La-aied Prapanthadara

High-throughput assays for detection of the F1534C mutation in the voltage-gated sodium channel gene in permethrin-resistant Aedes aegypti and the distribution of this mutation throughout Thailand.

Objectives  To develop rapid monitoring tools to detect the F1534C permethrin‐resistance mutation in domain IIIS6 of the Aedes aegypti voltage‐gated sodium channel gene and determine the frequency and distribution of this mutation in Thailand.

Identification, characterization and structure of a new Delta class glutathione transferase isoenzyme

The insect GST (glutathione transferase) supergene family encodes a varied group of proteins belonging to at least six individual classes. Interest in insect GSTs has focused on their role in conferring insecticide resistance. Previously from the mosquito malaria vector Anopheles dirus, two genes encoding five Delta class GSTs have been characterized for structural as well as enzyme activities. We have obtained a new Delta class GST gene and isoenzyme from A. dirus, which we name adGSTD5-5. The adGSTD5-5 isoenzyme was identified and was only detectably expressed in A. dirus adult females. A putative promoter analysis suggests that this GST has an involvement in oogenesis. The enzyme displayed little activity for classical GST substrates, although it possessed the greatest activity for DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] observed for Delta GSTs. However, GST activity was inhibited or enhanced in the presence of various fatty acids, suggesting that the enzyme may be modulated by fatty acids. We obtained a crystal structure for adGSTD5-5 and compared it with other Delta GSTs, which showed that adGSTD5-5 possesses an elongated and more polar active-site topology.

Purification and characterization of a major glutathione S-transferase from the mosquito Anopheles dirus (Species B)

The major form of glutathione S-transferase (GST) activity from the mosquito Anopheles dirus (species B), a vector of malaria in Thailand has been purified 421-fold. It constituted approx. 20% of the total measured CDNB conjugating activity in the homogenate. This enzyme appeared as a single band of 25.0 +/- 0.26 kDa on SDS-PAGE and was kinetically characterized with 10 substrates and 4 inhibitors. The enzyme is capable of catalysing dehydrochlorination of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) in vitro at a rate of 4.4 nmol of 1,1-dichloro-2,2-bis-(p-chlorophenyl)ethane (DDE) formation per mg protein. This is comparable to the rate of catalysis of the orthologous isoenzyme from An. gambiae reported previously. The IC50 plots of the inhibitor data (fractional velocity vs log [I]) for three of the inhibitors indicate the homogenous nature of this enzyme. However, inhibition by ethacrynic acid demonstrates more than a single affinity site for interaction. The six N-terminal amino acids of the purified enzyme are identical to a GST reported from Aedes aegypti, which was indicated to play a role in DDT-resistance in this species. The results suggest that the two enzymes may belong to the same class, however each possesses a different specificity.

Heterologous expression and characterization of alternatively spliced glutathione S-transferases from a single Anopheles gene.

Three cDNA sequences of glutathione S-transferase (GST), adgst1-2, adgst1-3 and adgst1-4, which are alternatively spliced products of the adgst1AS1 gene, were obtained from fourth instar larvae of Anopheles dirus mosquito by reverse transcriptase PCR reactions. The nucleotide sequences of these three cDNAs share >67% identity and the translated amino acid sequences share 61-64% identity. A comparison of the An. dirus to the An. gambiae enzymes shows that adGST1-2 versus agGST1-4, adGST1-3 versus agGST1-5 and adGST1-4 versus agGST1-3 have 85, 92 and 85% amino acid sequence identity, respectively, which confirms that orthologous isoenzymes occur across anopheline species. These three proteins were expressed at high levels, approximately 15-20 mg from 200 ml of E. coli culture. The recombinant enzymes were purified by affinity chromatography on an S-hexylglutathione agarose column. The subunit sizes of adGST1-2, adGST1-3 and adGST1-4 are 24.3, 23.9 and 25.1 kDa. The recombinant enzymes have high activities with 1-chloro-2,4-dinitrobenzene (CDNB), detectable activity with 1,2-dichloro-4-nitrobenzene but markedly low activity with ethacrynic acid and p-nitrophenethyl bromide. adGST1-3 was shown to be the most active enzyme from the kinetic studies. Permethrin inhibition of CDNB activity, at varying concentrations of CDNB, was significantly different, being uncompetitive for adGST1-2, noncompetitive for adGST1-3 and competitive for adGST1-4. In contrast, permethrin inhibition with varying glutathione concentrations was noncompetitive for all three GSTs. Despite the enzymes being splicing products of the same gene and sharing identical sequence in the N-terminal 45 amino acids, these GSTs show distinct substrate specificities, kinetic properties and inhibition properties modulated by the differences in the C-terminus.

