Characterisation of Dermanyssus gallinae glutathione S-transferases and their potential as acaricide detoxification proteins
© Bartley et al. 2015
Received: 5 May 2015
Accepted: 17 June 2015
Published: 26 June 2015
Glutathione S-transferases (GSTs) facilitate detoxification of drugs by catalysing the conjugation of the reduced glutathione (GSH) to electrophilic xenobiotic substrates and therefore have a function in multi-drug resistance. As a result, knowledge of GSTs can inform both drug resistance in, and novel interventions for, the control of endo- and ectoparasite species. Acaricide resistance and the need for novel control methods are both pressing needs for Dermanyssus gallinae, a highly economically important haematophagous ectoparasite of poultry.
A transcriptomic database representing D. gallinae was examined and 11 contig sequences were identified with GST BlastX identities. The transcripts represented by 3 contigs, designated Deg-GST-1, −2 and −3, were fully sequenced and further characterized by phylogenetic analysis. Recombinant versions of Deg-GST-1, −2 and −3 (rDeg-GST) were enzymically active and acaricide-binding properties of the rDeg-GSTs were established by evaluating the ability of selected acaricides to inhibit the enzymatic activity of rDeg-GSTs.
6 of the identified GSTs belonged to the mu class, followed by 3 kappa, 1 omega and 1 delta class molecules. Deg-GST-1 and −3 clearly partitioned with orthologous mu class GSTs and Deg-GST-2 partitioned with delta class GSTs. Phoxim, permethrin and abamectin significantly inhibited rDeg-GST-1 activity by 56, 35 and 17 % respectively. Phoxim also inhibited rDeg-2-GST (14.8 %) and rDeg-GST-3 (20.6 %) activities.
Deg-GSTs may have important roles in the detoxification of pesticides and, with the increased occurrence of acaricide resistance in this species worldwide, Deg-GSTs are attractive targets for novel interventions.
The haematophagous poultry red mite [Dermanyssus gallinae (De Geer, 1778)] is the most economically important parasite affecting commercial egg production facilities worldwide, causing an estimated annual production loss of €130 million in Europe alone [1, 2]. Infestation levels are estimated to range from a “normal” 50,000 mites per bird to over 500,000 mites per bird in severe infestations . The impact on bird health and behaviour can be severe and manifests as agitation and pecking, weight loss and anaemia; with production losses attributed to increase bird mortality, a decrease in egg quality and output, higher feed-conversion rate and mite control costs [2, 4, 5]. In addition, D. gallinae is as a potential vector for a number of diseases, some of which may also impact on human health; for example, Erisipelas and Salmonella spp. [6–11].
Chemical-based mite control is the usual approach employed in commercial premises and several organophosphate, pyrethroid, spinosyn and carbamate acaricides are licensed for use in the EU . Resistance to organophosphates, pyrethroids, carbamates and dichlorodiphenyltrichloroethane (DDT) has been identified in red mite populations across the EU [12–17]. However, the underlying mechanism(s) involved in acaricide detoxification and resistance in D. gallinae remain unknown. In addition to active site mutation, the development of acaricide resistance in other pest species may be attributed to the activities of detoxifying enzymes including members of the glutathione S-transferase (GST), cytochromeP450 (CYP450), and carboxylesterase (CBE) superfamilies and may be multi-factorial in nature . The CYP450 and CBEs function in phase I of detoxification (modification) by catalyzing the addition of polar groups to xenobiotics. Phase II (conjugation) most often involves the GST-catalysed conjugation of reduced glutathione (GSH) to the electrophilic xenobiotic . The glutathione conjugate acts as a flag, targeting the xenobiotic to the membrane transporters, whereupon it is excreted from the cell in phase III of the detoxification process .
GSTs belong to a large superfamily of versatile proteins with important functions in detoxification, amino acid catabolism, signal modulation, transportation . Cytosolic and mitochondrial GSTs are classified in several canonical classes (mu, kappa, alpha, pi, theta, sigma, zeta, omega, lambda, beta, tau, delta and epsilon) dependent on the structure of class-specific active site motifs, active site residues and binding affinities [22, 23].
