orientation within the pET3a vector, were induced with isopropyl
β-D-thiogalactoside. A 10 µl sample of whole cells was separated on a 12% (w/v) resolving,
5 % (w/v) stacking SDS/polyacrylamide gel and stained with Coomassie Blue R250. Left lane,
an E. coli culture containing aggst3-2 in the correct orientation ; middle lane, an E. coli culture
containing aggst3-2 in reverse orientation (negative control) ; right lane, molecular mass
standards (molecular masses indicated at the right). The subunit size of aggst3-2 is predicted
to be 24.9 kDa on the basis of its amino acid translation. A band of this approximate size is
clearly visible in the left lane.
18-19. These include the aggst1α, aggst1β and aggst1-2 genes
described previously [10,35] and the three class I GSTs described
here, namely aggst1-8, aggst1-9 and aggst1-10. Two possible sites
of hybridization for the single class II GST have been reported
[15] but only one of these, on division 38d, was confirmed by
further experiments. The class III GSTs are located on division
33c on chromosome 3R. This position coincides with the location
of one of two major quantitative trait loci associated with DDT
resistance in the ZAN\U strain of A. gambiae [36] (Figure 2).
Figure 5 HPLC analysis of DDT dehydrochlorinase activity by crude cell extracts expressing recombinant AgGST3-2
(A) Control insert in reverse orientation. (B) Insert in correct orientation; 50 µl of cell extract. (C) Insert in correct orientation ; 200 µl of cell extract.
Quantitative analysis of aggst3-2 expression
The co-localization of the A. gambiae class III genes with a major
gene conferring DDT resistance prompted us to study the relative
expression levels of members of this insect class in susceptible
and resistant insects by using real-time PCR technology. From
the standard curves shown in Figures 3(A) and 3(B) it was
possible to extrapolate the fluorescence values obtained with
Kisumu and ZAN\U cDNA and calculate the initial tem-
plate copy number. By dividing the copy number of aggst3-2 by
the copy number of S7 the values for GST expression in each
individual mosquito were standardized for variations in initial
cDNA concentrations so that the relative expression of aggst3-2
between the susceptible and resistant strains could be compared
(Figure 3C). The average ratio of aggst3-2 copy number to S7
copy number in the ZAN\U strain was (4.6p1.72)i10
−
$
com-
pared with (9.5p4.75)i10
−
%
in the Kisumu strain, representing
an approximate 5-fold overexpression in the resistant strain.
Expression in vitro
To verify that the A. gambiae class III GSTs encoded catalytically
active enzymes, we expressed aggst3-2 in E. coli (Figure 4) and
measured the CDNB- and DCNB-conjugating activity of the
crude protein homogenates. No CDNB-conjugating activity was
detectable in the control cultures but replicate crude cell extracts
from two separate E. coli cultures expressing recombinant
AgGST3-2 had a mean CDNB-conjugating activity of 2.879p
0.8 µmol\min per mg of crude protein and a mean DCNB-
conjugating activity of 5.74p2.7 µmol\min per mg of crude
protein.
DDT dehydrochlorinase activity of recombinant AgGST3-2
was measured as nmol of 1,1-dichloro-2,2-bis-(p-chlorophenyl)
ethane (DDE) detected by HPLC analysis after incubation of
the crude cell extract with 100 nmol of DDT as described in the
Experimental section. The recovery of DDT\DDE after extrac-
tion and analysis ranged from 43% to 58 %. Representative
results are shown in Figure 5. DDE was undetectable in the
control reactions containing the insert in the negative orientation
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300 H. Ranson and others
Table 1 Pairwise percentage similarities between derived amino acid sequences of A. gambiae GSTs
Sequences shown in bold are described here for the first time.
