Molecular Cloning, Biochemical Characterization, and Partial ...

INFECTION AND IMMUNITY, Apr. 2010, p. 1552–1563 0019-9567/10/$12.00 doi:10.1128/IAI.00848-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

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Molecular Cloning, Biochemical Characterization, and Partial Protective Immunity of the Heme-Binding Glutathione S-Transferases from the Human Hookworm Necator americanus䌤† Bin Zhan,1* Samirah Perally,2 Peter M. Brophy,2 Jian Xue,3 Gaddam Goud,1 Sen Liu,1 Vehid Deumic,1 Luciana M. de Oliveira,1 Jeffrey Bethony,1 Maria Elena Bottazzi,1 Desheng Jiang,1 Portia Gillespie,1 Shu-hua Xiao,3 Richi Gupta,1 Alex Loukas,4 Najju Ranjit,4 Sara Lustigman,5 Yelena Oksov,5 and Peter Hotez1* Department of Microbiology, Immunology and Tropical Medicine, The George Washington University, and Sabin Vaccine Institute, Washington, DC 200371; Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Aberystwyth, Wales SY23 3DA, United Kingdom2; National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai 200025, China3; Queensland Institute of Medical Research, Brisbane, Queensland 4006, Australia4; and Department of Virology and Parasitology, The Lindsley F. Kimball Research Institute of the New York Blood Center, New York, New York 100215 Received 28 July 2009/Returned for modification 24 August 2009/Accepted 6 January 2010

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. is also a leading cause of iron deficiency anemia in Latin America, especially Brazil (10), and Southeast Asia (47). Currently, the control of hookworm infection in developing countries depends on “deworming,” i.e., periodic anthelminthic treatment, typically with either single-dose albendazole or mebendazole (61). However, low efficacy of single-dose mebendazole has been demonstrated in the control of hookworm infection (6, 20, 35), possibly due to drug resistance (1) and/or rapid reinfection after treatment (2). These concerns have prompted international efforts to identify molecules that play crucial roles in the establishment of hookworm infection in hosts as targets for developing therapeutic and preventive vaccines (22, 31). The glutathione S-transferases (GSTs) are a versatile protein superfamily involved in cellular detoxification by either catalyzing toxin conjugation with glutathione (GSH) or passively binding to a wide range of endogenous/exogenous toxic molecules, including carcinogens, therapeutic drugs, environmental toxins, and products of oxidative stress (57). Parasite GSTs are believed to be involved in the detoxification of endogenously produced toxic compounds or host immune-initiated reactive oxygen species (ROS), as well as the transportation or metabolism of a variety of essential materials for

Human hookworm (Necator americanus) infection is considered one of the most important parasitic diseases of humans in developing countries, with up to 740 million cases worldwide (21) and resulting in as many as 22 million disability-adjusted life-years lost annually (18). The major pathology occurs as a consequence of adult hookworms that feed on blood in the human small intestine (52). The resulting chronic loss of blood represents a leading cause of iron deficiency anemia among populations in which hookworm is endemic, particularly for children and pregnant women (9, 30). For instance, among school-aged children in Zanzibar, an estimated 41% of iron deficiency anemia and 57% of moderate to severe anemia was attributable to hookworm infection (55). Hookworm infection

* Corresponding author. Mailing address: Department of Microbiology and Tropical Medicine, The George Washington University and Sabin Vaccine Institute, Ross Hall 736, 2300 Eye St. NW, Washington, DC 20037. Phone: (202) 994-3532. Fax: (202) 994-2913. E-mail for Bin Zhan: [email protected]. E-mail for Peter Hotez: mtmpjh@gwumc .edu. 䌤 Published ahead of print on 9 February 2010. † The authors have paid a fee to allow immediate free access to this article. 1552