CLONING, EXPRESSION AND CHARACTERIZATION OF AN INSECT CLASS I GLUTATHIONE S-TRANSFERASE FROM ANOPHELES DIRUS SPECIES B

Insect class I glutathione S-transferases (GSTs) were expressed from cDNA obtained from larvae of the Thai malaria vector. Anopheles dirus in a PCR RACE (rapid amplification of cDNA ends) reaction using a primer to the conserved N-terminal region of An. gambiae class I GSTs and a synthetic oligo d(T)-adaptor primer. Seven different plasmids, resulting from sub-cloning of an original single 0.7 Kb PCR band, were picked at random and sequenced. Four of these were clearly GSTs on the basis of putative amino acid sequence conservation. All the sequences had a conserved N-terminal region, but were highly divergent at the C-terminus. The variability in the PCR products suggests that there is a high level of GST class I isoenzyme variability in larval An. dirus. One of the subclones from the PCR reaction contained a full coding region of the cDNA for GST. This had a putative amino acid sequence which was 76 and 91% identity to the An. gambiae GST class I, agGST 1-5 and agGST 1-6 respectively, but only 48% identity to agGST 1-2. The catalytically active enzyme, expressed in Escherichia coli, was strongly immuno-cross reactive with antisera raised against the two An. gambiae class I GSTs. The expressed enzyme was purified to homogeneity from an E. coli cell lysate by S-hexylglutathione agarose affinity chromatography. The enzyme had a high specific activity with CDNB, and also used DCNB and ethacrynic acid as substrates. In addition, it had peroxidase and DDTase activity and its activity with CDNB, was strongly inhibited by a range of organophosphorus and pyrethroid insecticides. This is consistent with the predicted role of this GST class in insecticide resistance.

Isoenzymes of glutathione S-transferase from the mosquito Anopheles dirus species B: the purification, partial characterization and interaction with various insecticides

Previously we have purified and characterized a major glutathione S-transferase (GST) activity, GST-4a, from the Thai mosquito Anopheles dirus B, a model mosquito for study of anopheline malaria vectors [Prapanthadara, L. Koottathep, S., Promtet, N., Hemingway, J. and Ketterman, A.J. (1996) Insect Biochem. Mol. Biol. 26:3, 277-285]. In this report we have purified an isoenzyme, GST-4c, which has the greatest DDT-dehydrochlorinase activity. Three additional isoenzymes, GST-4b, GST-5 and GST-6, were also partially purified and characterized for comparison. All of the Anopheles GST isoenzymes preferred 1-chloro-2,4-dinitrobenzene (CDNB) as an electrophilic substrate. In kinetic studies with CDNB as an electrophilic substrate, the V(max) of GST-4c was 24.38 micromole/min/mg which was seven-fold less than GST-4a. The two isoenzymes also possessed different K(m)s for CDNB and glutathione. Despite being only partially pure GST-4b had nearly a four-fold greater V(max) for CDNB than GST-4c. In contrast, GST-4c possessed the greatest DDT-dehydrochlorinase specific activity among the purified insect GST isoenzymes and no activity was detected for GST-5. Seven putative GST substrates used in this study were not utilized by An. dirus GSTs, although they were capable of inhibiting CDNB conjugating activity to different extents for the different isoenzymes. Bromosulfophthalein and ethacrynic acid were the most potent inhibitors. The inhibition studies demonstrate different degrees of interaction of the An. dirus isoenzymes with various insecticides. The GSTs were inhibited more readily by organochlorines and pyrethroids than by the phosphorothioates and carbamate. In a comparison between An. dirus and previous data from An. gambiae the two anopheline species possess a similar pattern of GST isoenzymes although the individual enzymes differ significantly at the functional level. The available data suggests there may be a minimum of three GST classes in anopheline insects.

Single amino acid changes outside the active site significantly affect activity of glutathione S-transferases.