GSTs are highly important in the detoxification processes in haematophagous parasites, particularly in the elimination of toxic haemoglobin breakdown products [24–26]. GSTs have also been identified as major allergens in several parasite and pest species (e.g. scabies and house dust mites, parasitic nematodes and cockroaches), that provoke pathophysiology associated with allergy [27–31]. In addition, secreted GSTs have been implicated in immune-evasion of the nitric oxide response in helminths . Given their pivotal role in detoxification, drug resistance and host-parasite interaction, GSTs have been the focus of novel control strategies against a number of ecto- and endoparasites [33–38].
The identification of D. gallinae GSTs (Deg-GSTs) will therefore lead to a deeper understanding of acaricide resistance in this species and may lead to novel targeted control methods. We have previously produced a D. gallinae transcriptomic database  and, in the present study, we identify the repertoire of GSTs in D. gallinae by data-mining the database with GST search terms and investigate the interactions of 3 selected Deg-GSTs with acaricides commonly used in the poultry sector to characterise the potential for involvement of the Deg-GSTs in acaricide detoxification.
Generation of D. gallinae transcriptome dataset
Mixed stage and gender D. gallinae mites collected by scraping aggregated mites from the enriched-cage system of a commercial egg-laying unit in Scotland, UK. Mites were allowed to migrate away from the detritus and were then snap frozen in liquid nitrogen within 12 h of collection. Total RNA was purified using Trizol reagent (Life Technologies) as previously described Wright . Roche 454 sequencing was performed (Edinburgh Genomics, University of Edinburgh) following the manufacturer’s protocol [40, 41]. The raw read data were assembled using Newbler (version 2.3) into contig sequences and putative biological functions inferred by homology searching against the NCBI nr database (19 July 2013 release) using BLASTx . Where possible, the D. gallinae trancriptomic dataset was further annotated with Gene Ontology (GO) terms, Enzyme Commission (EC) codes and InterProScan (IPS) identities using the Blast2GO pipeline [43–45].
Amplification and sequencing of selected D. gallinae glutathione S-transferases (Deg-GSTs)
The complete coding sequences of selected Deg-GSTs were obtained using rapid amplification of cDNA ends (RACE) using the SMART™ RACE cDNA amplification kit (Clontech) according to the manufacturer’s touchdown protocol and where necessary or, where full length sequence was already represented in the assembled contigs, by direct amplification from cDNA by PCR using the Advantage® 2 PCR Kit (Clontech). Sequences of cloned cDNAs representing three GSTs, Deg-GST-1, −2 and −3 were determined following cloning in pGEM® T-Easy plasmid (Promega). Homology searching of translated Deg-GST sequences against the UniProtKB database (release 2013/06) was performed using blastp. Additionally, Signal P v4.0  was used to identify putative signal sequences and ScanProsite  to identify functional domains and protein signature sequences in the translated Deg-GST sequences. A selection of proteins with highest homology to the Deg-GSTs were aligned with the translated Deg-GST along with arthropod and eukaryotic GSTs belonging to selected GST classes, using ClustalX v2.0.11 . The constituent proteins were manually truncated at C and N terminus by up to 29 amino acids in order to eliminate regions of extensive gapping and poor alignment. The truncated alignment was imported into the TOPALi v2.5  and the Whelan & Goldman model (WAG) amino acid substitution model was applied to the alignment to calculate distances. A phylogenetic tree was constructed using the MrBayes Bayesian tree method  using default parameters. The integrity of each partition or clade (clade credibility) was assessed using posterior probabilities.