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CLONING AND CHARACTERIZATION OF NECATOR AMERICANUS GSTs

parasites (57). Due to their critical roles in parasite-host interactions, GSTs have been targeted for pharmaceutical and vaccine purposes and have demonstrated protective effects against some parasites (11, 17, 34). For instance, a GST from schistosomes is currently a lead vaccine candidate for human schistosomiasis caused by Schistosoma haematobium (7) and Schistosoma mansoni (4) and is undergoing phase II and phase III clinical trials (15, 41). Our previous studies have identified a nematode-specific class of parasitic GSTs exhibiting a high affinity for heme (59, 69). X-ray structural data suggest that heme binding is due to the association of tetrapyrrole compounds with a large hydrophobic binding pocket that forms during homodimerization of two hookworm GST subunits (3). These properties are considered to be an adaptation to either nematode blood-feeding activity or heme transportation and utilization, since nematodes are heme auxotrophic (51). When hookworms and other parasitic nematodes feed on blood, they lyse red blood cells and then degrade hemoglobin by an ordered cascade (63). Heme is released during the breakdown of host hemoglobin. Since heme is a potent enzyme inhibitor and generator of toxic ROS (44), it is possible that blood-feeding parasitic nematodes, like hookworms, employ GST proteins in heme detoxification (69). In contrast, insoluble cofactors like heme must also be transported by lipophilic proteins, such as GSTs, in order to be incorporated in essential enzymes, such as cytochrome c, peroxidases, and the numerous globins known to be present in nematode proteomes (45). Thus, blood-feeding worms like hookworms need to maintain a cytotoxic-metabolic requirement balance, and to this end, GST is a potential heme regulator. A GST expressed from the canine hookworm Ancylostoma caninum was shown to exhibit a strong affinity for heme, and vaccination with a recombinant form of this GST, Ac-GST-1, protected both dogs against A. caninum larval challenge infection (39% worm burden reduction) and hamsters against N. americanus larval challenge infection (51 to 54% worm burden reduction) (65, 69). Here, we report the cloning and recombinant expression of three GSTs, Na-GST-1, Na-GST-2, and Na-GST-3, from the human hookworm N. americanus, based on their sequence similarities to Ac-GST-1. In addition to biochemical characterization of the three Na-GSTs and confirmation of their heme-binding properties, we report on the protective immunity resulting from vaccination with recombinant Na-GST-1 adjuvanted with Alhydrogel in two vaccine trials conducted in a permissive hamster model of N. americanus and suggest a plausible mechanism by which Na-GST-1 vaccinations could elicit protective immunity. MATERIALS AND METHODS Immunoscreening of N. americanus cDNA library with anti-Ac-GST-1 sera. A total of 4 ⫻ 105 phages of an N. americanus third-stage larva (L3) cDNA ␭ZapII library (68) were immunoscreened with the rabbit antiserum against Ac-GST-1, a GST produced by A. caninum that induced protection against hookworm larva challenge in dog and hamster vaccine trials (69), by a method described previously (70). DNA cloning, sequencing, and analysis. PCR products of the positive clones were subjected to double-stranded DNA sequencing using vector flanking primers corresponding to the T3 and T7 promoter regions. The 5⬘ end of Na-gst-3 cDNA was isolated from first-strand cDNA of adult N. americanus by a modified RNA ligase-mediated rapid amplification technique (GeneRacer; Invitrogen) us-

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ing Na-gst-3 gene-specific primers as described previously (70). Nucleotide and deduced amino acid sequences were compared to existing sequences in GenBank by BLAST searching (http://www.ncbi.nlm.nih.gov). The software used for sequence analysis was ESEE version 3.1 (14). Sequences were aligned using CLUSTAL W (http://clustalw.genome.ad.jp) and prepared for display using BOXSHADE (http: //bioweb.pasteur.fr/seqanal/interfaces/boxshade-simple.html). Phylogenetic neighbor-joining bootstrap trees were produced and viewed using TreeView (48). RT-PCR. Reverse transcription-PCR (RT-PCR) was used to determine the life stages in which Na-gst-1, Na-gst-2, and Na-gst-3 mRNAs were transcribed, as described previously (67). The specific primers based on the nucleotide sequences of Na-gst-1 (bp 6 to 626), Na-gst-2 (bp 2 to 622), and Na-gst-3 (bp 28 to 648) were designed, synthesized, and employed to amplify their corresponding cDNAs from reverse-transcribed mRNAs of L3 and the adult stage of N. americanus. Primers (PKA3-1/PKA5-4) for the untranslated region of a constitutively expressed house-keeping gene of hookworm protein kinase A (PKA) (29) were used as a control. Expression and purification of the recombinant proteins. Full-length cDNA of Na-gst-1, Na-gst-2, and Na-gst-3 were cloned in-frame into the eukaryotic expression vector pPICZ␣A (Invitrogen), and the recombinant proteins were expressed in Pichia pastoris strain X33 by methanol induction and purified with SP-Sepharose FF cation exchange chromatography as described previously (26, 43). The purity of each GST molecule was confirmed by SDS-PAGE. Western blotting, two-dimensional gel electrophoresis, and immunolocalization. Polyclonal antisera against recombinant Na-GST-1, Na-GST-2, and NaGST-3 proteins were prepared in rabbits as previously described (70). In order to remove the antibodies that recognize cross-reacting epitopes among the hookworm GSTs, each polyclonal rabbit antiserum was absorbed twice with the other two recombinant N. americanus GSTs immobilized on nitrocellulose membranes. Each absorbed rabbit anti-Na-GST serum was then used to determine whether the corresponding native protein was present in larval or adult hookworms, by Western blotting as previously described (5, 58). The somatic extracts of N. americanus adult worms were also separated by two-dimensional gel electrophoresis (25, 59) and silver-stained or electrotransferred for Western blotting as described above. For immunolocalization studies, adult A. caninum worm sections were probed with each specific rabbit antiserum and then with Cy3-conjugated anti-rabbit IgG (BD Biosciences), as described previously (62). The fixed worms were also processed for immunoelectron microscopy using gold particle-labeled anti-rabbit IgG (Amersham Biosciences) as described previously (40). Assessment of enzymatic activity. The enzyme activities of recombinant NaGST-1 (rNa-GST-1), rNa-GST-2, and rNa-GST-3 were determined according to the method of Habig and Jakoby (27) with 1-chloro-2,4-dinitrobenzene (CDNB) and a panel of other model and potential natural GST substrates. The reaction of catalyzing the conjugation of reduced GSH was initiated by the addition of the substrates. The change in absorbance due to the formation of the glutathione conjugate was recorded once every minute at 25°C. The enzyme activity was expressed as nmol/min/mg protein. Ligand binding assays. Ligand binding to hookworm GSTs was determined by measuring changes in intrinsic protein fluorescence, as described previously (59). In the binding assays, 1 ␮M recombinant hookworm GST was added to 20 mM potassium phosphate buffer (pH 6.5) containing 100 mM sodium chloride at 25°C. Changes in fluorescence were recorded with a Shimadzu spectrofluorometer (RF-5301 PC) with excitation and emission wavelengths for intrinsic protein fluorescence (tryptophan) of 280 and 320 nm, respectively. Increasing concentrations of hematin and protoporphyrin IX were added and incubated for 3 min prior to measurement. Hamster vaccine trials. Pichia-derived recombinant Na-GST-1, Na-GST-2, and Na-GST-3 were tested in two separate trials for the protection they afforded against N. americanus L3 challenge in an adapted hamster model established at the Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (IPD-CCDCP) (66), under an approved protocol as described previously (65, 69). Because of limited capacity in the animal facility, which could not hold more than 60 hamsters at a time, Na-GST-1 and Na-GST-2/-3 were tested separately against an adjuvant-only negative control under the same conditions in each trial. Golden hamsters aged 5 weeks were obtained from the Shanghai Animal Center, Chinese Academy of Sciences. A total of 25 ␮g of each recombinant Necator GST formulated with Alhydrogel was used to immunize each of 20 hamsters intramuscularly once every 2 weeks for a total of three injections as previously described (65). Another group of 20 hamsters were immunized only with Alhydrogel as a negative control. One week after the last immunization, all the hamsters were challenged subcutaneously with 250 N. americanus L3. The L3 used for challenging hamsters were derived from the hamster model described previously (65, 66). Twenty-five days postchallenge, the

