molecular cloning, biochemical characterization, and ... · tion (39% worm burden reduction) and...

INFECTION AND IMMUNITY, Apr. 2010, p. 1552–1563 Vol. 78, No. 40019-9567/10/$12.00 doi:10.1128/IAI.00848-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Molecular Cloning, Biochemical Characterization, and PartialProtective Immunity of the Heme-Binding Glutathione

S-Transferases from the Human HookwormNecator americanus�†

Bin Zhan,1* Samirah Perally,2 Peter M. Brophy,2 Jian Xue,3 Gaddam Goud,1 Sen Liu,1Vehid 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,4Najju 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; andDepartment 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 andrepresent potential targets for vaccination. Three cDNAs, each encoding a full-length GST protein from thehuman hookworm Necator americanus (and designated Na-GST-1, Na-GST-2, and Na-GST-3, respectively) wereisolated from cDNA based on their sequence similarity to Ac-GST-1, a GST from the dog hookworm Ancylos-toma caninum. The open reading frames of the three N. americanus GSTs each contain 206 amino acids with51% to 69% sequence identity between each other and Ac-GST-1. Sequence alignment with GSTs from otherorganisms shows that the three Na-GSTs belong to a nematode-specific nu-class GST family. All threeNa-GSTs, when expressed in Pichia pastoris, exhibited low lipid peroxidase and glutathione-conjugating enzy-matic activities but high heme-binding capacities, and they may be involved in the detoxification and/ortransport of heme. In two separate vaccine trials, recombinant Na-GST-1 formulated with Alhydrogel elicited32 and 39% reductions in adult hookworm burdens (P < 0.05) following N. americanus larval challenge relativeto the results for a group immunized with Alhydrogel alone. In contrast, no protection was observed in vaccinetrials with Na-GST-2 or Na-GST-3. On the basis of these and other preclinical data, Na-GST-1 is underpossible consideration for further vaccine development.

Human hookworm (Necator americanus) infection is consid-ered one of the most important parasitic diseases of humans indeveloping countries, with up to 740 million cases worldwide(21) and resulting in as many as 22 million disability-adjustedlife-years lost annually (18). The major pathology occurs as aconsequence of adult hookworms that feed on blood in thehuman small intestine (52). The resulting chronic loss of bloodrepresents a leading cause of iron deficiency anemia amongpopulations in which hookworm is endemic, particularly forchildren and pregnant women (9, 30). For instance, amongschool-aged children in Zanzibar, an estimated 41% of irondeficiency anemia and 57% of moderate to severe anemia wasattributable to hookworm infection (55). Hookworm infection

is also a leading cause of iron deficiency anemia in LatinAmerica, especially Brazil (10), and Southeast Asia (47).

Currently, the control of hookworm infection in developingcountries depends on “deworming,” i.e., periodic anthelmin-thic treatment, typically with either single-dose albendazole ormebendazole (61). However, low efficacy of single-dose me-bendazole has been demonstrated in the control of hookworminfection (6, 20, 35), possibly due to drug resistance (1) and/orrapid reinfection after treatment (2). These concerns haveprompted international efforts to identify molecules that playcrucial roles in the establishment of hookworm infection inhosts as targets for developing therapeutic and preventive vac-cines (22, 31).

The glutathione S-transferases (GSTs) are a versatile pro-tein superfamily involved in cellular detoxification by eithercatalyzing toxin conjugation with glutathione (GSH) or pas-sively binding to a wide range of endogenous/exogenous toxicmolecules, including carcinogens, therapeutic drugs, environ-mental toxins, and products of oxidative stress (57). ParasiteGSTs are believed to be involved in the detoxification of en-dogenously produced toxic compounds or host immune-initi-ated reactive oxygen species (ROS), as well as the transporta-tion or metabolism of a variety of essential materials for

* Corresponding author. Mailing address: Department of Microbi-ology and Tropical Medicine, The George Washington University andSabin Vaccine Institute, Ross Hall 736, 2300 Eye St. NW, Washington,DC 20037. Phone: (202) 994-3532. Fax: (202) 994-2913. E-mail for BinZhan: [email protected]. E-mail for Peter Hotez: [email protected].

� Published ahead of print on 9 February 2010.† The authors have paid a fee to allow immediate free access to this

article.

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parasites (57). Due to their critical roles in parasite-host inter-actions, GSTs have been targeted for pharmaceutical and vac-cine purposes and have demonstrated protective effects againstsome parasites (11, 17, 34). For instance, a GST from schisto-somes is currently a lead vaccine candidate for human schis-tosomiasis caused by Schistosoma haematobium (7) and Schis-tosoma mansoni (4) and is undergoing phase II and phase IIIclinical trials (15, 41).