Similarity (%)
Sequence agGST1-3 agGST1-4 agGST1-5 agGST1-6 agGST1-7 agGST1-8 agGST1-9 agGST1-10 agGST2-1 agGST3-1 agGST3-2
agGST1-2 47.4 46.4 49.3 49.8 36.4 39.2 37.3 27.8 12.9 27.8 30.1
agGST1-3 58.4 61.2 60.8 33.5 34.2 27.9 29.4 11.0 27.9 29.2
agGST1-4 63.2 65.6 39.6 42.4 34.6 29.4 11.1 30.9 32.7
agGST1-5 82.8 43.1 41.1 35.9 35.4 14.8 33.5 37.3
agGST1-6 45.5 43.1 35.4 35.9 14.4 34.9 38.8
agGST1-7 39.9 32.1 29.4 11.5 30.3 34.4
agGST1-8 37.7 29.9 11.9 26.8 29.9
agGST1-9 28.9 16.1 24.5 26.4
agGST1-10 12.8 28.0 27.5
agGST2-1 10.1 11.9
agGST3-1 63.8
Figure 6 Dendrogram illustrating the relationship between insect GSTs
Amino acid sequences were aligned by using CLUSTAL W and the tree was constructed with the neighbour-joining method program from a similarity matrix of pairwise comparisons made by
using the Jukes–Cantor algorithm. Selected bootstrap values from 500 replicate trees are shown (as percentage values) at the dendrogram nodes. The sequences denoted by CG were obtained
from the Drosophila genome annotations (http://flybase.bio.indiana.edu) as described in the text. All other sequences were retrieved from GenBank2 or are described in this study. The tree was
rooted with the rat gene encoding Kappa GST (GenBank2 accession number S83436). Abbreviations : Dm, D. melanogaster ; Md, M. domestica ; Lucil, Lucilia cuprina ; Ag, A. gambiae ; Cv, Culicodies
variipennis ; Ae, Ae. aegypti ; Plx, P. xylostella ; Msex, Ma. sexta.
(Figure 5A) and in the absence of GSH, indicating that DDT
dehydrochlorinase activity in the assay was dependent on both
enzyme and GSH. In the experimental assays expressing recombi-
nant AgGST3-2, the percentage conversion of DDT to DDE was
dependent on the amount of crude extract used in the assay.
For example, in the experiment shown in Figure 5(B), 50 µlof
extract was used ; 24 nmol of DDE and 33 nmol of DDT were
detected by HPLC. When the volume of cell extract was increased
to 200 µl (Figure 5C) 43 nmol of DDE was recovered, repre-
senting a 92% conversion of DDT to DDE. If these values are
expressed as nmol of DDE\µg of protein, values of 12.5 and 5.5
are obtained for the experiments shown in Figures 5(B) and 5(C)
respectively, suggesting that the concentration of DDT was rate-
limiting in the experiment shown in Figure 5(C).
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301Glutathione S-transferases and DDT resistance in Anopheles gambiae
Figure 7 Dendrogram illustrating the relationship between Drosophila GSTs and mammalian representatives from each of the evolutionarily distinct GST
classes
See the legend to Figure 6 for details.
Phylogenetic analysis
Table 1 shows the percentage similarity between the deduced
amino acid sequences of all known A. gambiae GSTs. Previous
classifications have designated GSTs as being members of the
same class if their amino acid sequences are more than 40 %
identical [1]. By this criterion, only aggst1-8 of the five newly
described A. gambiae GSTs would be classified as belonging to
the insect class I family and none of these genes would be
classified as class II. This suggests that the present classification
of insect GSTs into only two classes might need re-evaluating.
We therefore conducted a phylogenetic analysis of all the known
insect GST sequences, including 28 sequences retrieved from the
Drosophila genome database.
Figure 6 shows a phylogenetic tree illustrating the relationships
between these sequences, based on a CLUSTAL W amino acid
alignment. The available insect GST sequences can be split into
at least seven subdivisions on the basis of this phylogeny. Only
three of these contain sequences with confirmed GST activity.
These are the pre-existing classes I and class II plus a third clade,
which we have called class III, in line with the existing no-
menclature for insect GSTs, containing 20 sequences including
aggst3-1 and aggst3-2 and the published genes DmGST-3 from
D. melanogaster, GST-3 from Plutella xylostella and MsGST1
from Manduca sexta [37–39]. The low support for the monophyly
of class I and of class III, indicated by the bootstrap values in
Figure 6, perhaps suggests that these classes should be further
subdivided. We therefore used amino acid distance matrices
(results not shown) to examine the support for this classification.
In this approach, we classified a gene for GST as belonging to a
particular class if it satisfied the following two criteria: (1) at
least 40% sequence similarity to a member of this class from a
different species, and (2) less than 40 % sequence similarity to all
other classes of insect GST. The assignment of genes to classes II
and III is supported by these criteria but ambiguities arose in the
classification of class I. In Figure 6, and in the choice of
nomenclature, we have classified aggst1-9 and aggst1-10 (plus the
putative Drosophila GST, cg10065) as class I GSTs. However,
these sequences show less than 40% sequence similarity to other
members of this group and therefore do not belong to this class
on the basis of previously published criteria [1]. Nevertheless,
because these genes show the greatest levels of identity with class
I GSTs out of all currently known insect GSTs, we propose to
assign these GSTs to class I at present.