Expression, purification and activity of recombinant Deg-GSTs (rDeg-GSTs)
The complete coding sequences of the Deg-GSTs were subcloned into the pETSUMO champion plasmid vector (Invitrogen, primer sequences and conditions available on request from authors). For recombinant protein expression, purified pETSUMO-Deg-GST plasmid DNAs were transformed into Escherichia coli BL21-CodonPlus® (DE3)-RIL competent cells (Stratagene) and protein expression, purification and quantification performed as described previously . Purified rDeg-GSTs were used for electrophoresis on NuPAGE® Bis-Tris 4-12 % gels under reducing conditions and stained with SimplyBlue™ (Invitrogen). To confirm identity of the rDeg-GSTs, the single visible bands were excised, destained and subjected to reductive alkylation using DTT and iodoacetamide. Gel pieces were digested overnight at 37 °C in trypsin and digests analysed on an Ultraflex II MALDI-ToF-ToF mass spectrometer (Bruker Daltonics). The masses obtained were used for database searching with the MASCOT search engine using Swiss-Prot and local databases with a 50 ppm mass tolerance window. Significant matches from the Peptide Mass Fingerprint data were confirmed by MS/MS analysis using the search criteria above and an MS/MS tolerance window of 0.5 Da.
where the At1 and At2 are the mean adjusted A340nm at the initial and final time points; Δε the molar extinction coefficient of CDNB (Δε =9.6). t2 – t1 is the time (in minutes) between At2 and At1; b is the path length of the spectrophotometer (cm = 0.286); m is the quantity of rDeg-GST per well (in mg) and Vtot is the total volume per well (litres).
Acaricide inhibition assays
5 mM stocks of the acaricides: spinosad, permethrin, phoxim and abamectin (PESTANAL® analytical standards, Sigma) and 50 mM 4-nitrobenzyl chloride (NBC) were prepared in ethanol. Acaricide, NBC (+ve control) or ethanol (no treatment control) was placed (2.4 μl) into triplicate wells of a 96-well microtitre plate and mixed with 20 μl rDeg-GST [containing defined quantities of rDeg-GSTs in Tris/NaCl buffer (10 mM Tris, 0.5 M NaCl, pH 7.4)]. The plate was incubated for 10 mins at R.T. with agitation prior to the addition of 100 μl per well of 1 mM CDNB substrate in substrate buffer. GST activity was measured as detailed above and adjusted for background absorbance of the different acaricides at A340nm. The assay was repeated on 3 separate occasions and the mean enzyme activities analysed using a one-way ANOVA followed by pairwise comparisons using Tukey’s post-hoc analysis to assess significance.
Identification and characterisation of Dermanyssus gallinae GST transcripts
The annotated D. gallinae transcriptome containing 13,363 contiguous sequences (contigs)  was interrogated using glutathione S-transferase keywords and 11 contigs were identified with GST terms. These 11 contigs ranged from 208 to 1030 bp in length and, based on BLASTx descriptions, 6 were putatively identified as mu class, 3 as mitochondrial kappa class, 1 omega and 1 delta class.
The 3 longest contigs with the highest number of constituent sequences and top hit BLASTx e values were selected for further study. The contig forming Deg-GST-1 was 1016 bp in length, comprised of 58 overlapping sequences and had a 48 % identity (over 237 amino acid residues, E = 8e-72) with a putative mu-1 like GST from the highly related predatory mite species Metaseiulus occidentalis (accession number XP_003747409). The 1030 bp contig representing Deg-GST-2 comprised of 33 overlapping sequences and possessed 77 % identity (over 208 amino acid residues, 1.0E-77) with a putative M. occidentalis GST isoform C-like GST (accession number XP_003743487). The contig representing Deg-GST −3 was formed from 5 overlapping sequences, had a total length of 399 bp and a 76 % identity (over 66 amino acid residues, 2.0E-25) with a putative M. occidentalis mu-1 like GST (accession number XP_003742682).