Our previous studies have identified a nematode-specificclass of parasitic GSTs exhibiting a high affinity for heme (59,69). X-ray structural data suggest that heme binding is due tothe association of tetrapyrrole compounds with a large hydro-phobic binding pocket that forms during homodimerization oftwo hookworm GST subunits (3). These properties are consid-ered to be an adaptation to either nematode blood-feedingactivity or heme transportation and utilization, since nema-todes are heme auxotrophic (51). When hookworms and otherparasitic nematodes feed on blood, they lyse red blood cellsand 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 toxicROS (44), it is possible that blood-feeding parasitic nema-todes, like hookworms, employ GST proteins in heme detoxi-fication (69). In contrast, insoluble cofactors like heme mustalso be transported by lipophilic proteins, such as GSTs, inorder to be incorporated in essential enzymes, such as cyto-chrome c, peroxidases, and the numerous globins known to bepresent in nematode proteomes (45). Thus, blood-feedingworms like hookworms need to maintain a cytotoxic-metabolicrequirement balance, and to this end, GST is a potential hemeregulator.

A GST expressed from the canine hookworm Ancylostomacaninum was shown to exhibit a strong affinity for heme, andvaccination with a recombinant form of this GST, Ac-GST-1,protected both dogs against A. caninum larval challenge infec-tion (39% worm burden reduction) and hamsters against N.americanus larval challenge infection (51 to 54% worm burdenreduction) (65, 69). Here, we report the cloning and recombi-nant expression of three GSTs, Na-GST-1, Na-GST-2, andNa-GST-3, from the human hookworm N. americanus, basedon their sequence similarities to Ac-GST-1. In addition tobiochemical characterization of the three Na-GSTs and con-firmation of their heme-binding properties, we report on theprotective immunity resulting from vaccination with recombi-nant Na-GST-1 adjuvanted with Alhydrogel in two vaccinetrials conducted in a permissive hamster model of N. america-nus and suggest a plausible mechanism by which Na-GST-1vaccinations could elicit protective immunity.

MATERIALS AND METHODS

Immunoscreening of N. americanus cDNA library with anti-Ac-GST-1 sera. Atotal of 4 � 105 phages of an N. americanus third-stage larva (L3) cDNA �ZapIIlibrary (68) were immunoscreened with the rabbit antiserum against Ac-GST-1,a GST produced by A. caninum that induced protection against hookworm larvachallenge in dog and hamster vaccine trials (69), by a method described previ-ously (70).

DNA cloning, sequencing, and analysis. PCR products of the positive cloneswere subjected to double-stranded DNA sequencing using vector flanking prim-ers corresponding to the T3 and T7 promoter regions. The 5� end of Na-gst-3cDNA was isolated from first-strand cDNA of adult N. americanus by a modifiedRNA ligase-mediated rapid amplification technique (GeneRacer; Invitrogen) us-

ing Na-gst-3 gene-specific primers as described previously (70). Nucleotide anddeduced amino acid sequences were compared to existing sequences in GenBank byBLAST searching (http://www.ncbi.nlm.nih.gov). The software used for sequenceanalysis 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 neigh-bor-joining bootstrap trees were produced and viewed using TreeView (48).

RT-PCR. Reverse transcription-PCR (RT-PCR) was used to determine thelife stages in which Na-gst-1, Na-gst-2, and Na-gst-3 mRNAs were transcribed, asdescribed previously (67). The specific primers based on the nucleotide se-quences of Na-gst-1 (bp 6 to 626), Na-gst-2 (bp 2 to 622), and Na-gst-3 (bp 28 to648) were designed, synthesized, and employed to amplify their correspondingcDNAs from reverse-transcribed mRNAs of L3 and the adult stage of N. ameri-canus. Primers (PKA3-1/PKA5-4) for the untranslated region of a constitutivelyexpressed house-keeping gene of hookworm protein kinase A (PKA) (29) wereused as a control.

Expression and purification of the recombinant proteins. Full-length cDNA ofNa-gst-1, Na-gst-2, and Na-gst-3 were cloned in-frame into the eukaryotic expres-sion vector pPICZ�A (Invitrogen), and the recombinant proteins were expressedin Pichia pastoris strain X33 by methanol induction and purified with SP-Sepha-rose FF cation exchange chromatography as described previously (26, 43). Thepurity of each GST molecule was confirmed by SDS-PAGE.

Western blotting, two-dimensional gel electrophoresis, and immunolocaliza-tion. Polyclonal antisera against recombinant Na-GST-1, Na-GST-2, and Na-GST-3 proteins were prepared in rabbits as previously described (70). In order toremove the antibodies that recognize cross-reacting epitopes among the hook-worm GSTs, each polyclonal rabbit antiserum was absorbed twice with the othertwo recombinant N. americanus GSTs immobilized on nitrocellulose membranes.Each absorbed rabbit anti-Na-GST serum was then used to determine whetherthe corresponding native protein was present in larval or adult hookworms, byWestern blotting as previously described (5, 58). The somatic extracts of N.americanus adult worms were also separated by two-dimensional gel electro-phoresis (25, 59) and silver-stained or electrotransferred for Western blotting asdescribed above.

