(Received for publication, August 14, 1996, and in revised form, October 25, 1996)
From the Department of Pure and Applied Biology,
University of Wales, Cardiff CF1 3TL, Wales, United Kingdom, the
§ Entomology Branch, Division of Parasitic Diseases, Centers
for Disease Control and Prevention, Chamblee, Georgia 30341, and the
¶ Universite Paul Sabatier, Labatoire d'Entomologie appliquee,
31062 Toulouse Cedex, France
1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) resistance in both adults and larvae of Anopheles gambiae is mediated by stage-specific glutathione S-transferases (GSTs). On the basis of their biochemical characteristics the larval resistance-associated GSTs are likely to be insect class I GSTs. Aggst1-2, a class I GST gene, which is expressed in larvae, has been cloned from the malaria vector A. gambiae. The gene was inserted into a bacterial expression system, and the detection of 1-chloro-2,4-dinitrobenzene (CDNB) conjugating activity in Eschericia coli expressing the recombinant enzyme confirmed that aggst1-2 encodes a catalytically active GST. The gene encodes a 209 amino acid protein with 46% sequence similarity to a Drosophila melanogaster class I GST (GST-D1), 44% similarity with a Musca domestica class I GST (MdGST-1), but only low levels of homology with class II insect GSTs, including the adult specific AgGST2-1 from A. gambiae. Southern analysis of genomic DNA indicated that A. gambiae has multiple class I GSTs. In situ hybridization of class I genomic and cDNA clones to polytene chromosomes identified a single region of complementarity on chromosome 2R division 18B, suggesting that these class I GSTs in A. gambiae are arranged sequentially in the genome. Three positive overlapping recombinant clones were identified from an A. gambiae genomic library. Mapping and partial sequencing of these clones suggests that there are several GSTs and truncated GST pseudogenes within the 30kb of DNA that these clones span.
Glutathione S-transferases (GSTs1; EC 2.5.1.18) are a large family of enzymes abundant in most organisms (1-6). They protect the cell from attack by a wide range of reactive electrophilic compounds by conjugating these compounds with the tripeptide, glutathione, thereby increasing their solubility and aiding excretion from the cell. GSTs are also involved in the glutathione-dependent dehydrochlorination of insecticides (7). They can also act as binding proteins and may be important in the intracellular transport and excretion of hydrophobic compounds (8). In mammals, five cytosolic classes (alpha, mu, pi, sigma, and theta) of GSTs have been distinguished on the basis of amino acid sequence and substrate specificity (6, 9, 10). The GSTs are differentially regulated with specific enzymes being expressed in different tissues during each developmental stage and in response to various xenobiotics. In insects, GSTs are classified as either class I or class II on the basis of their immunological cross-reactivity and amino acid sequences (11).
Elevated levels of GST activity are associated with insecticide resistance in many insects. Extensive past and current use of the organochlorine insecticide DDT in malaria control programs has selected for DDT-resistant mosquito populations throughout the world. In the principal sub-saharan malaria vector, Anopheles gambiae, this resistance is mediated by GST-based metabolism of DDT (12). Partial purification of the GSTs from this species resolved multiple peaks of GST activity with differential abilities to metabolise DDT (13). Further characterization of A. gambiae GSTs was complicated by the presence of multiple GST isoenzymes, each with overlapping substrate specificities and physical properties that made purification to homogeneity difficult to achieve. We therefore set out to characterize the genes encoding A. gambiae GSTs in order to determine the genetic changes that have occurred in response to exposure to insecticides. We targeted the A. gambiae class I GSTs since earlier biochemical analysis (14) and Northern blots suggested that class I enzymes were more likely candidates for involvement in resistance.
Two strains of A. gambiae sensu stricto were used. G3 was colonized from The Gambia in 1975 and has been maintained in the United Kingdom since then without exposure to insecticides. Suakoko 2La, originated from Suakoko in Liberia. This strain was used since it is now being employed internationally as the reference strain for A. gambiae mapping work (15).