The contigs representing Deg-GST-1 and −2 were both predicted to contain the full coding sequence (CDS) of their respective GSTs (Accession numbers KR337505 and KR337506) and this was confirmed using PCR amplification. The contig representing Deg-GST-3 encoded a partial open reading frame (ORF) and the full length sequence (Accession Number KR337507) was obtained using RACE. The ORF of Deg-GST-1 was 735 bp in length, encoding a 244 amino-acid (aa) protein; the ORF of Deg-GST-2 was 633 bp encoding a 210 aa protein and the ORF of Deg-GST-3 was 669 bp encoding a 222 aa protein.
Deg-GST-2 partitioned into a large clade containing delta and epsilon GSTs. The representative epsilon GST species formed a cluster discrete from the delta GSTs which, in turn, formed one cluster containing mainly acarine-derived sequences and one cluster containing exclusively insect-derived sequences. As with the Deg-GST mu class GSTs, the Deg-GST-2 also partitioned most closely with M. occidentalis.
Interaction of Deg-GSTs with acaricides
The effect of selected acaricides on the ability of rDeg-GSTs to catalyse the conjugation of glutathione to the colorimetric substrate CDNB
% reduction in Deg-GST activity ± SEM
rDeg-GST-1 (0.3 μg GST/well)
rDeg-GST-2 (1.5 μg GST/well)
rDeg-GST-3 (1.5 μg GST/well)
50.84 ± 1.4b
18.1 ± 1.7b
33.3 ± 2.1b
−17.46 ± 3.6
1.7 ± 5.5
−18.8 ± 5.7
35.39 ± 0.9b
0.5 ± 3.0
−0.30 ± 0.3
56.38 ± 3.0b
14.8 ± 1.5a
20.56 ± 2.5a
17.31 ± 2.1a
4.3 ± 4.2
−0.57 ± 9.6
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
In this study we have identified the repertoire of Deg-GSTs in a transcriptomic dataset and selected and 2 mu class and one delta class Deg-GST for further characterisation. We have shown that several commonly used poultry acaricides have the potential to affect the function of recombinant versions of the selected Deg-GSTs. Control of Dermanyssus gallinae is becoming increasingly difficult due to the emergence of acaricide resistance coupled with the legislated withdrawal of previously effective acaricides , for example, the organophosphate fenitrothion is now banned in the UK . In addition, the application of acaricides, sometime illegally, has resulted in acaricide residues in eggs, meat and feed at concentrations greater than the maximum residue limits in the EU and elsewhere [54–56]. Our understanding of the mechanisms involved in acaricide resistance in D. gallinae are in their infancy, though organophosphate resistance has clearly been linked with increased acetylcholinesterase (AChE) activity . Molecular studies underpinning our fundamental understanding of poultry red mite biology were also lacking until very recently, when large quantities of transcriptomic data were generated [39, 58, 59]. In the transcriptomic dataset interrogated here, the most frequent BLASTx term associated with known detoxification pathways was gluthionine S-transferase. Of the 11 contigs with associated GST terms, 6 were putatively ascribed to the cytosolic mu class. The bias towards mu class GSTs in D. gallinae was also evident in the D. gallinae transcriptomic data published by Schichts et al. , where of the 32 isotigs identified with associated GST terms, 22 were putatively classified as mu. The high representation of mu class GSTs is in agreement with genomic analyses of another haematophagous parasitic acarine species, Ixodes scapularis, where in silico genome analysis revealed that mu was the predominant GST class followed by delta, epsilon, omega, zeta and kappa . This is in contrast to the insect species, Tribolium castaneum and Anopheles gambiae, which lack the mu class entirely; with the mu class replaced by an expanded delta class [61, 62]. Delta class GSTs were canonically defined as insect-specific [19, 63], however, delta class GSTs have now been described here from D. gallinae and previously from several other species of the Acari (e.g. Sarcoptes scabiei, I. scapularis [60, 64] and P. ovis [S Burgess, personal communication]. Epsilon and zeta class Deg-GSTs were not identified in the red mite transcriptomic data sets, which was unexpected because they have been identified in other acarine species genomes  and epsilon and delta classes are often expanded in other haematophagous arthropods . However, the apparent absence of these classes from the red mite transcriptomic databases may be due to very low levels of gene expression and thus representation in acarine transcriptomic datasets: for example, the P. ovis transcriptome (12,160 contig/isotig assembly) has only 2 zeta class GST isotigs and no epsilon class GSTs (personal communication S Burgess). Transcripts representing sigma class GSTs were also absent from the D. gallinae transcriptomic data, which is in agreement with other Acari species e.g. the I. scapularis genome  and the P. ovis transcriptome [S. Burgess personal communication].