For immunolocalization studies, adult A. caninum worm sections were probedwith each specific rabbit antiserum and then with Cy3-conjugated anti-rabbit IgG(BD Biosciences), as described previously (62). The fixed worms were alsoprocessed for immunoelectron microscopy using gold particle-labeled anti-rabbitIgG (Amersham Biosciences) as described previously (40).

Assessment of enzymatic activity. The enzyme activities of recombinant Na-GST-1 (rNa-GST-1), rNa-GST-2, and rNa-GST-3 were determined according tothe 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 reactionof catalyzing the conjugation of reduced GSH was initiated by the addition of thesubstrates. The change in absorbance due to the formation of the glutathioneconjugate was recorded once every minute at 25°C. The enzyme activity wasexpressed as nmol/min/mg protein.

Ligand binding assays. Ligand binding to hookworm GSTs was determined bymeasuring changes in intrinsic protein fluorescence, as described previously (59).In the binding assays, 1 �M recombinant hookworm GST was added to 20 mMpotassium phosphate buffer (pH 6.5) containing 100 mM sodium chloride at25°C. Changes in fluorescence were recorded with a Shimadzu spectrofluorom-eter (RF-5301 PC) with excitation and emission wavelengths for intrinsic proteinfluorescence (tryptophan) of 280 and 320 nm, respectively. Increasing concen-trations of hematin and protoporphyrin IX were added and incubated for 3 minprior 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 affordedagainst N. americanus L3 challenge in an adapted hamster model established atthe Institute of Parasitic Diseases, Chinese Center for Disease Control andPrevention (IPD-CCDCP) (66), under an approved protocol as described pre-viously (65, 69). Because of limited capacity in the animal facility, which couldnot hold more than 60 hamsters at a time, Na-GST-1 and Na-GST-2/-3 weretested separately against an adjuvant-only negative control under the same con-ditions in each trial. Golden hamsters aged 5 weeks were obtained from theShanghai Animal Center, Chinese Academy of Sciences. A total of 25 �g of eachrecombinant Necator GST formulated with Alhydrogel was used to immunizeeach of 20 hamsters intramuscularly once every 2 weeks for a total of threeinjections as previously described (65). Another group of 20 hamsters wereimmunized only with Alhydrogel as a negative control. One week after the lastimmunization, all the hamsters were challenged subcutaneously with 250 N.americanus L3. The L3 used for challenging hamsters were derived from thehamster 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 andprepared for display using BOXSHADE. Identical amino acids are shaded in black, and similar amino acids in gray. The percentage of sequenceidentity between any two hookworm GSTs is shown at the end of each sequence. (B) Neighbor-joining tree representing phylogenetic relationshipsbetween 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 recov-ered from the intestines. The t test was used for statistical analysis.

Measurement of humoral immune responses of hamsters. Hamsters were bledprior to each vaccination. The sera were separated and used to measure thelevels of the three anti-hookworm GST IgG antibodies by a modified indirectenzyme-linked immunosorbent assay as described previously (32). A standardreference calibration curve of hamster serum was made from combined aliquotsof sera from recombinant Na-GST-1-immunized hamsters by 4-parameter logis-tic 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 byinterpolating the optical density reading at 492 nm into the standard refer-ence calibration curve to obtain the corresponding arbitrary units using Soft-Max Pro 5.0.

Nucleotide sequence accession numbers. The full-length nucleotide sequencesof Na-gst-1, Na-gst-2, and Na-gst-3 have been submitted to GenBank with acces-sion numbers FJ711440, FJ711440, and FJ711440, respectively.

RESULTS

Cloning of Na-gst-1, Na-gst-2, and Na-gst-3. A total of 12positive clones were obtained by immunoscreening 4 � 105

phage plaques of the N. americanus L3 cDNA expression li-brary with anti-Ac-GST-1 rabbit serum. Sequencing resultsshowed that eight of these clones were identical and encodedan open reading frame that shared 69% amino acid identity toAc-GST-1 and was therefore named Na-GST-1. Two of thepositive clones encoded an open reading frame with 64%amino acid identity to Ac-GST-1 and were designated Na-GST-2. One of the positive clones, named Na-GST-3, shared54% identity to Ac-GST-1. The three Na-GSTs shared 51% to67% amino acid sequence identity with each other (Fig. 1A).The full-length Na-GST-1, Na-GST-2, and Na-GST-3 proteinscontained 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-EBIdemonstrated that the three N. americanus GSTs belonged tothe GST superfamily, containing typical GST N-terminal do-main (IRP004045) and C-terminal domain (IRP004046) struc-tures, including conserved tyrosine residues in the N-terminaldomain (Tyr-4 and Tyr-8). Greater variations in amino acidsequences were observed at the putative substrate bindingpocket (H-site) located at the C termini in the hookwormGSTs, consistent with interactions between different substrates