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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,

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hamsters in each group were euthanized and the adult hookworms were recovered from the intestines. The t test was used for statistical analysis. Measurement of humoral immune responses of hamsters. Hamsters were bled prior to each vaccination. The sera were separated and used to measure the levels of the three anti-hookworm GST IgG antibodies by a modified indirect enzyme-linked immunosorbent assay as described previously (32). A standard reference calibration curve of hamster serum was made from combined aliquots of sera from recombinant Na-GST-1-immunized hamsters by 4-parameter logistic log modeling using SoftMax Pro 5.0 (Molecular Devices). Anti-Na-GST-1, -Na-GST-2, and -Na-GST-3 IgG levels in hamster sera were calculated by interpolating the optical density reading at 492 nm into the standard reference calibration curve to obtain the corresponding arbitrary units using SoftMax Pro 5.0. Nucleotide sequence accession numbers. The full-length nucleotide sequences of Na-gst-1, Na-gst-2, and Na-gst-3 have been submitted to GenBank with accession numbers FJ711440, FJ711440, and FJ711440, respectively.

RESULTS Cloning of Na-gst-1, Na-gst-2, and Na-gst-3. A total of 12 positive clones were obtained by immunoscreening 4 ⫻ 105 phage plaques of the N. americanus L3 cDNA expression library with anti-Ac-GST-1 rabbit serum. Sequencing results showed that eight of these clones were identical and encoded an open reading frame that shared 69% amino acid identity to Ac-GST-1 and was therefore named Na-GST-1. Two of the positive clones encoded an open reading frame with 64% amino acid identity to Ac-GST-1 and were designated NaGST-2. One of the positive clones, named Na-GST-3, shared 54% identity to Ac-GST-1. The three Na-GSTs shared 51% to 67% amino acid sequence identity with each other (Fig. 1A). The full-length Na-GST-1, Na-GST-2, and Na-GST-3 proteins contained 206 amino acids with predicted molecular masses/ pIs of 23,679.38 Da/5.74, 23,633.20 Da/8.43, and 23,610.15 Da/ 6.01, respectively. InterPro database searching at EMBL-EBI demonstrated that the three N. americanus GSTs belonged to the GST superfamily, containing typical GST N-terminal domain (IRP004045) and C-terminal domain (IRP004046) structures, including conserved tyrosine residues in the N-terminal domain (Tyr-4 and Tyr-8). Greater variations in amino acid sequences were observed at the putative substrate binding pocket (H-site) located at the C termini in the hookworm GSTs, consistent with interactions between different substrates