PCR Amplification of A. gambiae Class I GSTsTotal RNA was
extracted from A. gambiae G3 fourth instar larvae as
described previously (16). mRNA was isolated using the Poly(A)Tract
mRNA isolation system IV (Promega). Reverse transcription of
mRNA to cDNA was achieved with the Promega Riboclone cDNA
synthesis system using an oligo(dT) adaptor primer
(5-GACTCGAGTCGACATCGA(dT)17-3
). Degenerate primers for
insect class I GSTs were designed based on the sequence of Musca
domestica GST-1 (11), and these were used to amplify a homologous
region from A. gambiae cDNA. The 50-µl PCR reaction
contained 20 ng of first strand cDNA, 50 ng of each primer
(5
-TA(C/T)AAAAGCTT(C/T)GCNGA(C/T)TA(C/T)TA(C/T)TA(C/T)CC-3
and
5
-TCNGCNACNGTNA(A/G)N(C/G)(A/T)(A/G)TCNCCNGCAGC(A/G)TA(C/T)TC(A/G)TG-3
), 0.5 mM dNTPs, 2 mM MgCl2, 1.5 units
of Taq DNA polymerase, and Taq DNA polymerase
buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25 °C), 0.1% Triton X-100). Thirty-five cycles of amplification were carried out (95 °C for 1 min, 56 °C for 2 min, and 72 °C for 3 min). The 155-bp PCR product was subcloned into
pBluescript (Stratagene) and sequenced in both directions.
An 18-bp antisense oligonucleotide (5
-GTCCAGGAAGCTGTTCAG-3
) specific
to the 155-bp GST fragment was designed and used in a modified 5
-RACE
(rapid amplification of cDNA ends) procedure (16) to obtain the
5
-end of the cDNA. The 546-bp product, aggst1-1, was
subcloned and sequenced. For each plasmid insert, both strands of DNA
were sequenced at least twice. Alignments of the nucleotide sequences
were carried out using the LASERGENE package (DNASTAR, Inc.). This
package was also used to align multiple insect GSTs using the clustal method.
Genomic DNA was isolated from G3 as described previously (16). Separate 10 µg aliquots of G3 genomic DNA were digested to completion with SalI, EcoRV, and HindIII, separated on an 0.8% agarose gel, transferred to a nylon membrane (Amersham Life Sciences) and hybridized with a 32P-labeled aggst1-1 probe (specific activity > 1 × 109 dpm/µg) at 60 °C overnight in hybridization buffer (6 × SSC (20 × SSC contains 175.3 g of NaCl and 88.2 g of sodium citrate in 1 liter of water adjusted to pH 7.0 with NaOH), 0.1% (w/v) SDS, 0.1% (w/v) sodium pyrophosphate, 5% (w/v) polyethylene glycol 8000, and 1% blocking reagent (Boehringer Mannheim)).
A genomic library was made from the Suakoko 2La strain by partially digesting genomic DNA with Sau3A-1 and ligating into BamHI digested arms of Lambda Dash II (Stratagene). The library (150,000 plaque forming units) was screened with 32P-labeled aggst1-1 according to standard procedures (17). Three positive recombinant clones were obtained: Ag_B1, Ag_B2 and Ag_B3. Recombinant bacteriophage DNA was obtained as described by Chang and Natori (18). The restriction digested DNA was hybridized with aggst1-1 as described above.
An open reading frame encoding aggst1-2, a GST which was
related to but distinct from aggst1-1, was identified in
the recombinant clone Ag_B1. A 22 bp primer [5
CGTGGCA
TTGGATTTTTAC 3
] encompassing the predicted
initiation codon (underlined) of aggst1-2 was constructed. This was used to amplify a cDNA containing the full length open reading frame of aggst1-2 from A. gambiae G3 in
a PCR reaction utilizing the oligo-(dT) adaptor primer described above.
The PCR reaction was performed as described above with the following
modifications: the Taq DNA polymerase was added after an
initial denaturation step at 94 °C for 5 min, the annealing
temperature was dropped to 50 °C and the extension time was
decreased to 1 min.