and the large and wide binding cavities found previously on theX-ray crystal structure (3). One putative N-linked glycosylationsite (Asn-Ala-Thr) was located between amino acids 164 and166 of Na-GST-1; none were found in Na-GST-2 and Na-GST-3. Phylogenetic analysis based on the alignment of hook-worm GSTs with the representative GSTs of different classesdemonstrated that hookworm GSTs, as well as other parasiticnematode GSTs, such as Hc-GST1, form a unique nematode-specific class termed the nu class (Fig. 1B) (54).

Expression of recombinant Na-GST-1, Na-GST-2, and Na-GST-3 in yeast. The full-length recombinant Na-GST-1, Na-GST-2, and Na-GST-3 proteins were expressed in Pichia pas-toris X-33 following induction with methanol and then purifiedwith SP Sepharose chromatography. In addition, a second Q-Sepharose column chromatographic step was required for thepurification of Na-GST-2. The purified recombinant Na-GST-1, Na-GST-2, and Na-GST-3 appeared as a single band atapproximately 24 kDa by SDS-PAGE (Fig. 2).

RT-PCR analysis. A 621-bp cDNA product was amplifiedfrom L3 and adult cDNAs of N. americanus for each of thethree GSTs (Fig. 3). However, transcription of Na-gst-1 andNa-gst-2 was more abundantly represented in the adult stagethan in L3, while Na-gst-3 was transcribed equally in both L3and the adult stage of N. americanus. As a positive control, theconstitutive PKA gene was detected in both stages of hook-worm at similar intensities. No product was amplified from thecontrol without template.

Detection and immunolocalization of native Na-GST-1, Na-GST-2, and Na-GST-3 in adult N. americanus. Cross-reactionamong the three Necator GSTs and Ac-GST-1 was observedwith sera from rabbits immunized with purified rNa-GST-1,rNa-GST-2, and rNa-GST-3 (data not shown). However, spe-cific rabbit serum absorbed with the other two Necator GSTsshowed no cross-reaction. These specific sera were then usedto detect the corresponding native protein expressed in differ-ent stages of N. americanus.

Western blots showed that the absorbed anti-Na-GST-1 rab-bit serum recognized a band with an apparent Mr of 24 kDa notonly in the extracts of adult worms but also in L3 of N. ameri-canus (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 glutathionemetabolism) proteins. Bootstrap values are indicated at the nodes (1,000 replicates). The tree was constructed from a multiple sequence alignmentperformed using ClustalW and viewed using TreeView. The protein sequences used in the tree include heme-binding Ac-GST-1 (SwissProtaccession number AAT37718), Hc-GST-1 (SwissProt accession number AAF81283), and a selection of C. elegans GST sequences referred to bytheir Wormbase protein codes, which begin with “CE” (http://www.wormbase.org/). Other sequences used are listed below, with their GenBankor SwissProt accession numbers: Lumbricus rubellus alpha GST (LrubGSTA), LRP02027; human alpha GST (homoGSTA1), NP_66583; mousealpha GST (musGSTA1), NP_032207; Dermatophagoides pteronyssinus mu GST (DerGSTM1), AAB32224.1; Fasciola hepatica mu GST (Fhep-GSTM27), 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; Drosophilamelanogaster sigma GST (DmelGSTS), AAF57901; Octopus vulgaris S-crystallins, CAA52850.1; Ommastrephes sloani sigma GST (SquidS-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. moriomega 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 (musG-STT1), 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. melanogastermicrosomal 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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kDa was also recognized by anti-Na-GST-1 antibody, indi-cating the possible presence of a low-molecular-mass GST-1homologue or partially degraded Na-GST-1. In adult N. ameri-canus excretory-secretory (ES) products, anti-Na-GST-1 stronglyrecognized two bands with approximate molecular masses of30 and 32 kDa, as well as the predicted 24-kDa protein. Rec-ognition of Na-GST-1 in adult ES products indicates that Na-GST-1 may be secreted by the parasite. The higher-molecular-mass forms of Na-GST-1 (30 and 32 kDa) detected in ESproducts may represent a glycosylated form of the enzyme,given that one N-linked glycosylation site is predicted in thesequence of Na-GST-1. No cross-reaction was observed withother identified hookworm GSTs (Na-GST-2, Na-GST-3, andAc-GST-1) or an irrelevant recombinant protein, rNa-ASP-2(26).

