Characterization and expression analysis of a newly identified glutathione S-transferase of the hard tick Haemaphysalis longicornis during blood-feeding

Abstract

Background: Ticks are obligate hematophagous parasites important economically and to health. Ticks consume large amounts of blood for their survival and reproduction; however, large amounts of iron in blood could lead to oxidative stress. Ticks use several molecules such as glutathione S-transferases (GSTs), ferritins, and peroxiredoxins to cope with oxidative stress. This study aimed to identify and characterize the GSTs of the hard tick Haemaphysalis longicornis in order to determine if they have a role in coping with oxidative stress.

Methods: Genes encoding GSTs of H. longicornis were isolated from the midgut CDNA library. Genes have been cloned and recombinant GSTs have been expressed. The enzymatic activities, enzyme kinetic constants, and optimal pH of the recombinant GSTs toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined. The gene transcription and protein expression profiles were determined in the whole ticks and internal organs, and developmental stages using real time RT-PCR and Western blotting during blood feeding. The localization of GST proteins in organs was also observed using immunofluorescent antibody test (IFAT).

Results: We have isolated two genes encoding GSTs (HlGST and HlGST2). The enzymatic activity toward CDNB is 9.75 ± 3.04 units/mg protein for recombinant HlGST and 11.63 ± 4.08 units/mg protein for recombinant HlGST2. Kinetic analysis of recombinant HlGST showed K _m values of 0.82 ± 0.14 mM and 0.64 ± 0.32 mM for the function of CDNB and GSH, respectively. Meanwhile, recombinant HlGST2 has K _m values of 0.61 ± 0.20 mM and 0.53 ± 0.02 mM for the function of CDNB and GSH, respectively. The optimum pH of recombinant HlGST and recombinant HlGST2 activity was 7.5-8.0. Transcription of both GSTs increases in different developmental stages and organs during blood-feeding. GST proteins are upregulated during blood-feeding but decreased upon engorgement in whole ticks and in some organs, such as the midgut and hemocytes. Interestingly, salivary glands, ovaries, and fat bodies showed decreasing protein expression during blood-feeding to engorgement. Varying localization of GSTs in the midgut, salivary glands, fat bodies, ovaries, and hemocytes was observed depending on the feeding state, especially in the midgut and salivary glands.

Conclusions: In summary, a novel GST of H. longicornis has been identified. Characterization of the GSTs showed that GSTs have positive correlation with the degree and localization of oxidative stress during blood-feeding. This could indicate their protective role during oxidative stress.

Keywords: Blood-feeding; Glutathione S-transferases; Haemaphysalis longicornis; Oxidative stress; Tick.

Fig. 1

Multiple sequence alignment of the deduced amino acid sequences of HlGST and HlGST2 with other tick GSTs. Identical residues are shaded black, while similar residues are shaded gray. The percent identities with HlGST and HlGST2 are placed at the end of the sequences. The GenBank accession numbers for GST sequences are as follows: Haemaphysalis longicornis (AAQ74441.1), Dermacentor variabilis (ACF35504.1), Rhipicephalus sanguineus (AGK29895.1), Ixodes scapularis A (XP_002401749.1) and Ixodes scapularis B (XP_002434207.1)

Fig. 2

Phylogenetic tree of GSTs from different species of ticks, selected vertebrates, and invertebrates. A dendogram was created by the neighbor-joining method based on the deduced amino acid sequence of GSTs. Bootstrap values are placed at the nodes. GenBank accession numbers are as follows: Aedes aegypti (Theta), Q16X19; Homo sapiens (Alpha), P08263; Ixodes scapularis A, XP_002401749.1; Ixodes scapularis B, XP_002434207.1; Rattus norvegicus, NP_803175.1; Mus muculus, NP_032209.1; Haemaphysalis longicornis, AAQ7444.1; Dermacentor variabilis, ACF35504.1; Rhipicephalus (Boophilus) annulatus, ABR24785.1; Rhipicephalus sanguineus, AGK29895.1; Anopheles gambiae (Delta), Q8MUS1; Drosophila melanogaster, (Epsilon) Q7KK90; Homo sapiens, (Zeta) O43708; and Proteus mirabilis, (Beta) P15214

