Molecular cloning, biochemical characterization, and partial protective immunity of the heme-binding glutathione S-transferases from the human hookworm Necator americanus

Abstract

Hookworm glutathione S-transferases (GSTs) are critical for parasite blood feeding and survival and represent potential targets for vaccination. Three cDNAs, each encoding a full-length GST protein from the human hookworm Necator americanus (and designated Na-GST-1, Na-GST-2, and Na-GST-3, respectively) were isolated from cDNA based on their sequence similarity to Ac-GST-1, a GST from the dog hookworm Ancylostoma caninum. The open reading frames of the three N. americanus GSTs each contain 206 amino acids with 51% to 69% sequence identity between each other and Ac-GST-1. Sequence alignment with GSTs from other organisms shows that the three Na-GSTs belong to a nematode-specific nu-class GST family. All three Na-GSTs, when expressed in Pichia pastoris, exhibited low lipid peroxidase and glutathione-conjugating enzymatic activities but high heme-binding capacities, and they may be involved in the detoxification and/or transport of heme. In two separate vaccine trials, recombinant Na-GST-1 formulated with Alhydrogel elicited 32 and 39% reductions in adult hookworm burdens (P < 0.05) following N. americanus larval challenge relative to the results for a group immunized with Alhydrogel alone. In contrast, no protection was observed in vaccine trials with Na-GST-2 or Na-GST-3. On the basis of these and other preclinical data, Na-GST-1 is under possible consideration for further vaccine development.

FIG. 1.

(A) Alignment of the deduced amino acid sequences of identified hookworm GSTs. Sequences were aligned using CLUSTAL W and prepared for display using BOXSHADE. Identical amino acids are shaded in black, and similar amino acids in gray. The percentage of sequence identity between any two hookworm GSTs is shown at the end of each sequence. (B) Neighbor-joining tree representing phylogenetic relationships between nematode-specific nu-class GSTs (Ac-GST-1, Na-GST-1, Na-GST-2, Na-GST-3, and Hc-GST-1); representative GSTs from alpha, mu, pi, sigma, omega, and zeta; elongation factors with GST domains; and MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) proteins. Bootstrap values are indicated at the nodes (1,000 replicates). The tree was constructed from a multiple sequence alignment performed using ClustalW and viewed using TreeView. The protein sequences used in the tree include heme-binding Ac-GST-1 (SwissProt accession number AAT37718), Hc-GST-1 (SwissProt accession number AAF81283), and a selection of C. elegans GST sequences referred to by their Wormbase protein codes, which begin with “CE” (http://www.wormbase.org/). Other sequences used are listed below, with their GenBank or SwissProt accession numbers: Lumbricus rubellus alpha GST (LrubGSTA), LRP02027; human alpha GST (homoGSTA1), NP_66583; mouse alpha GST (musGSTA1), NP_032207; Dermatophagoides pteronyssinus mu GST (DerGSTM1), AAB32224.1; Fasciola hepatica mu GST (FhepGSTM27), P31670; human mu GST (homoGSTM1) CAG46666; mouse mu GST (musGSTM1), AAH91763; Unio tumidus pi GST (UtumGSTP), AAX20373.1; human pi GST (homoGSTP1), AAH10915; mouse pi GST (musGSTP1), P19157; Onchocerca volvulus pi GST (OvolGSTP), AAA53575; Xenopus laevis sigma GST (XlaeGSTS), AAH53774.1; Gallus gallus sigma GST (chickGSTS), NP_990342.1; Bombyx mori sigma GST (BmorGSTS), BAD911071; human sigma GST (homoGSTPGD2), NP_055300; mouse sigma GST (musGSTPGD2), NP_062328; Drosophila melanogaster sigma GST (DmelGSTS), AAF57901; Octopus vulgaris S-crystallins, CAA52850.1; Ommastrephes sloani sigma GST (Squid S-crystallins), P46088; human zeta GST (homoGSTZ1), AAH31777; mouse zeta GST (musGSTZ1), NP_034493; D. melanogaster zeta GST (DmelGSTZ1), AAC28280.2; B. mori zeta GST (BmorGSTZ4), ABC79691; Schistosoma mansoni omega GST (SmanGSTO), AA049385; B. mori omega GST (BmorGSTO2), NP_001037406; human omega GST (homoGSTO1), NP_004823; mouse omega GST (musGSTO1), AAH85165; Strongylocentrotus purpuratus theta GST (SpurGSTT1), XP_790223.1; human theta GST (homoGSTT1), NP_000844; mouse theta GST (musGSTT1), NP_032211; S. purpuratus elongation factor 1B (SpurEF1B), NP_001020382; D. melanogaster elongation factor 1B (DmelEF1B), NP_504808; human elongation factor 1G (homoEF1G), NP_001396; mouse elongation factor 1G (musEF1G), AAH99413; D. melanogaster microsomal GST-like protein (DmelMAPEG), AAC98692.1; S. purpuratus predicted microsomal GST-like protein (SpurMAPEG1), XP_793864; human microsomal GST-like protein (homoMAPEG1), NP_002404; and mouse microsomal GST-like protein (musMAPEG1), NP_064330.

