Comprehensive analysis of the secreted proteome of adult Necator americanus hookworms.

The human hookworm Necator americanus infects more than 400 million people worldwide, contributing substantially to the poverty in these regions. Adult stage N. americanus live in the small intestine of the human host where they inject excretory/secretory (ES) products into the mucosa. ES products have been characterized at the proteome level for a number of animal hookworm species, but until now, the difficulty in obtaining sufficient live N. americanus has been an obstacle in characterizing the secretome of this important human pathogen. Herein we describe the ES proteome of N. americanus and utilize this information along with RNA Seq data to conduct the first proteogenomic analysis of a parasitic helminth, significantly improving the available genome and thereby generating a robust description of the parasite secretome. The genome annotation resulted in a revised prediction of 3,425 fewer genes than initially reported, accompanied by a significant increase in the number of exons and introns, total gene length and the percentage of the genome covered by genes. Almost 200 ES proteins were identified by LC-MS/MS with SCP/TAPS proteins, 'hypothetical' proteins and proteases among the most abundant families. These proteins were compared to commonly used model species of human parasitic infections, including Ancylostoma caninum, Nippostrongylus brasiliensis and Heligmosomoides polygyrus. SCP/TAPS proteins are immunogenic in nematode infections, so we expressed four of those identified in this study in recombinant form and showed that they are all recognized to varying degrees by serum antibodies from hookworm-infected subjects from a disease-endemic area of Brazil. Our findings provide valuable information on important families of proteins with both known and unknown functions that could be instrumental in host-parasite interactions, including protein families that might be key for parasite survival in the onslaught of robust immune responses, as well as vaccine and diagnostic targets.

Randomized, Placebo Controlled Trial of Experimental Hookworm Infection for Improving Gluten Tolerance in Celiac Disease.

INTRODUCTION: Celiac disease is an autoimmune disorder where intestinal immunopathology arises after gluten consumption. Previous studies suggested that hookworm infection restores gluten tolerance; however, these studies were small (n = 12) and not placebo controlled. METHODS: We undertook a randomized, placebo-controlled trial of hookworm infection in 54 people with celiac disease. The 94-week study involved treatment with either 20 or 40 Necator americanus third-stage larvae (L3-20 or L3-40) or placebo, followed by escalating gluten consumption (50 mg/d for 12 weeks, 1 g intermittent twice weekly for 12 weeks, 2 g/d sustained for 6 weeks, liberal diet for 1 year). RESULTS: Successful study completion rates at week 42 (primary outcome) were similar in each group (placebo: 57%, L3-20: 37%, and L3-40: 44%; P = 0.61), however gluten-related adverse events were significantly reduced in hookworm-treated participants: Median (range) adverse events/participant were as follows: placebo, 4 (1-9); L3-20, 1 (0-9); and L3-40, 0 (0-3) (P = 0.019). Duodenal villous height:crypt depth deteriorated similarly compared with their enrolment values in each group (mean change [95% confidence interval]: placebo, -0.6 [-1.3 to 0.2]; L3-20, -0.5 [-0.8 to 0.2]; and L3-40, -1.1 [-1.8 to 0.4]; P = 0.12). A retrospective analysis revealed that 9 of the 40 L3-treated participants failed to establish hookworm infections. Although week 42 completion rates were similar in hookworm-positive vs hookworm-negative participants (48% vs 44%, P = 0.43), quality of life symptom scores were lower in hookworm-positive participants after intermittent gluten challenge (mean [95% confidence interval]: 38.9 [33.9-44] vs 45.9 [39.2-52.6]). DISCUSSION: Hookworm infection does not restore tolerance to sustained moderate consumption of gluten (2 g/d) but was associated with improved symptom scores after intermittent consumption of lower, intermittent gluten doses.

Lipopeptide-Based Oral Vaccine Against Hookworm Infection.