Glutathione S-transferases (GSTs: E.C. 2.5.1.18) are a multigene family of multifunctional dimeric proteins that play a central role in detoxication. Four allelic forms of the mosquito Anopheles dirus GST, adGST1-1, were cloned, expressed and characterized. The one or two amino acid changes in each allelic form was shown to confer different kinetic properties. Based on an available crystal structure, several of the residue changes were not in the putative substrate-binding pocket. Modeling showed that these insect Delta class GSTs also possess a hydrophobic surface pocket reported for Alpha, Mu and Pi class GSTs. The atom movement after replacement and minimization showed an average atom movement of about 0.1 A for the 0 to 25 A distance from the alpha carbon of the single replaced residue. This does not appear to be a significant movement in a static modeled protein structure. However, 200-500 atoms were involved with movements greater than 0.2 A. Dynamics simulations were performed to study the effects this phenomenon would exert on the accessible conformations. The data show that residues affecting nearby responsive regions of tertiary structure can modulate enzyme specificities, possibly through regulating attainable configurations of the protein.

Genomic organization and putative promoters of highly conserved glutathione S-transferases originating by alternative splicing in Anopheles dirus

The genomic DNA of a GST class I alternative splicing gene has been characterized from Anopheles dirus, a Thai malaria vector. This gene organization is highly conserved in An. dirus and Anopheles gambiae (aggst1alpha), with >80% nucleotide identity in the coding region. Their gene organization contains six exons for four mature GST transcripts, which share exon 1 and exon 2 but vary between four different exon 3 sequences (exon 3A-3D). The deduced amino acid sequence of the GST transcripts from these two genes also shows very high conservation, with 85-93% identity for each orthologous gene. Two putative promoters and possible regulatory elements were predicted by a combination of the TSSW and MatInspector programs. The Ad214 promoter is proposed to be involved in developmental stage regulation. The Ad2112 promoter would appear to respond to intra- or extracellular stimuli. These two Anopheline species appear to have diverged in the distant past based on gene neighbors and phylogenetic data, yet these GST genes are still conserved. Therefore it is highly probable that this GST gene organization has one or more important roles.

Selection of Anopheles dirus for refractoriness and susceptibility to Plasmodium yoelii nigeriensis

Two lines of the Oriental malaria vector mosquito Anopheles dirus species A (Diptera: Culicidae), one fully refractory and one fully susceptible to Plasmodium yoelii nigeriensis (an African rodent malaria parasite), were established after 17 generations of mass selection, followed by single female selection for one or two generations. Prior to selection, the stock colony of An. dirus was 17% refractory. Both lines of An. dirus produced abundant ookinetes that started to invade the midgut within 24 h post‐infection, as seen in histological sections. In most of the refractory mosquitoes, oocysts stopped development < 12 h post‐invasion, indicating a rapid defence mechanism. Dead P. y. nigeriensis parasites were apparently localized as small melanized spots (2–5 μm) seen in wet preparations of mosquito midguts dissected 5–7 days post infective bloodmeal. In some refractory An. dirus females, apart from the spots, a small number of totally encapsulated oocysts (c. 10 μm) were also present. These larger melanized parasites predominated in a few females: they appeared 2–3 days post‐infection as a secondary delayed defence mechanism. The progeny of reciprocal matings between susceptible and refractory lines had ∼50% susceptibility. Backcrosses of F1 hybrids with susceptible or refractory lines increased or decreased the susceptibility of backcross progeny accordingly. Overall, these results suggest polygenic control of susceptibility to P. y. nigeriensis infection. The refractory line of An. dirus showed normal susceptibility to natural infections of the human malarias P. falciparum and P. vivax from local patients.

Expression and Characterization of Three New Glutathione Transferases, an Epsilon (AcGSTE2- 2), Omega (AcGSTO1- 1), and Theta (AcGSTT1- 1) From Anopheles cracens (Diptera: Culicidae), a Major Thai Malaria Vector

ABSTRACT Glutathione transferases (GSTs) (E.C.2.5.1.18) are multifunctional enzymes involved in the detoxification of many exogenous and endogenous compounds. This study aimed to characterize several new GSTs from Anopheles cracens, a major Thai malaria vector formerly known as Anopheles dirus. The three recombinant enzymes obtained were from the epsilon, theta and omega classes. They showed 80–93% identity to orthologous An. gambiae GSTs. AcGSTE2-2 possessed peroxidase activity that cannot be detected for the An. gambiae AgGSTE2-2. AcGSTT1-1 had high activity toward several substrates that are specific for mammalian theta class. The AcGSTO1-1 can use 1-chloro-2,4-dinitrobenzene, dichloroacetic acid, and hydroxyethyl disulfide substrates. The enzymes bound but did not metabolize the organophosphate temephos. The epsilon AcGSTE2-2 functioned as a peroxidase and DDT metabolizing enzyme. The theta AcGSTT1-1 functioned not only as peroxidase but also acted as a binding protein for organophosphates. The omega GST had thiol transferase activity suggesting a role in oxidative stress response.