Phylogenetic analyses of the 3 selected Deg-GSTs consistently placed the Deg-GSTs within the Parasitiformes superorder. Mites belonging to the Acariformes superorder (e.g. the Astigmid mite species P. ovis and S. scabiei) partitioned in a separate clade. In all cases, Deg-GSTs partitioned most closely with M. occidentalis, which is unsurprising as both species belong to the suborder Dermanyssina and M. occidentalis is the most closely taxonomically related species included in the phylogenetic analysis.
All 3 rDeg-GSTs exhibited activity with CDNB; a ubiquitous substrate for all GST classes except theta class , however, rDeg-GST-1 was 18 and 26 times more active than rDeg-GST-2 and rDeg-GST-3 respectively. The variation in Deg-GST activity is likely to be the result of underlying primary sequence differences affecting the affinity for CDNB, as even single amino acid substitutions in or near to the active sites of GSTs have been shown to dramatically alter CDNB binding ability . Variation in GST structure arising from allelic variation also impacts on detoxification in vivo and such allelic variation has been associated with altered binding specificity of GSTs to DDT in the mosquito Anopheles gambiae . The generation of alleleic variants provides a mechanism whereby differential pesticide-binding specificities can arise and result in pesticide-tolerant phenotypes. However, no evidence for GST-allelic variation associated with acaricide resistance in Acarid populations has been reported to date.
Here, we tested the ability of a range of commonly-used poultry sector acaricides to interact with rDeg-GSTs and the greatest reduction in GST activity was measured when rDeg-GST-1 activity was inhibited in the presence of the organophosphate compound phoxim. Phoxim was also the only acaricide that produced a statistically significant reduction in activity in all 3 rDeg-GSTs indicating that the 3 rDeg-GSTs tested here may play a role in phoxim detoxification. Whether the binding between the rDeg-GSTs and phoxim is a functional interaction resulting in a competitive inhibition of GST activity with the CDNB substrate (i.e. phoxim acts as a reducible substrate for GST-mediated GSH conjugation) or whether phoxim is non-competitive inhibitor (i.e. phoxim binds to, and directly inhibits the function of the rDeg-GSTs with conjugation to GSH) is not yet clear. Phoxim, and other organophosphates, irreversibly binds to acetylcholinesterase (AChE), thus preventing the AChE-mediated breakdown of acetylcholine (ACh) and the natural cessation of nervous impulses . The resulting overstimulation of the acarine nervous system eventually leads to paralysis and death. In addition to the interaction of phoxim with Deg-GSTs shown here, several other commercially-available organophosphates have been shown to interact in vitro with GSTs derived from other acarine species [68–70], indicating a potential role for GSTs in the detoxification or inactivation of organophosphate pesticides in the Acari. A five-fold increase in the expression of a GST transcript in the tick Rhipicephalus microplus following exposure to coumaphos [71, 72] and a ten-fold increase in GST activity in organophosphate-resistant Tetranychus urticae mite extract  adds weight to the argument that over-expression of GSTs may play an important role in organophosphate detoxification and resistance in the Acari.