BACKGROUND: The human hookworm, Necator americanus, is a parasite that infects almost half a billion people worldwide. Although treatment is available, vaccination is favorable to combat the spread of this parasite due to its wide distribution and continuous reinfection cycle in endemic communities. METHODS: We have designed a lipopeptide oral delivery system using a B-cell epitope derived from the aspartic protease Na-APR-1 from N americanus, attached to a T-helper epitope. Lipopeptides were self-assembled into nanoparticles or entrapped in liposomes that were electrostatically coated with alginate and trimethyl chitosan polymer shields. The adjuvant-free vaccine candidates were orally administered to mice and generated a humoral immune response against both peptide antigen, and the parent protein in the hookworm gut. RESULTS: The vaccine candidates were evaluated in a rodent hookworm challenge model, resulting in up to 98% and 99% decreases in mean intestinal worm and egg burdens in immunized mice, respectively. CONCLUSIONS: Lipopeptide survived the gastrointestinal conditions, induced humoral immune responses and drived protection against parasite challenge infection.

Transcriptomic profiling of nematode parasites surviving vaccine exposure.

Some nematode species are economically important parasites of livestock, while others are important human pathogens causing some of the most important neglected tropical diseases. In both humans and animals, anthelmintic drug administration is the main control strategy, but the emergence of drug-resistant worms has stimulated the development of alternative control approaches. Among these, vaccination is considered to be a sustainable and cost effective strategy. Currently, Barbervax® for the ruminant strongylid Haemonchus contortus is the only registered subunit vaccine for a nematode parasite, although a vaccine for the human hookworm Necator americanus is undergoing clinical trials (HOOKVAC consortium). As both these vaccines comprise a limited number of proteins, there is potential for selection of nematodes with altered sequences or expression of the vaccine antigens. Here we compared the transcriptome of H. contortus populations from sheep vaccinated with Barbervax® with worms from control animals. Barbervax® antigens are native integral membrane proteins isolated from the brush border of the intestinal cells of the adult parasite and many of those are proteases. Our findings provide no evidence for changes in expression of genes encoding Barbervax® antigens in the surviving parasite populations. However, surviving parasites from vaccinated animals showed increased expression of other proteases and regulators of lysosome trafficking, and displayed up-regulated lipid storage and defecation abilities that may have circumvented the effect of the vaccine. Implications for other potential vaccines for human and veterinary nematodes are discussed.

Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus.

Fatty acid and retinol-binding proteins (FARs) comprise a family of unusual α-helix rich lipid-binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein [C. elegans FAR-7 (Ce-FAR-7)] is from a subfamily of FARs that does not appear to be important at the host/parasite interface. We have therefore examined [Necator americanus FAR-1 (Na-FAR-1)] from the blood-feeding intestinal parasite of humans, N. americanus. The 3D structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by NMR (nuclear magnetic resonance) spectroscopy and X-ray crystallography respectively, reveals an α-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand-binding cavity and an additional C-terminal α-helix. Titration of apo-Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein-ligand complexes can be formed. Na-FAR-1 and possibly other FARs may have a wider repertoire for hydrophobic ligand binding, as confirmed in the present study by our finding that a range of neutral and polar lipids co-purify with the bacterially expressed recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male.

Probing the equatorial groove of the hookworm protein and vaccine candidate antigen, Na-ASP-2.

Hookworm activation-associated secreted proteins can be structurally classified into at least three different groups. The hallmark feature of Group 1 activation-associated secreted proteins is a prominent equatorial groove, which is inferred to form a ligand binding site. Furthermore, a conserved tandem histidine motif is located in the centre of the groove and believed to provide or support a yet to be determined catalytic activity. Here, we report three-dimensional crystal structures of Na-ASP-2, an L3-secreted activation-associated secreted protein from the human hookworm Necator americanus, which demonstrate transition metal binding ability of the conserved tandem histidine motif. We further identified moderate phosphohydrolase activity of recombinant Na-ASP-2, which relates to the tandem histidine motif. By panning a random 12-mer peptide phage library, we identified a peptide with high similarity to the human calcium-activated potassium channel SK3, and confirm binding of the synthetic peptide to recombinant Na-ASP-2 by differential scanning fluorimetry. Potential binding modes of the peptide to Na-ASP-2 were studied by molecular dynamics simulations which clearly identify a preferred topology of the Na-ASP-2:SK3 peptide complex.