Relationship between mammalian and insect GSTs
The remaining four subdivisions shown in Figure 6 consist solely
of sequences with GST-like domains, retrieved from the Droso-
phila genome database, and have not been experimentally verified
as functional GST enzymes. CG4688 and CG11784 have the
greatest similarity to MdGST6A (31.5% and 35.1% respectively)
but, because these levels of similarity are below the arbitrary cut-
off value, they have been classified as belonging to a separate
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302 H. Ranson and others
subdivision in Figure 6. CG9362 and CG9363 are closely related
(62.6% similarity to each other) but have less than 20 % similarity
to any other known insect GST sequence. Similarly, the amino
acid similarities support the existence of a distinct subdivision
containing CG6773A, CG6773B, CG6781, CG6662 and CG6776
and a separate subdivision containing CG1702, CG12930A and
CG12930B; however, the members of both these subdivisions
have very low similarity to previously characterized insect GSTs.
To investigate the relationship between the insect and mam-
malian GST classes, we took a representative member of each
of the eight cytosolic classes of mammalian GST and used
CLUSTAL W to align these sequences with the putative
Drosophila GST sequences. This alignment was used to generate
the phylogenetic tree shown in Figure 7. Previous classifications
have denoted all insect GSTs as belonging to the Theta class [34]
but the phylogeny shown in Figure 7 does not support this
conclusion. Although this tree is not intended to resolve the true
phylogenetic relationship between GST families, it is interesting
to note that representatives from the Theta, Sigma, Omega and
Zeta class are present in Drosophila. For example, the Drosophila
sequences CG9362 and CG9363 possess 56–58% identity with
the human Zeta GST, GSTZ1-1, and these putative insect GSTs
contain N-terminal motifs closely related to the SSCXWR-
VRIAL (single-letter amino acid codes) motif found in Zeta class
GSTs from plants, nematodes and mammals [4] (the predicted
translations for CG9362 and CG9363 contain the motifs SSCS-
WRVRVAL and SSCSWRVRIAM respectively). A further
subdivision of insect GSTs shows high similarity to the Omega-
class human GST GST O1-1. All five putative Drosophila
sequences within this subdivision, which are clustered on
chromosome 3L, also contain a cysteine residue flanked by
phenylalanine and proline residues at the proposed active site [7].
Finally, as noted recently [40], the insect class II enzymes are
phylogenetically related to the Sigma class.
DISCUSSION
Evolutionary relationship between insect GSTs
We have identified five A. gambiae genes encoding GSTs, at least
four of which are actively transcribed in adult mosquitoes. The
fifth gene, aggst1-9, seems to be transcriptionally silent in adult
and larval mosquitoes. It is not known whether this gene is
expressed during earlier life stages or whether it is a pseudogene.
A comparison of amino acid sequence similarities found that
four of the genes described here were below the threshold for
inclusion in either insect class I or class II, suggesting that the
current classification system for insect GSTs is inadequate. This
observation is supported by biochemical data from A. gambiae
that identified at least eight fractions with GST activity, only one
of which was immunologically related to the class I GST family
[20]. The publication of the first draft of the Drosophila genome
[41] prompted us to re-examine the classification of insect GSTs.
Our analysis supported the existence of seven possible classes of
protein with GST-like subunits. The existing class I and II
families were resolved by our analysis and five additional classes
were proposed. Of these five classes, only the largest, which we
have named class III, includes insect proteins with confirmed
GST activity ([38] and the present study). In our choice of
Drosophila sequences to include in the analysis we selected only
those encoding peptides of the approximate size of GST subunits
(approx. 25 kDa) and showing significant similarity throughout
the entire sequence rather than just at the N-terminus. Never-
theless, we acknowledge that some genes might have GST-like
domains but not possess GST activity and likewise that some
proteins might have acquired GST activity as a result of
convergent evolution [42]. Hence we might have inadvertently
included sequences that are not part of the same phylogenetic
gene tree.
A preliminary investigation into the relationship between the
insect GST classes and the previously characterized mammalian
cytosolic GST classes revealed that four of the subgroups of
insect GSTs have significant sequence similarity to confirmed
GST classes (Theta, Sigma, Omega and Zeta). The class I
insect GST subgroup has also been referred to as the Delta class
and the insect class II as Sigma [9], in line with the nomenclature
of the existing GST classes. The insect class III family, described
for the first time here, does not belong to the Delta class or to any
of the other existing GST families on the basis of established
criteria. Therefore, to maintain consistency with the proposed
nomenclature for GST classes [9], the insect class III would
perhaps be more appropriately denoted by a Greek character.
We suggest that hereafter the insect class III family be referred to
as Epsilon (ε).