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and the large and wide binding cavities found previously on the X-ray crystal structure (3). One putative N-linked glycosylation site (Asn-Ala-Thr) was located between amino acids 164 and 166 of Na-GST-1; none were found in Na-GST-2 and NaGST-3. Phylogenetic analysis based on the alignment of hookworm GSTs with the representative GSTs of different classes demonstrated that hookworm GSTs, as well as other parasitic nematode GSTs, such as Hc-GST1, form a unique nematodespecific class termed the nu class (Fig. 1B) (54). Expression of recombinant Na-GST-1, Na-GST-2, and NaGST-3 in yeast. The full-length recombinant Na-GST-1, NaGST-2, and Na-GST-3 proteins were expressed in Pichia pastoris X-33 following induction with methanol and then purified with SP Sepharose chromatography. In addition, a second QSepharose column chromatographic step was required for the purification of Na-GST-2. The purified recombinant NaGST-1, Na-GST-2, and Na-GST-3 appeared as a single band at approximately 24 kDa by SDS-PAGE (Fig. 2). RT-PCR analysis. A 621-bp cDNA product was amplified from L3 and adult cDNAs of N. americanus for each of the three GSTs (Fig. 3). However, transcription of Na-gst-1 and Na-gst-2 was more abundantly represented in the adult stage than in L3, while Na-gst-3 was transcribed equally in both L3 and the adult stage of N. americanus. As a positive control, the constitutive PKA gene was detected in both stages of hookworm at similar intensities. No product was amplified from the control without template. Detection and immunolocalization of native Na-GST-1, NaGST-2, and Na-GST-3 in adult N. americanus. Cross-reaction among the three Necator GSTs and Ac-GST-1 was observed with sera from rabbits immunized with purified rNa-GST-1, rNa-GST-2, and rNa-GST-3 (data not shown). However, specific rabbit serum absorbed with the other two Necator GSTs showed no cross-reaction. These specific sera were then used to detect the corresponding native protein expressed in different stages of N. americanus. Western blots showed that the absorbed anti-Na-GST-1 rabbit serum recognized a band with an apparent Mr of 24 kDa not only in the extracts of adult worms but also in L3 of N. americanus (Fig. 4A). In L3, another lower band of approximately 14

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.

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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.

kDa was also recognized by anti-Na-GST-1 antibody, indicating the possible presence of a low-molecular-mass GST-1 homologue or partially degraded Na-GST-1. In adult N. americanus excretory-secretory (ES) products, anti-Na-GST-1 strongly recognized two bands with approximate molecular masses of 30 and 32 kDa, as well as the predicted 24-kDa protein. Recognition of Na-GST-1 in adult ES products indicates that NaGST-1 may be secreted by the parasite. The higher-molecularmass forms of Na-GST-1 (30 and 32 kDa) detected in ES products may represent a glycosylated form of the enzyme, given that one N-linked glycosylation site is predicted in the sequence of Na-GST-1. No cross-reaction was observed with other identified hookworm GSTs (Na-GST-2, Na-GST-3, and Ac-GST-1) or an irrelevant recombinant protein, rNa-ASP-2 (26). For Na-GST-2, the absorbed specific serum recognized a strong band only in the adult extracts and in the adult ES products of N. americanus and not in the extracts of L3 (Fig. 4B), indicating that the specific expression of Na-GST-2 is restricted to the adult worm. Na-GST-3 was expressed in both the adult stage and L3 of the worm (Fig. 4C). The weak band of 30 kDa detected in adult ES products for Na-GST-2 and

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-NaGST-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, rNaGST-3, rAc-GST-1, and nonrelevant rNa-ASP-2 proteins were used as controls.

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.

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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.

Na-GST-3 may represent cross-reactivity of residual antibodies against shared epitopes of Na-GST-1. Two-dimensional Western blotting of somatic extracts of adult N. americanus demonstrated that as many as eight forms of GSTs expressed in adult worms were recognized by anti-AcGST-1 antiserum (Fig. 5). This observation may indicate different GSTs or GSTs with different posttranslational modifications. Immunolocalization studies revealed that specific anti-rNaGST-1 antiserum bound strongly to the esophagus, muscle, hypodermis, and gut of adult N. americanus (Fig. 6A). Subsequent immunoelectron microscopic examination with gold-labeled secondary antibody also showed that Na-GST-1 was located in the basal layer of the cuticle and hypodermis of adult worms (Fig. 6B). Specific rabbit anti-Na-GST-2 and Na-GST-3 sera bound strongly to the buccal capsule and weakly to cuticle and gut (Fig. 6A). No significant staining was observed using normal rabbit serum. Functional analysis. The enzymatic activities of recombinant Na-GST-1, Na-GST-2, and Na-GST-3, as well as rAc-