FIG. 2.

SDS-PAGE of purified recombinant Na-GST-1, Na-GST-2, and Na-GST-3. Two micrograms of each recombinant protein was loaded.

FIG. 3.

Developmental transcription of Na-gst-1, Na-gst-2, and Na-gst-3 mRNAs. RT-PCR was performed on total RNA isolated from L3 (lane 1) and adult worms (lane 2) of N. americanus. Specific primers for Na-gst-1, Na-gst-2, and Na-gst-3 were used for amplification. Primers (PKA3-1/PKA5-4) for protein kinase A were used as a positive control. Distilled water (dH₂O) was included instead of DNA for each PCR set as a negative (no template) control.

FIG. 4.

Developmental expression of N. americanus GST proteins identified with specific rabbit antisera absorbed with the other two Na-GST recombinant proteins. (A) Rabbit anti-Na-GST-1 serum absorbed with rNa-GST-2 and rNa-GST-3. (B) Rabbit anti-Na-GST-2 serum absorbed with rNa-GST-1 and rNa-GST-3. (C) Rabbit anti-Na-GST-3 serum absorbed with rNa-GST-1 and rNa-GST-2. Five-micrograms of extracts of L3 (lane 1), adult (lane 2), and ES products of adult N. americanus (lane 3) were homogenized in SDS-PAGE sample buffer, subjected to electrophoresis, and transferred to polyvinylidene fluoride membrane. Five nanograms of rNa-GST-1, rNa-GST-2, rNa-GST-3, rAc-GST-1, and nonrelevant rNa-ASP-2 proteins were used as controls.

FIG. 5.

Visualization of GST subunits of Necator americanus adult worms using two-dimensional gel electrophoresis and Western blot analysis. (A) Two-dimensional gel electrophoresis of N. americanus whole extracts. GST proteins are circled in red. (B) Western blot of adult cytosolic extract with rabbit anti-Ac-GST-1 serum.

FIG. 6.

Immunolocalization of Na-GST-1, Na-GST-2, and Na-GST-3 in sections of adult N. americanus. (A) Fluorescence detection with specific antisera followed by Cy3-conjugated anti-rabbit IgG serum. (B) Immunoelectron micrograph (EM) showing expression of Na-GST-1 in the basal layer of the cuticle (cu) and hypodermis (hy) of adult worms using rabbit anti-rNa-GST-1 followed by gold-labeled anti-rabbit IgG.

FIG. 7.

Plot showing geometric mean arbitrary units (GMU) of IgG against rNa-GST-1, rNa-GST-2, and rNa-GST-3 detected at different time points of the vaccine study. V, vaccination; C, larval challenge; N, necropsy. Results for hamsters injected with Alhydrogel only were set as adjuvant control.