Of all known A. gambiae genes encoding GSTs, aggst1-2 and
aggst1-9 are unique in that their open reading frames are
uninterrupted by introns, although the presence of introns in the
5h non-coding sequence, as found in aggst1β, MdGST1 and
DmGST1 [10,43], cannot be discounted. The remaining class I
and class III GSTs in A. gambiae all possess an intron at the
identical position in the 5h coding region and aggst3-1 and
aggst3-2 also have a second intron within the centre of the open
reading frame. This contrasts with the situation in Drosophila,in
which none of the class I GSTD genes clustered on chromosome
3R division 87B [12] have introns within their open reading
frames, and furthermore an analysis of the 10 putative class III
GSTs on chromosome 2R division 55C retrieved from the
Drosophila genome database predicts that these genes are also
intronless [41]. However, it is not true that all Drosophila GST
genes are intronless. For example, the class II Drosophila
GST DmGST-2 is interrupted by two introns [14].
The conservation of intron\exon boundaries across the class I
and class III genes in A. gambiae and the absence of introns in the
homologous gene families in Drosophila might suggest that
the duplication events that occurred to produce the class I and
class III lineages occurred after the divergence of the Nematocera
and Brachycera Dipteran suborders. This hypothesis, however,
is not supported when all members of the class I and class III
families are considered. For example, a homologue of the A.
gambiae class I gene aggst1-7 has been identified in Drosophila
(CG17639) and both of these genes possess two introns at
identical sites (H. Ranson and N. Roberts, unpublished work),
perhaps suggesting a common ancestor for these two genes.
Role of A. gambiae GSTs in insecticide resistance
An association between elevated GST activity and insecticide
resistance has been observed in many insect species but there
have been very few reports describing the individual enzymes
involved. GST-2 from the mosquito Aedes aegypti is over-
expressed in a DDT-resistant strain [16] but the ability of this
enzyme to metabolize DDT has not been established and there-
fore the significance of this result is not clear. In addition,
there have been reports that expression of housefly MdGST-3 is
positively correlated with resistance and that recombinant
MdGST-3 is able to degrade the insecticide dimethylparathion
[11,44]. However, the genomic organization of this gene seems
to be extremely complex with a variant gene copy number
in different strains and hence the exact role of this enzyme in
insecticide resistance is difficult to ascertain [11]. To our know-
ledge there have been only two substantiated reports of a direct
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303Glutathione S-transferases and DDT resistance in Anopheles gambiae
relationship between GST overexpression and resistance. The
first is in the diamondback moth, P. xylostella. In this insect,
increased expression of the PxGST3 gene, which encodes an
enzyme capable of degrading organophosphorous insecticides, is
strongly correlated with resistance [38]. The second example is in
D. melanogaster, in which a recombinant GST enzyme, GST D1,
exhibiting DDTase activity was found at elevated levels in a
DDT-resistant strain [45].
With our results from A. gambiae we have shown that members
of the class I family of insect GSTs are not of major importance
in DDT resistance [21] and the expression profile of class II also
discounts a major role for this gene in resistance [15]. To
establish the identity of the GST enzymes responsible for
resistance, we conducted a genome-wide scan to identify regions
of the genome associated with resistance to DDT. We identified
two major loci, the first, rtd1, on chromosome 3R between
divisions 32c and 34c, and the second, rtd2, on chromosome 2L
in close proximity to division 21 [36]. Two of the genes encoding
GST described in this study map to chromosome 3R, division
33c, i.e. in the exact midpoint of the boundaries defined by rtd1,
invoking the hypothesis that this resistance locus is a cis-acting
regulatory element controlling the expression of these class III
GSTs. In support of this we have now shown that aggst3-2 is
overexpressed in the resistant strain and that recombinant
aggst3-2 is very efficient at metabolizing DDT. DDT dehydro-
chlorinase activity has previously been reported for recombi-
nant GSTs from A. gambiae [21] but this is the first definitive
demonstration that a GST with DDT dehydrochlorinase activity
is overexpressed in a DDT-resistant strain of mosquitoes. By
analogy with Drosophila, in which 10 class III genes for GST
are tightly clustered within approx. 14 kb of DNA [41], it is likely
that the class III family in A. gambiae extends beyond the two
members described here. If multiple members of this gene family
are under the control of a common regulatory factor, a mutation
in this factor could account for the elevated activity of several
different GST enzymes observed in our earlier biochemical studies
[21,46].
We acknowledge the work of the Drosophila genome-sequencing community for
generating the data referenced here and the assistance of Dr John Vontas in HPLC
analysis. C. W. R. acknowledges support from the Institut Pasteur, the Centre
National de la Recherche Scientifique and Genoscope-Centre national de se
!
quenc
:
age,
France. This work was partly funded by Wellcome Trust, Royal Society and
Leverhulme fellowships (to H. R.) and a grant from the John D. and Catherine T.
MacArthur Foundation (to F. H. C.).
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