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GST-1, were investigated using a panel of model and potential natural GST substrates. Relatively high GST conjugation activity to the conventional substrate CDNB was observed in the three N. americanus GSTs, confirming their function as GSTs in vitro (Table 1). In each case, the specific activity of the Na-GST was higher than that of Ac-GST-1. However, all three N. americanus GSTs and Ac-GST-1 had limited activities with other model substrates and predicted parasite GST natural substrates (the reactive carbonyls), possibly indicating their minor roles as peroxidases. All N. americanus GSTs exhibited very high heme-/hematinbinding activities. The recombinant Na-GST-1, Na-GST-2, and Na-GST-3, as well as Ac-GST-1, were observed to bind hematin (heme oxidized to the Fe3⫹ state) and its precursor protoporphyrin IX following quenching of intrinsic fluorescence assays. The dissociation constant (Kd) values for hookworm GSTs binding iron-containing hematin were 4 to 25 times higher than those for the structurally related but iron-deficient precursor protoporphyrin IX (Table 2). The 50% inhibitory concentrations (IC50s) of GSH-CDNB conjugation activity were determined for the heme-related compounds and confirmed that GST affinity for hematin was significantly higher than that for its precursor protoporphyrin IX, which lacks an iron center (Table 2). The IC50 of hematin for rNa-GST-2conjugating activity was 3- to 5-fold lower than those of the other hookworm GSTs, implying that Na-GST-2 exhibited the highest affinity for heme. The hookworm GSTs’ heme-binding Kd values were about 10-fold larger than their corresponding IC50s, suggesting that quenching of intrinsic fluorescence detects a heme-binding site distinct from the active site, as seen for Hc-GST-1 from Haemonchus contortus (59). Hamster antibody responses to rNa-GST-1, rNa-GST-2, and rNa-GST-3 immunization. Hamster sera from the second vaccine trial were available for screening of anti-Na-GST IgG response. Vaccination of hamsters with rNa-GST-1 formulated with Alhydrogel induced a strong and persistent IgG response based on the calculated geometric mean arbitrary units (GMU) (Fig. 7). Immunization with rNa-GST-2 and rNa-GST-3 also induced significant specific-IgG responses compared to the response to immunization with the adjuvant-only control (Fig. 7). A higher IgG antibody response was observed for NaGST-1 immunization than Na-GST-2 and Na-GST-3 immunization; however, higher antibody backgrounds were also observed in preimmune hamsters and the adjuvant control group of the Na-GST-1 trial. The highest GMUs were observed after the third vaccination with the three hookworm GSTs. The antibody titer declined after hamsters were challenged with L3. Postchallenge reduction in hookworm burden. Two individual trials were performed under the same conditions. In the first trial, hamsters vaccinated with rNa-GST-1 demonstrated a 32.2% reduction in adult worms recovered from the small intestines in comparison to the number recovered from hamsters immunized with adjuvant alone. The reduction rate was statistically significant (P ⬍ 0.05) (Table 3). No protection was observed in groups vaccinated with rNa-GST-2 or rNa-GST-3. In a second trial, significant worm reduction was also shown in the group vaccinated with rNa-GST-1 (38.6%, P ⬍ 0.05) (Table 3), but only 9.5% worm reduction was observed in the group vaccinated with rNa-GST-3, with no statistical signifi-

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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.

cance. There was no worm reduction seen in the group vaccinated with rNa-GST-2. DISCUSSION It has been more than a decade since glutathione S-transferase (GST) activities were first identified in the extracts and ES products of N. americanus adult worms and the GST proteins were isolated from worm extracts (12, 13). In this study, for the first time, three GSTs were cloned from N. americanus by immunoscreening a cDNA expression library with anti-

serum against Ac-GST-1, a GST from the canine hookworm A. caninum that was also shown to be a protective molecule against L3 challenge in animal vaccine trials (69). Sequence comparison with GSTs from other organisms demonstrated that GSTs from hookworms are from a novel family of nematode-specific GSTs designated the nu class (54). The nematode-specific nu-class GSTs are functionally and structurally different from other classes of GSTs and are characterized by a high-affinity binding site for heme and its related products and limited activity with other substrates (59, 69). The crystal structures of nu-class GSTs contain long/deep and large clefts

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TABLE 1. Specific activities of recombinant N. americanus GSTs toward model and natural GST substrates Amt (mM) of: Substrate

pH Substrate

1-Chloro-2,4-dinitrobenzene Trans-2-nonenal Trans,trans-2,4-decadienal Cumene hydroperoxide Ethacrynic acid 1,2-Dichloro-4-nitrobenzene Trans-4, phenyl-3-buten-2-one 1,2-Epoxy-3-(p-nitrophenoxy) propane 4-Hydroxy-2-nonenal a b c

GSH

␭maxc (nm)

1 0.023 0.023 0.064 0.08 1 0.05 1

1 1 1 1 1 5 0.25 5

6.5 6.5 6.5 7 6.5 7.5 6.5 6.5

340 225 280 340 270 345 290 360

0.1

0.5

6.5

224

Sp act/nmol/min/mg proteinb

⌬εa

Na-GST-1

Na-GST-2

Na-GST-3

Ac-GST-1

⫺1

9.6 ⫻ 10 cm mol 83,940 ⫾ 1,380 11,990 ⫾ 460 12,920 ⫾ 2,240 3,010 ⫾ 120 ⫺19.2 mM⫺1 cm⫺1 224 ⫾ 20 172 ⫾ 9 708 ⫾ 21 288 ⫾ 20 ⫺1 ⫺1 ⫺29.7 mM cm ⬍20 240.6 ⫾ 10.3 709.0 ⫾ 45.3 ⬍20 6 2 ⫺1 6.22 ⫻ 10 cm mol 332 ⫾ 12 25 ⫾ 2 456 ⫾ 17 409 ⫾ 57 5.0 mM⫺1 cm⫺1 306 ⫾ 46 ⬍20 142 ⫾ 50 ⬍20 9.6 ⫻ 106 cm2 mol⫺1 ⬍20 ⬍20 180 ⫾ 20 ⬍20 ⫺1 ⫺1 ⫺24.8 mM cm 58 ⫾ 8 ⬍20 ⬍20 58 ⫾ 5 4.5 mM⫺1 cm⫺1 ND ND ND ND 6