In vitro Expression of aggst1-2. Aggst1-2 was expressed
in vitro by inserting the cDNA isolated above into the
plasmid expression vector, pET 3a (Novagen) which contains the
bacteriophage T7 promoter. The coding region of aggst1-2
was reamplified in a PCR reaction using primers which contained the
initiation and termination codons of the gene preceded by
BamHI sites. The 650 bp PCR product obtained was digested
with BamHI, ligated into the BamHI site of pET3a and the resultant plasmid was used to transform E. coli
BL21(DE3)pLysS. Colonies containing the appropriate insert in the
correct orientation were identified by restriction digestion and grown
at 30 °C to an O.D. of 0.6. Expression of the recombinant enzyme was
induced by the additon of 0.4 mM isopropyl
-D-thiogalactopyranoside (IPTG) and the incubation was
continued for a further 3 h at 30 °C. The cells were harvested
by centrifugation for 10 min at 5000g, freeze thawed,
resuspended in 50 mM Tris HCl pH 7.4, 1 mM
EDTA, 10 mM
mercaptoethanol, and disrupted by
sonication. After the addition of 10 mM DTT, the cell
debris was removed by centrifugaion (30000g 20 min) and the
supernatants were assayed. Protein concentration was determined using
Bio-Rad protein reagent (19) and GST activity was assayed
spectrophotometrically by measuring the conjugation of glutathione to
the standard GST substrate, 1-chloro-2,4 dinitrobenzene (CDNB)
(20).
In situ hybridizations
were conducted on A. gambiae ovarian nurse cell polytene
chromosomes as described earlier (21). Separate hybridizations were
conducted using the recombinant bacteriophage clone, Ag_B1, and the
5-GST cDNA clone, aggst1-1.
Alignment of the 155-bp fragment from A. gambiae G3
cDNA, isolated by PCR using degenerate primers for insect class I
GSTs, to the sequence of M. domestica GST-1 (11) indicated
this encoded a partial GST cDNA (Fig. 1). An
antisense oligonucleotide based on the sequence of the PCR product was
used to obtain the 5-end of the cDNA by RACE techniques. A 546-bp
cDNA, aggst1-1, with an open reading frame of 426-bp
was obtained (Fig. 2). This partial cDNA encoded a
protein whose sequence shared 70-75% homology with other insect class
I GSTs, indicating that aggst1-1 is part of a class I GST
gene (Table I). A comparison of the nucleotide sequence
of aggst1-1 with the sequence of the initial 155-bp PCR fragment (Fig. 1) revealed several differences between the two clones,
suggesting that these cDNAs represent different alleles or are the
products of different genes.
|
To determine whether multiple class I GST genes occur in the
insecticide-susceptible G3 strain of A. gambiae, a Southern
blot of EcoRV-, SalI-, and
HindIII-digested genomic DNA was probed with a class I
cDNA (Fig. 3). Multiple bands were detected with all
three restriction enzymes. The size and number of the bands detected
suggests that either aggst1-1 is part of a large gene containing one or more introns or that multiple class I GST genes are
present. In order to decide between these two interpretations, the
genomic organization of class I GST genes from A. gambiae was investigated. To identify the structural gene encoding agGST1-1 and to determine whether this forms part of a contiguous gene family,
an A. gambiae 2La genomic library was screened
with the cDNA probe. Arrangement of the A. gambiae class
I GST genes was studied in the 2La strain rather than G3
since this is the standard reference strain for large scale A. gambiae genome work (15).
Three recombinant bacteriophage clones were isolated, and partial
restriction mapping showed that they spanned a contiguous region
of > 30 kb of A. gambiae 2La genomic DNA.
The clone Ag_B1 was mapped and partially sequenced. A 3-kb
SalI fragment contained two partial GST sequences, and a
5-kb ScaI fragment encompassing the SalI fragment
was then sequenced. Three open reading frames, which encoded GSTs, were
identified within this fragment. The arrangement of these genes is
shown in Fig. 4. Aggst1-3 and
aggst1-4 are thought to be pseudogenes since, based on
homology with other class I GST genes, they are truncated at the
5-end. Aggst1-2 encodes a full-length GST. The nucleotide
sequence and deduced amino acid sequences of aggst1-2 are
shown in Fig. 5. The open reading frame contains no
introns and encodes a 209 amino acid protein with a predicted molecular
mass of 23.5 kDa. Two putative polyadenylation signals were
identified.
The isolation, by 3-RACE PCR techniques, of a full-length open reading
frame of aggst1-2 from A. gambiae G3 larval
cDNA confirmed that this gene is actively transcribed. To establish
whether aggst1-2 encoded a catalytically active GST, the
cDNA was expressed in vitro, and the CDNB conjugating
activity of crude protein homogenates containing the recombinant
protein was compared with similar homogenates containing the
non-recombinant pET vector. Growth of the E. coli cultures
containing the expression constructs at 37 °C resulted in all the
recombinant protein being produced in insoluble inclusion bodies.