For Na-GST-2, the absorbed specific serum recognized astrong band only in the adult extracts and in the adult ESproducts of N. americanus and not in the extracts of L3 (Fig.4B), indicating that the specific expression of Na-GST-2 isrestricted to the adult worm. Na-GST-3 was expressed in boththe adult stage and L3 of the worm (Fig. 4C). The weak bandof 30 kDa detected in adult ES products for Na-GST-2 and

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 (lane1) 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 setas a negative (no template) control.

FIG. 4. Developmental expression of N. americanus GST proteinsidentified with specific rabbit antisera absorbed with the other twoNa-GST recombinant proteins. (A) Rabbit anti-Na-GST-1 serum ab-sorbed with rNa-GST-2 and rNa-GST-3. (B) Rabbit anti-Na-GST-2serum 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-micro-grams of extracts of L3 (lane 1), adult (lane 2), and ES products ofadult N. americanus (lane 3) were homogenized in SDS-PAGE samplebuffer, subjected to electrophoresis, and transferred to polyvinylidenefluoride membrane. Five nanograms of rNa-GST-1, rNa-GST-2, rNa-GST-3, rAc-GST-1, and nonrelevant rNa-ASP-2 proteins were used ascontrols.



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Na-GST-3 may represent cross-reactivity of residual antibodiesagainst shared epitopes of Na-GST-1.

Two-dimensional Western blotting of somatic extracts ofadult N. americanus demonstrated that as many as eight formsof GSTs expressed in adult worms were recognized by anti-Ac-GST-1 antiserum (Fig. 5). This observation may indicate dif-ferent GSTs or GSTs with different posttranslational modifi-cations.

Immunolocalization studies revealed that specific anti-rNa-GST-1 antiserum bound strongly to the esophagus, muscle,hypodermis, and gut of adult N. americanus (Fig. 6A). Subse-quent immunoelectron microscopic examination with gold-la-beled secondary antibody also showed that Na-GST-1 was lo-cated in the basal layer of the cuticle and hypodermis of adultworms (Fig. 6B). Specific rabbit anti-Na-GST-2 and Na-GST-3sera bound strongly to the buccal capsule and weakly to cuticleand gut (Fig. 6A). No significant staining was observed usingnormal rabbit serum.

Functional analysis. The enzymatic activities of recombi-nant Na-GST-1, Na-GST-2, and Na-GST-3, as well as rAc-

GST-1, were investigated using a panel of model and potentialnatural GST substrates. Relatively high GST conjugation ac-tivity to the conventional substrate CDNB was observed in thethree N. americanus GSTs, confirming their function as GSTsin vitro (Table 1). In each case, the specific activity of theNa-GST was higher than that of Ac-GST-1. However, all threeN. americanus GSTs and Ac-GST-1 had limited activities withother model substrates and predicted parasite GST naturalsubstrates (the reactive carbonyls), possibly indicating theirminor roles as peroxidases.

All N. americanus GSTs exhibited very high heme-/hematin-binding activities. The recombinant Na-GST-1, Na-GST-2, andNa-GST-3, as well as Ac-GST-1, were observed to bind hema-tin (heme oxidized to the Fe3� state) and its precursor proto-porphyrin IX following quenching of intrinsic fluorescence as-says. The dissociation constant (Kd) values for hookwormGSTs binding iron-containing hematin were 4 to 25 timeshigher than those for the structurally related but iron-deficientprecursor protoporphyrin IX (Table 2). The 50% inhibitoryconcentrations (IC50s) of GSH-CDNB conjugation activitywere determined for the heme-related compounds and con-firmed that GST affinity for hematin was significantly higherthan that for its precursor protoporphyrin IX, which lacks aniron center (Table 2). The IC50 of hematin for rNa-GST-2-conjugating activity was 3- to 5-fold lower than those of theother hookworm GSTs, implying that Na-GST-2 exhibited thehighest affinity for heme. The hookworm GSTs’ heme-bindingKd values were about 10-fold larger than their correspondingIC50s, suggesting that quenching of intrinsic fluorescence de-tects a heme-binding site distinct from the active site, as seenfor Hc-GST-1 from Haemonchus contortus (59).

Hamster antibody responses to rNa-GST-1, rNa-GST-2, andrNa-GST-3 immunization. Hamster sera from the second vac-cine trial were available for screening of anti-Na-GST IgGresponse. Vaccination of hamsters with rNa-GST-1 formulatedwith Alhydrogel induced a strong and persistent IgG responsebased on the calculated geometric mean arbitrary units (GMU)(Fig. 7). Immunization with rNa-GST-2 and rNa-GST-3 alsoinduced significant specific-IgG responses compared to theresponse to immunization with the adjuvant-only control (Fig.7). A higher IgG antibody response was observed for Na-GST-1 immunization than Na-GST-2 and Na-GST-3 immuni-zation; however, higher antibody backgrounds were also ob-served in preimmune hamsters and the adjuvant control groupof the Na-GST-1 trial. The highest GMUs were observed afterthe third vaccination with the three hookworm GSTs. Theantibody titer declined after hamsters were challenged with L3.