2

13.75 mM⫺1 cm⫺1

1,504 ⫾ 82

1,140 ⫾ 26

690 ⫾ 59

361 ⫾ 2

⌬ε, extinction coefficient. Values are the averages ⫾ standard deviations of 4 replicates. ND, no activity detected under standard assay conditions. ␭max, wavelength of the most intense absorption.

at the H-site compared with those of other GSTS, providing potential sites for heme and other ligands (3, 54). Similar to GSTs from the blood-feeding nematodes H. contortus and A. caninum, the three GSTs produced by N. americanus in this study revealed only limited glutathione-dependent peroxidase activity or conjugating activity toward cytotoxic carbonyl products of lipid peroxidation. Therefore, in contrast to GSTs produced by cestodes and other parasites, it is less likely that hookworm GSTs are involved in the detoxification of reactive oxygen species (ROS) initiated by host immune responses (11, 13). Instead, hookworms and other parasitic nematodes may express GSTs with specific physiological roles involved in blood feeding or transport of lipophilic compounds. The significant heme-binding property of nematode-specific nu-class GSTs suggests that they have specific physiological roles in the detoxification and trafficking of heme or its related compounds produced during blood feeding. Due to its oxidative iron in the molecular structure, free heme is a potent enzyme inhibitor and generator of toxic reactive oxygen species, catalyzing the formation of lipid peroxides and damaging DNA via oxidative stress (44). The nematode-specific hemebiding GSTs, such as the N. americanus GSTs in this study, may act as a carrier to bind/detoxify heme by conjugating it to reduced GSH, thereby protecting nematodes from the attack of ROS induced by the excess free heme. The GST from blood-feeding Plasmodium falciparum has also been shown to bind and detoxify heme compounds (8, 28). Despite its oxidative iron, heme is also a key cofactor for multiple biological processes, including detoxification of endogenous/exogenous

TABLE 2. Kd and IC50s of GSTs for heme-related compoundsa Kd value (␮M) for:

IC50 (␮M) for:

Recombinant GST

Hematin

Protoporphyrin IX

Hematin

Na-GST-1 Na-GST-2 Na-GST-3 Ac-GST-1

3.970 ⫾ 0.3973 2.905 ⫾ 0.1174 3.691 ⫾ 0.2388 2.174 ⫾ 0.1948

25.44 ⫾ 1.415 24.38 ⫾ 1.322 13.78 ⫾ 0.5693 54.38 ⫾ 1.596

0.23 ⫾ 0.01 0.07 ⫾ 0.01 0.32 ⫾ 0.04 0.38 ⫾ 0.01

Protoporphyrin IX 324 ⫾ 15 387 ⫾ 18 14 ⫾ 1

a Values are the averages ⫾ standard deviations of 4 replicates. Dissociation constants (Kd) for heme-related compounds were determined following quenching of intrinsic fluorescence of recombinant hookworm GSTs, and IC50s of heme-related compounds were determined for hookworm GST-catalyzed GSHCDNB conjugation.

toxic molecules (37, 60), biological sensors (53), cellular differentiation (46), protein expression regulation (19, 56), mitochondrial protein transport (36), and protein degradation (50). However, free-living worms and parasitic helminths are unable to synthesize heme even though these worms contain essential hemoproteins (51). Therefore, nematode worms must rely on external heme capture from blood and tissue feeding or ingestion of bacteria and fungi. Analysis of available worm genome databases confirms that nematode genomes lack orthologous genes involved in the heme biosynthetic pathway (33, 51). The human filarial parasitic nematode Brugia malayi requires Wolbachia, an endosymbiotic proteobacterium, to synthesize and provide heme for worm survival. Inhibition of Wolbachia’s heme biosynthesis pathway seriously inhibited and damaged the filarial parasite when cultured in vitro (64). Nematodes need external heme for their important biological functions. However, free heme is toxic and hydrophobic. As such, the nematode-specific heme-binding GSTs, such as the Na-GSTs in this study, may act as soluble carriers to detoxify the free heme and to carry the reduced heme to biological sites. In the absence of a completed genome project for N. americanus, the total number of different GST forms produced by this parasite is not known. By two-dimensional electrophoresis, a total of eight forms of GST were identified in N. americanus extracts, although we cannot rule out the possibility that some of these GSTs represent isoforms or the same GSTs with different posttranslational modifications. The GSTs from N. americanus include the three nematode-specific nu-class GSTs cloned and expressed in this study, but they may also include more-generic GST classes with high peroxidase activity. Indeed, previous studies demonstrated the high levels of glutathione-dependent lipid peroxidase activity in glutathione affinity-purified fractions from adult N. americanus (12, 13). An analysis of the C. elegans genome database reveals more than 40 different GSTs in the free-living worm (16). Some of the C. elegans GSTs show high levels of glutathione-dependent lipid peroxidase activity, while others exhibit high affinity for heme (49, 59), similar to that described here for the hookworm GSTs. The three Na-GSTs exhibited significant similarities in their amino acid sequences (51 to 67% identity) and were highly expressed in adult hookworms, a finding consistent with the

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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.