Fig. 3

SDS-PAGE (a) and Western blotting (b) of recombinant GSTs. a The column farthest left includes markers of molecular weights. Lane 1: bacterial lysate of empty vector; Lane 2: bacterial lysate of HlGST after induction by 1 mM IPTG; Lane 3: bacterial lysate of HlGST after purification by HisTrap affinity chromatography; Lane 4: bacterial lysate of HlGST2 after induction by 1 mM IPTG; Lane 5: bacterial lysate of HlGST2 after purification by HisTrap affinity chromatography. The lysates were run on a 12% SDS-PAGE gel. Gels were stained using Coomassie Blue staining solution. b The column farthest left indicates molecular weight markers. Lane 1: recombinant HlGST; Lane 2: recombinant HlGST2. Bands were visualized using 5-Bromo-4-chloro-3-indolylphosphate/Nitroblue Tetrazolium (BCIP/NBT) Calbiochem® (Merck KGaA, Darmstadt, Germany). Arrowheads indicate the bands for recombinant HlGST and HlGST2 proteins

Fig. 4

Effect of pH on the enzymatic activity of recombinant HlGST (a) and HlGST2 (b). Buffers were 0.1 M citrate for pH 5.0–5.5 and Tris-HCl for pH 6.5–8.5. Error bars represent the standard deviation of three replicates, and the remaining activities were recorded as percentages relative to the highest activity

Fig. 5

Transcription profiles of GST genes in different stages and tissues of ticks during blood-feeding. Total mRNA was prepared from different stages and tissues after dissection. P0 and L23 primers were used as controls for whole ticks and tissues, respectively. Error bar represents the mean ± standard deviation. ^*P < 0.05, significantly different by Student's t-test as compared to the unfed at the same stage. P-values and t-values are indicated in Additional file 6: Table S1

Fig. 6

Expression profiles of GSTs in different tick stages (a) and organs (b) during blood-feeding. Proteins were prepared from different stages and tissues after dissection. Antiserum against tubulin was used as a control for Western blotting. Western blotting results are shown as representative data of three separate experiments showing the same trend. Since no band can be seen using the tubulin antisera on hemocytes, the protein concentration was determined using Micro BCA and maintained at 390 ng before loading for Western blotting

Fig. 7

Localization of GSTs in tissues of partially fed adult ticks. Immunofluorescent antibody test (IFAT) was used to determine the localization of the GSTs in the different tissues of ticks. Antiserum against HlGST or HlGST2 was used for the primary antibody, anti-mouse IgG conjugated with Alexa 488 was used for the secondary antibody, and nuclei were visualized using DAPI. Normal mouse serum was used for a control. The tissues were visualized using confocal microscope. Abbreviations: Salivary glands (A, acinus; D, salivary ducts); Midgut (E, enterocytes; L, lumen; Fat bodies (T, tracheal complex; Fb, fat body cells); Ovary (O, oocyte; Pd, pedicel; Ow, ovarian wall). Arrows show positive GST fluorescence. Scale-bars: 20 μm

Fig. 8

Examination of HlGST (a) and HlGST2 (b) in selected tissues during blood-feeding. The salivary glands, midgut, and ovary were observed during blood-feeding of adult ticks by indirect immunofluorescent antibody test (IFAT) using a confocal laser scanning microscope. Antiserum against HlGST or HlGST2 was used for the primary antibody, anti-mouse IgG conjugated with Alexa 488 was used for the secondary antibody, and nuclei were visualized using DAPI. Normal mouse serum was used for a control. Abbreviations: Salivary glands (A, acinus; D, salivary ducts); Midgut (E, enterocytes; L, lumen); Ovary (O, oocyte; Pd, pedicel; Ow, ovarian wall). Arrows show the positive fluorescence of GST. Scale-bars: 20 μm