Lowering the incubation temperature to 30 °C reduced but did not
eliminate this sequestration, making comparisons between separate
cultures difficult. Nevertheless, the GST activity of replicate
cultures expressing recombinant agGST1-2 was always >15-fold higher
than the GST activity of control cultures. The results of a
representative experiment are shown in Fig. 6.
An alignment of the agGST1-2 amino acid sequence with other insect GSTs was used to calculate percentage similarities (Table I). AgGST1-2 has 41-46% similarity indices with class I GSTs from other insect species but less than 14% similarity indices with class II GSTs, indicating that agGST1-2 is a class I insect GST. The four invariant residues that appear to be of critical importance in ensuring the correct folding of all GSTs (Pro-54, Leu-143, Gly-151, and Asp-158) are conserved in agGST1-2 (indicated with an asterisk in Fig. 5). In addition, agGST1-2 possesses a serine residue near the N terminus (Ser-10) and an asparagine (Asn-48) (shown in bold in Fig. 5) near the invariant proline, both of which are characteristics of insect class I GSTs (22).
To determine the physical location of class I GSTs in A. gambiae, the aggst1-1 cDNA and genomic clones
containing aggst1-2 were independently hybridized to adult
ovarian nurse cell polytene chromosomes. All the probes hybridized to a
single region on chromosome 2R, division 18B (Fig.
7).
The GSTs of A. gambiae are primarily of interest because of their role in DDT resistance. Multiple GSTs have been partially purified from this mosquito, and resistance is associated with both qualitative and quantitative changes in a number of different GST enzymes (13). The biochemical characteristics of these enzymes suggested they were likely to be insect class I GSTs. This prediction is supported by the metabolism of DDT by a recombinant Drosophila class I GST (GST D1) (23), the only expressed GST to date where DDT metabolism has been demonstrated. We have now cloned and sequenced the first class I GST gene, aggst1-2 from A. gambiae. The gene encodes a catalytically active GST as demonstrated by the ability of recombinant agGST1-2 to conjugate glutathione to the general GST substrate, CDNB. The open reading frame of aggst1-2 is uninterrupted by introns, which have also been observed in the Drosophila class I GSTs (24). The aggst1-2 gene is distinct from the aggst2-1 reported earlier (25). The two genes belong to different GST classes as determined by sequence homology. They also map to different regions of the polytene chromosome.
In the ZANDS resistant strain of A. gambiae, although DDT resistance in both adults and larvae is GST based, the same enzymes are not involved in the different life stages (13). The class II GST cDNA, aggst2-1, was isolated from an adult female cDNA library (25). Attempts in our laboratory to isolate aggst2-1 cDNA from A. gambiae larvae were unsuccessful and Northern analysis using an AgGST2-1 probe provided by R. Reiss (University of Irvine, CA) detected an AgGST2-1 transcript in adult mosquitoes but not in larvae. This suggests that AgGST2-1 is an adult specific cDNA. In contrast, the agGST1-2 is clearly expressed in larvae as demonstrated by our ability to amplify it by PCR from larval derived cDNA. This stage specific expression of GSTs is not unique to A. gambiae. In D. melanogaster, certain class I GSTs are not expressed in adults but are expressed in earlier developmental stages (24).
In D. melanogaster, a cluster of class I GSTs, the GST D genes, are closely linked within a 60-kb DNA region. This GST family comprises at least eight genes although two of these are likely to be pseudogenes (24). Similarly, the genes encoding the multiple class I GSTs present in the housefly, Musca domestica, are thought to be arranged sequentially within the genome (26). Southern blots of A. gambiae genomic DNA suggested that multiple class I GST genes are also present in this species. The identification of three overlapping genomic clones spanning an area >30 kb, which were shown by hybridization experiments to contain class I GSTs, indicated that the mosquito genes are also closely linked.
In situ hybridization with A. gambiae class I GST cDNAs
or genomic clones highlighted a single region on chromosome arm 2R. The
failure to detect any other region of complementarity with any of the
probes indicated that the class I GST genes in A. gambiae are arranged sequentially along chromosome 2R. As in
Drosophila, this region also contains pseudogenes as two 5
truncated class I GST genes were found upstream of the full-length
aggst1-1 gene.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Z71480[GenBank] and Z71481[GenBank].