Postchallenge reduction in hookworm burden. Two individ-ual trials were performed under the same conditions. In thefirst trial, hamsters vaccinated with rNa-GST-1 demonstrated a32.2% reduction in adult worms recovered from the smallintestines in comparison to the number recovered from ham-sters immunized with adjuvant alone. The reduction rate wasstatistically significant (P � 0.05) (Table 3). No protection wasobserved in groups vaccinated with rNa-GST-2 or rNa-GST-3.In a second trial, significant worm reduction was also shown inthe group vaccinated with rNa-GST-1 (38.6%, P � 0.05) (Ta-ble 3), but only 9.5% worm reduction was observed in thegroup vaccinated with rNa-GST-3, with no statistical signifi-

FIG. 5. Visualization of GST subunits of Necator americanus adultworms using two-dimensional gel electrophoresis and Western blotanalysis. (A) Two-dimensional gel electrophoresis of N. americanuswhole extracts. GST proteins are circled in red. (B) Western blot ofadult cytosolic extract with rabbit anti-Ac-GST-1 serum.



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cance. There was no worm reduction seen in the group vacci-nated with rNa-GST-2.

DISCUSSION

It has been more than a decade since glutathione S-trans-ferase (GST) activities were first identified in the extracts andES products of N. americanus adult worms and the GST pro-teins were isolated from worm extracts (12, 13). In this study,for the first time, three GSTs were cloned from N. americanusby 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 moleculeagainst L3 challenge in animal vaccine trials (69). Sequencecomparison with GSTs from other organisms demonstratedthat GSTs from hookworms are from a novel family of nema-tode-specific GSTs designated the nu class (54). The nema-tode-specific nu-class GSTs are functionally and structurallydifferent from other classes of GSTs and are characterized bya high-affinity binding site for heme and its related productsand limited activity with other substrates (59, 69). The crystalstructures of nu-class GSTs contain long/deep and large clefts

FIG. 6. Immunolocalization of Na-GST-1, Na-GST-2, and Na-GST-3 in sections of adult N. americanus. (A) Fluorescence detection withspecific antisera followed by Cy3-conjugated anti-rabbit IgG serum. (B) Immunoelectron micrograph (EM) showing expression of Na-GST-1 inthe 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.



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at the H-site compared with those of other GSTS, providingpotential sites for heme and other ligands (3, 54). Similar toGSTs from the blood-feeding nematodes H. contortus and A.caninum, the three GSTs produced by N. americanus in thisstudy revealed only limited glutathione-dependent peroxidaseactivity or conjugating activity toward cytotoxic carbonyl prod-ucts of lipid peroxidation. Therefore, in contrast to GSTs pro-duced by cestodes and other parasites, it is less likely thathookworm GSTs are involved in the detoxification of reactiveoxygen species (ROS) initiated by host immune responses (11,13). Instead, hookworms and other parasitic nematodes mayexpress GSTs with specific physiological roles involved inblood feeding or transport of lipophilic compounds.

The significant heme-binding property of nematode-specificnu-class GSTs suggests that they have specific physiologicalroles in the detoxification and trafficking of heme or its relatedcompounds produced during blood feeding. Due to its oxida-tive iron in the molecular structure, free heme is a potentenzyme inhibitor and generator of toxic reactive oxygen spe-cies, catalyzing the formation of lipid peroxides and damagingDNA via oxidative stress (44). The nematode-specific heme-biding GSTs, such as the N. americanus GSTs in this study, mayact as a carrier to bind/detoxify heme by conjugating it toreduced GSH, thereby protecting nematodes from the attackof ROS induced by the excess free heme. The GST fromblood-feeding Plasmodium falciparum has also been shown tobind and detoxify heme compounds (8, 28). Despite its oxida-tive iron, heme is also a key cofactor for multiple biologicalprocesses, including detoxification of endogenous/exogenous

toxic molecules (37, 60), biological sensors (53), cellular dif-ferentiation (46), protein expression regulation (19, 56), mito-chondrial protein transport (36), and protein degradation (50).However, free-living worms and parasitic helminths are unableto synthesize heme even though these worms contain essentialhemoproteins (51). Therefore, nematode worms must rely onexternal heme capture from blood and tissue feeding or inges-tion of bacteria and fungi. Analysis of available worm genomedatabases confirms that nematode genomes lack orthologousgenes involved in the heme biosynthetic pathway (33, 51). Thehuman filarial parasitic nematode Brugia malayi requires Wol-bachia, an endosymbiotic proteobacterium, to synthesize andprovide heme for worm survival. Inhibition of Wolbachia’sheme biosynthesis pathway seriously inhibited and damagedthe filarial parasite when cultured in vitro (64). Nematodesneed external heme for their important biological functions.However, free heme is toxic and hydrophobic. As such, thenematode-specific heme-binding GSTs, such as the Na-GSTsin this study, may act as soluble carriers to detoxify the freeheme and to carry the reduced heme to biological sites.