TABLE 3. Results of vaccine trials with rNa-GST1, rNa-GST2, and rNa-GST3 in hamsters challenged with N. americanus L3

Trial

Vaccine antigen

Mean no. of adult worms ⫾ SD (no. of hamsters) recovered from: Vaccine group

Control group

8.0 ⫾ 5.0 (20)

11.8 ⫾ 4.6 (19)

% Worm reduction

P value

32.2

⬍0.05

1

rNa-GST-1

2

rNa-GST-2 18.3 ⫾ 12.4 (20) 13.9 ⫾ 7.5 (20) rNa-GST-3 13.2 ⫾ 6.7 (20) 13.9 ⫾ 7.5 (20)

3

rNa-GST-1 10.5 ⫾ 8.8 (23)

17.1 ⫾ 6.3 (19)

38.6

⬍0.05

4

rNa-GST-2 14.9 ⫾ 6.3 (19) rNa-GST-3 13.3 ⫾ 7.4 (20)

14.7 ⫾ 10.6 (20) 14.7 ⫾ 10.6 (20)

9.5

⬎0.05

role of these enzymes in blood feeding. However, some important differences were also noted among the three GST proteins. Localization of Na-GST-1 revealed its widespread distribution within parasite tissues, including the cuticle/ hypodermis, muscle, gut, and esophagus. The presence of the enzyme in the gut and esophagus is consistent with its secreted role and possible extracellular detoxification/transporting of heme-related compounds. In addition, the distribution of all three N. americanus GSTs in the cuticle and hypodermis suggests that the worm may acquire exogenous heme or its related compounds not only from gut but also from the surface of the worm, possibly through the connection channels on the cuticle. The cuticular distribution of GST was also observed in the canine hookworm (69). By Western blotting, Na-GST-1 was detectable in both larval and adult extracts, although the mRNA of Na-GST-1 was found to be of low abundance in

VOL. 78, 2010


larvae compared to the level in adults. In contrast to NaGST-1, both Na-GST-2 and Na-GST-3 are localized more specifically to the adult parasite buccal capsule, suggesting a more restricted role for these two enzymes in either attachment or feeding of the adult parasite. The immunolocalization of these two molecules was similar to that previously found for N. americanus C-type lectin (39). Na-GST-2 protein was detected only in adult hookworms, although the mRNA was weakly transcribed in L3. Na-GST-3 mRNA and the Na-GST-3 protein were found in both hookworm larvae and adults. Recently, recombinant Na-GST-3 has been found to induce the synthesis of prostaglandin, an important mediator of inflammation, indicating its potential role in immunomodulation (P. M. Brophy, unpublished data). An important vaccine strategy for the Human Hookworm Vaccine Initiative (HHVI), an international product development partnership with a mission to produce and test recombinant vaccines, relies on targeting adult parasite blood feeding (22, 38). Because of the putative role of Na-GSTs in heme detoxification, the ability of these molecules to function as potential drug or vaccine targets was studied. However, a major hurdle for human hookworm vaccine development is the absence of laboratory animal models that adequately reproduce human infections with N. americanus (22, 38). For instance, canine hookworm infections are permissive only with hookworms of the genus Ancylostoma, e.g., A. caninum and A. ceylanicum, while in hamsters, the number of N. americanus L3 that develop into egg-laying adult hookworms is highly variable and inconsistent. Therefore, the HHVI considers preclinical vaccine testing in dogs and hamsters just one of several criteria for advancing candidates into the clinic, in addition to human immunoepidemiological studies and molecular and immunological mechanisms of action (38). In previous studies, Ac-GST-1 elicited almost 40% worm burden reductions in dogs challenged with A. caninum (69), while in hamsters, Ac-GST-1 immunizations achieved more than 50% protection (65, 69). In this study, immunization of hamsters with recombinant Na-GST-1, Na-GST-2, and NaGST-3 formulated with Alhydrogel resulted in adequate IgG responses. A higher IgG response to immunization with NaGST-1 than to immunization with Na-GST2 and Na-GST3 was observed; however, the background levels in the preimmune sera and the adjuvant control group were also higher in the trial with Na-GST-1. The highest antibody titer was detected after the third immunization with the three Necator GSTs. The antibody titer in the sera of hamsters declined after they were challenged with L3. An antibody titer reduction following larval challenge is a common phenomenon for hookworm vaccine trials (43, 69), putatively because of antibody absorption by specific antigen produced by the challenged hookworms. No efforts were made to further characterize the cytokine responses, although some hamster reagents are beginning to become available (24, 42). Only hamsters vaccinated with rNa-GST-1 achieved protection levels comparable to those reported previously with Ac-GST-1 (65, 69). In contrast, no significant vaccine protection was observed either for Na-GST-2 or Na-GST-3. Further investigations are under way to identify whether the protection of Na-GST-1 immunization is related to neutralization of the enzymatic or heme-binding activities with hamster-specific antibodies and if the lack of