The inhibitory effect of permethrin on rDeg-GST activity was limited to rDeg-GST-1, where a 35 % decrease in activity was observed in the acaricide inhibition assay. The interaction between GSTs and permethrin and the inhibition of GST function has also been identified in other tick and mite species using similar acaricide inhibition assays [68–70]. In addition, the increase in GST transcription in response to sublethal pyrethroid treatments in several mite species [74–76] and the increased susceptibility of Rhipicephalus sanguineus sensu lato ticks to the effects of permethrin when an abundant mu class GST is inhibited via RNAi  indicates an important role of GSTs in the detoxification processes of pyrethroids in the Acari. The exact nature of the interaction of permethrin with GSTs and their role in detoxification remains unclear: the interaction between the insect Tenebrio molitor GST and decamethrin involved direct binding to the enzyme’s active site, but did not result in conjugation of the pesticide with GSH, suggesting sequestration of the decamethrin, thereby preventing it’s interaction with the target ligand . Mutations in and/or increased expression of the effector molecules of detoxification pathways are often associated with pesticide resistance and pyrethroid resistance mediated by esterase and CYP450 over-expression as well as SNPs in the voltage-sensitive sodium channel have all been documented in the Acari [72, 74, 79–81].
The increase in GST gene expression and GST activity in response to treatment with pyrthroids (as discussed above) suggests that increased GST activity is also likely to be part of a multi-factorial detoxification system involved in pyrethroid resistance in the Acari. There have been numerous reports of pyrethroid resistance in D. gallinae [12, 13, 15, 17] and the potential existence of point mutations in GSTs associated with pyrethroid resistance, allowing increased affinity for pesticides, has been explored in other mites, but thus far none have been identified . The mechanism by which GSTs function in pyrethroid resistance may therefore revolve around increased expression of the enzymes rather than alterations in their structure; thus an increase in the transcription of 6 GSTs in the citrus red mite (Panonychus citri) following exposure to a sublethal dose of fenpropathrin . In S. scabiei, an increase in GST activity in permethrin-resistant mites when compared to permethrin-sensitive mites is associated with mite survival following exposure to permethrin [74, 75]. Furthermore, when pyrethroid-resistant scabies mites were treated with the GST inhibitor and acaricide synergist diethyl malate (DEM), the resistant phenotype was abolished and sensitivity to permethrin restored .
Spinosad (Elector®) has recently been registered for use in poultry pest control and has shown to be very effective against all motile stages of D. gallinae [83, 84]. No significant change in activity of the 3 rDeg-GSTs was observed following incubation with spinosad suggesting that these particular Deg-GSTs do not have high affinity for this acaricide. Abamectin is a macrocyclic lactone-containing pesticide, which was approved for commercial D. gallinae control in the UK in 2012 . The effect of abamectin on rDeg-GST activity was limited to 17 % reduction in rDeg-GST-1 activity, and, although spinosad and abamectin do not appear to interact to any great extent with the rDeg-GSTs tested here; this does not preclude the possibility of other Deg-GSTs functioning in the detoxification of these compounds.
We have shown that mu and delta class GSTs are present in D. gallinae and interact with phoxim, permethrin and abamectin acaricide compounds and they therefore may have important roles in the detoxification of several classes of pesticides. To further characterise the role(s) of Deg-GSTs in detoxification and resistance, it would be constructive to perform in vivo studies with mites quantifying the Deg-GST activity levels and gene expression profiles following exposure to acaricides in mite populations with and without defined acaricide exposure and resistance. Identifying Deg-GSTs involved in the detoxification of acaricides may allow the development of targeted treatment design to disrupt detoxification and hinder the mites’ ability to survive acaricide treatment. Selectively targeting and disrupting Deg-GST function could therefore potentially work in synergy with existing acaricides to increase their efficacy and prolong their use in the field by overcoming resistance.
The authors gratefully acknowledge funding for this project from the British Egg Marketing Board Trust and from DEFRA (grant number AW 00186). We would like to also acknowledge continued support from Mr. John Campbell, Glenrath Farms Ltd. for his continued enthusiasm and help in our red mite research. We would also like to thank Professor Mark Blaxter (Edinburgh Genomics) for the 454 transcriptome sequencing, The Moredun Proteomics Group for the MALDI-ToF-ToF and Dr Stewart Burgess for the P. ovis transcriptome data.
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