In the absence of a completed genome project for N. ameri-canus, the total number of different GST forms produced bythis parasite is not known. By two-dimensional electrophoresis,a total of eight forms of GST were identified in N. americanusextracts, although we cannot rule out the possibility that someof these GSTs represent isoforms or the same GSTs withdifferent posttranslational modifications. The GSTs from N.americanus include the three nematode-specific nu-class GSTscloned and expressed in this study, but they may also includemore-generic GST classes with high peroxidase activity. In-deed, previous studies demonstrated the high levels of gluta-thione-dependent lipid peroxidase activity in glutathione affin-ity-purified fractions from adult N. americanus (12, 13). Ananalysis of the C. elegans genome database reveals more than40 different GSTs in the free-living worm (16). Some of the C.elegans GSTs show high levels of glutathione-dependent lipidperoxidase activity, while others exhibit high affinity for heme(49, 59), similar to that described here for the hookwormGSTs.

The three Na-GSTs exhibited significant similarities in theiramino acid sequences (51 to 67% identity) and were highlyexpressed in adult hookworms, a finding consistent with the

TABLE 1. Specific activities of recombinant N. americanus GSTs toward model and natural GST substrates

SubstrateAmt (mM) of:

pH �maxc

(nm) �εaSp act/nmol/min/mg proteinb

Substrate GSH Na-GST-1 Na-GST-2 Na-GST-3 Ac-GST-1

1-Chloro-2,4-dinitrobenzene 1 1 6.5 340 9.6 � 106 cm2 mol1 83,940 1,380 11,990 460 12,920 2,240 3,010 120Trans-2-nonenal 0.023 1 6.5 225 19.2 mM1 cm1 224 20 172 9 708 21 288 20Trans,trans-2,4-decadienal 0.023 1 6.5 280 29.7 mM1 cm1 �20 240.6 10.3 709.0 45.3 �20Cumene hydroperoxide 0.064 1 7 340 6.22 � 106 cm2 mol1 332 12 25 2 456 17 409 57Ethacrynic acid 0.08 1 6.5 270 5.0 mM1 cm1 306 46 �20 142 50 �201,2-Dichloro-4-nitrobenzene 1 5 7.5 345 9.6 � 106 cm2 mol1 �20 �20 180 20 �20Trans-4, phenyl-3-buten-2-one 0.05 0.25 6.5 290 24.8 mM1 cm1 58 8 �20 �20 58 51,2-Epoxy-3-(p-nitrophenoxy)

propane1 5 6.5 360 4.5 mM1 cm1 ND ND ND ND

4-Hydroxy-2-nonenal 0.1 0.5 6.5 224 13.75 mM1 cm1 1,504 82 1,140 26 690 59 361 2

a �ε, extinction coefficient.b Values are the averages standard deviations of 4 replicates. ND, no activity detected under standard assay conditions.c �max, wavelength of the most intense absorption.

TABLE 2. Kd and IC50s of GSTs for heme-related compoundsa

RecombinantGST

Kd value (�M) for: IC50 (�M) for:

Hematin ProtoporphyrinIX Hematin Protoporphyrin

IX

Na-GST-1 3.970 0.3973 25.44 1.415 0.23 0.01 324 15Na-GST-2 2.905 0.1174 24.38 1.322 0.07 0.01 387 18Na-GST-3 3.691 0.2388 13.78 0.5693 0.32 0.04 14 1Ac-GST-1 2.174 0.1948 54.38 1.596 0.38 0.01

a Values are the averages standard deviations of 4 replicates. Dissociationconstants (Kd) for heme-related compounds were determined following quench-ing of intrinsic fluorescence of recombinant hookworm GSTs, and IC50s ofheme-related compounds were determined for hookworm GST-catalyzed GSH-CDNB conjugation.



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role of these enzymes in blood feeding. However, someimportant differences were also noted among the three GSTproteins. Localization of Na-GST-1 revealed its widespreaddistribution within parasite tissues, including the cuticle/hypodermis, muscle, gut, and esophagus. The presence of theenzyme in the gut and esophagus is consistent with its secretedrole and possible extracellular detoxification/transporting ofheme-related compounds. In addition, the distribution of allthree N. americanus GSTs in the cuticle and hypodermis sug-gests that the worm may acquire exogenous heme or its relatedcompounds not only from gut but also from the surface of theworm, possibly through the connection channels on the cuticle.The cuticular distribution of GST was also observed in thecanine hookworm (69). By Western blotting, Na-GST-1 wasdetectable in both larval and adult extracts, although themRNA of Na-GST-1 was found to be of low abundance in

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 timepoints of the vaccine study. V, vaccination; C, larval challenge; N, necropsy. Results for hamsters injected with Alhydrogel only were set as adjuvantcontrol.