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protection with Na-GST-2 and Na-GST-3 immunization resulted from the insufficient antibody response. Furthermore, a recent study revealed that a related GST from the liver fluke Fasciola hepatica modulates host immunity by suppressing immune responses associated with chronic inflammation to benefit the parasites’ survival within the host (23). Whether hookworm GST is also involved in this protective mechanism is also under investigation. In order to enhance the immune response, further optimization of Na-GSTs for vaccination is needed, including using different adjuvants and immunization routes. Pending these results, the possibility of developing Na-GST-1 as a human hookworm vaccine will undergo further analysis and consideration. ACKNOWLEDGMENTS This study was supported by the Sabin Vaccine Institute’s Human Hookworm Vaccine Initiative, which is funded by the Bill & Melinda Gates Foundation. P. M. Brophy acknowledges the support of the BBSRC, United Kingdom. REFERENCES 1. Albonico, M., Q. Bickle, M. Ramsan, A. Montresor, L. Savioli, and M. Taylor. 2003. Efficacy of mebendazole and levamisole alone or in combination against intestinal nematode infections after repeated targeted mebendazole treatment in Zanzibar. Bull. World Health Organ. 81:343–352. 2. Albonico, M., P. G. Smith, E. Ercole, A. Hall, H. M. Chwaya, K. S. Alawi, and L. Savioli. 1995. Rate of reinfection with intestinal nematodes after treatment of children with mebendazole or albendazole in a highly endemic area. Trans. R. Soc. Trop. Med. Hyg. 89:538–541. 3. Asojo, Q. A., K. Homma, M. Sedlacek, M. Ngamelue, G. N. Goud, B. Zhan, V. Deumic, O. Asojo, and P. J. Hotez. 2007. X-ray structures of Na-GST-1 and Na-GST-2 two glutathione s-transferase from the human hookworm Necator americanus. BMC Struct. Biol. 7:42–53. 4. Balloul, J. M., J. M. Grzych, R. J. Pierce, and A. Capron. 1987. A purified 28,000 dalton protein from Schistosoma mansoni adult worms protects rats and mice against experimental schistosomiasis. J. Immunol. 138:3448–3453. 5. Basavaraju, S., B. Zhan, M. W. Kennedy, Y. Liu, J. Hawdon, and P. J. Hotez. 2003. Ac-FAR-1, a 20 kDa fatty acid- and retinol-binding protein secreted by adult Ancylostoma caninum hookworms: gene transcription pattern, ligand binding properties and structural characterisation. Mol. Biochem. Parasitol. 126:63–71. 6. Bennett, A., and H. Guyatt. 2000. Reducing intestinal nematode infection: efficacy of albendazole and mebendazole. Parasitol. Today 16:71–74. 7. Boulanger, D., A. Warter, B. Sellin, V. Lindner, R. J. Pierce, J. P. Chippaux, and A. Capron. 1999. Vaccine potential of a recombinant glutathione Stransferase cloned from Schistosoma haematobium in primates experimentally infected with a homologous challenge. Vaccine 17:319–326. 8. Boyer, T. D., and E. Olsen. 1991. Role of glutathione S-transferases in heme transport. Biochem. Pharmacol. 42:188–190. 9. Brooker, S., P. J. Hotez, and D. A. P. Bundy. 2008. Hookworm-related anaemia among pregnant women: a systematic review. PLoS Negl. Trop. Dis. 2:e291. 10. Brooker, S., A. Jardim-Botelho, R. J. Quinnell, S. M. Geiger, I. R. Caldas, F. Fleming, P. J. Hotez, R. Correa-Oliveira, L. C. Rodrigues, and J. M. Bethony. 2007. Age-related changes in hookworm infection, anemia and iron deficiency in an area of high Necator americanus hookworm transmission in southeastern Brazil. Trans. R. Soc. Trop. Med. Hyg. 101:146–154. 11. Brophy, P. M., and J. Barrett. 1990. Glutathione transferase in helminths. Parasitology 100:345–349. 12. Brophy, P. M., L. H. Patterson, A. Brown, and D. I. Pritchard. 1995. Glutathione S-transferase (GST) expression in the human hookworm Necator americanus: potential roles for excretory-secretory forms of GST. Acta Trop. 59:259–263. 13. Brophy, P. M., and D. I. Pritchard. 1992. Metabolism of lipid peroxidation products by the gastro-intestinal nematodes Necator americanus, Ancylostoma ceylanicum and Heligmosomoides polygyrus. Int. J. Parasitol. 22:1009– 1012. 14. Cabot, E. L., and A. T. Beckenbach. 1989. Simultaneous editing of multiple nucleic acid and protein sequences with ESEE. Comput. Appl. Biosci. 5:233– 234. 15. Capron, A., G. Riveau, M. Capron, and F. Trottein. 2005. Schistosomes: the road from host-parasite interactions to vaccines in clinical trials. Trends Parasitol. 21:143–149. 16. C. elegans Sequencing Consortium. 1998. Genome sequence of the nematode C. elegans, a platform for investigating biology. Science 282:2012–2018.

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68. Zhan, B., J. Hawdon, Q. Shan, H. Ren, H. Qiang, S. H. Xiao, T. H. Li, Z. Feng, and P. Hotez. 2000. Construction and analysis of cDNA library of the third stage larvae of Necator americanus. Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi 18:26–28. 69. Zhan, B., S. Liu, S. Perally, J. Xue, R. Fujiwara, P. M. Brophy, S. Xiao, Y. Liu, J. Feng, A. Williamson, Y. Wang, L. L. Bueno, S. Mendez, G. Goud, J. M. Bethony, J. M. Hawdon, A. Loukas, K. Jones, and P. J. Hotez. 2005.

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