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

Trial Vaccineantigen

Mean no. of adult worms SD(no. of hamsters) recovered

from:% Wormreduction

Pvalue

Vaccine group Control group

1 rNa-GST-1 8.0 5.0 (20) 11.8 4.6 (19) 32.2 �0.05

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) 14.7 10.6 (20)rNa-GST-3 13.3 7.4 (20) 14.7 10.6 (20) 9.5 �0.05



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larvae compared to the level in adults. In contrast to Na-GST-1, both Na-GST-2 and Na-GST-3 are localized more spe-cifically to the adult parasite buccal capsule, suggesting a morerestricted role for these two enzymes in either attachment orfeeding of the adult parasite. The immunolocalization of thesetwo molecules was similar to that previously found for N.americanus C-type lectin (39). Na-GST-2 protein was detectedonly in adult hookworms, although the mRNA was weaklytranscribed in L3. Na-GST-3 mRNA and the Na-GST-3 pro-tein were found in both hookworm larvae and adults. Recently,recombinant Na-GST-3 has been found to induce the synthesisof prostaglandin, an important mediator of inflammation, in-dicating its potential role in immunomodulation (P. M. Bro-phy, unpublished data).

An important vaccine strategy for the Human HookwormVaccine Initiative (HHVI), an international product develop-ment partnership with a mission to produce and test recombi-nant vaccines, relies on targeting adult parasite blood feeding(22, 38). Because of the putative role of Na-GSTs in hemedetoxification, the ability of these molecules to function aspotential drug or vaccine targets was studied. However, a ma-jor hurdle for human hookworm vaccine development is theabsence of laboratory animal models that adequately repro-duce human infections with N. americanus (22, 38). For in-stance, canine hookworm infections are permissive only withhookworms of the genus Ancylostoma, e.g., A. caninum and A.ceylanicum, while in hamsters, the number of N. americanus L3that develop into egg-laying adult hookworms is highly variableand inconsistent. Therefore, the HHVI considers preclinicalvaccine testing in dogs and hamsters just one of several criteriafor advancing candidates into the clinic, in addition to humanimmunoepidemiological studies and molecular and immuno-logical mechanisms of action (38).

In previous studies, Ac-GST-1 elicited almost 40% wormburden reductions in dogs challenged with A. caninum (69),while in hamsters, Ac-GST-1 immunizations achieved morethan 50% protection (65, 69). In this study, immunization ofhamsters with recombinant Na-GST-1, Na-GST-2, and Na-GST-3 formulated with Alhydrogel resulted in adequate IgGresponses. A higher IgG response to immunization with Na-GST-1 than to immunization with Na-GST2 and Na-GST3 wasobserved; however, the background levels in the preimmunesera and the adjuvant control group were also higher in thetrial with Na-GST-1. The highest antibody titer was detectedafter the third immunization with the three Necator GSTs. Theantibody titer in the sera of hamsters declined after they werechallenged with L3. An antibody titer reduction followinglarval challenge is a common phenomenon for hookwormvaccine trials (43, 69), putatively because of antibody ab-sorption by specific antigen produced by the challengedhookworms. No efforts were made to further characterizethe cytokine responses, although some hamster reagents arebeginning to become available (24, 42). Only hamsters vacci-nated with rNa-GST-1 achieved protection levels comparableto those reported previously with Ac-GST-1 (65, 69). In con-trast, no significant vaccine protection was observed either forNa-GST-2 or Na-GST-3. Further investigations are under wayto identify whether the protection of Na-GST-1 immunizationis related to neutralization of the enzymatic or heme-bindingactivities with hamster-specific antibodies and if the lack of

protection with Na-GST-2 and Na-GST-3 immunization re-sulted from the insufficient antibody response. Furthermore, arecent study revealed that a related GST from the liver flukeFasciola hepatica modulates host immunity by suppressing im-mune responses associated with chronic inflammation to ben-efit the parasites’ survival within the host (23). Whether hook-worm GST is also involved in this protective mechanism is alsounder 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-1as a human hookworm vaccine will undergo further analysisand consideration.

ACKNOWLEDGMENTS

This study was supported by the Sabin Vaccine Institute’s HumanHookworm Vaccine Initiative, which is funded by the Bill & MelindaGates Foundation. P. M. Brophy acknowledges the support of theBBSRC, United Kingdom.

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