¹H, ¹³C and ¹5N chemical shift assignments of Na-FAR-1, a helix-rich fatty acid and retinol binding protein of the parasitic nematode Necator americanus.

The fatty acid and retinol-binding (FAR) proteins are a family of unusual helix-rich lipid binding proteins found exclusively in nematodes, and are secreted by a range of parasites of humans, animals and plants. Na-FAR-1 is from the parasitic nematode Necator americanus, an intestinal blood-feeding parasite of humans. Sequence-specific (1)H, (13)C and (15)N resonance assignments have been obtained for the recombinant 170 amino acid protein, using three-dimensional triple-resonance heteronuclear magnetic resonance experiments. Backbone assignments have been obtained for 99.3% of the non-proline HN/N pairs (146 out of 147). The amide resonance of T45 was not observed, probably due to rapid exchange with solvent water. A total of 96.9% of backbone resonances were identified, while 97.7% assignment of amino acid sidechain protons is complete. All Hα(166), Hß(250) and Hγ(160) and 98.4% of the Hδ (126 out of 128) atoms were assigned. In addition, 99.4% Cα (154 out of 155) and 99.3% Cß (143 out of 144) resonances have been assigned. No resonances were observed for the NH(n) groups of R93 NεHε, arginine, N(η1)H2, N(η2)H2, histidine N(δ1)H(δ1), N(ε1)H(ε1) and lysine N(ζ3)H3. Na-FAR-1 has a similar overall arrangement of α-helices to Ce-FAR-7 of the free-living Caeorhabditis elegans, but with an extra C-terminal helix.

Systems metabolic effects of a necator americanus infection in Syrian hamster.

Hookworms (Ancylostoma duodenale and Necator americanus) are blood-feeding intestinal nematodes that infect approximately 700 million people worldwide. To further our understanding of the systems metabolic response of the mammalian host to hookworm infection, we employed a metabolic profiling strategy involving the combination of (1)H NMR spectroscopic analysis of urine and serum and multivariate data analysis techniques to investigate the biochemical consequences of a N. americanus infection in the hamster. The infection was characterized by altered energy metabolism, consistent with hookworm-induced anemia. Additionally, disturbance of gut microbiotal activity was associated with a N. americanus infection, manifested in the alterations of microbial-mammalian cometabolites, including phenylacetylglycine, p-cresol glucuronide, 4-hydroxy-3-methyl-phenylpropionic acid, hippurate, 4-hydroxyphenylactate, and dimethylamine. The correlation between worm burden and metabolite concentrations also reflected a changed energy metabolism and gut microbial state. Furthermore, elevated levels of urinary 2-aminoadipate was a characteristic feature of the infection, which may be associated with the documented neurological consequences of hookworm infection.

Identification of the nuclear receptor DAF-12 as a therapeutic target in parasitic nematodes.

Nematode parasitism is a worldwide health problem resulting in malnutrition and morbidity in over 1 billion people. The molecular mechanisms governing infection are poorly understood. Here, we report that an evolutionarily conserved nuclear hormone receptor signaling pathway governs development of the stage 3 infective larvae (iL3) in several nematode parasites, including Strongyloides stercoralis, Ancylostoma spp., and Necator americanus. As in the free-living Caenorhabditis elegans, steroid hormone-like dafachronic acids induced recovery of the dauer-like iL3 in parasitic nematodes by activating orthologs of the nuclear receptor DAF-12. Moreover, administration of dafachronic acid markedly reduced the pathogenic iL3 population in S. stercoralis, indicating the potential use of DAF-12 ligands to treat disseminated strongyloidiasis. To understand the pharmacology of targeting DAF-12, we solved the 3-dimensional structure of the S. stercoralis DAF-12 ligand-binding domain cocrystallized with dafachronic acids. These results reveal the molecular basis for DAF-12 ligand binding and identify nuclear receptors as unique therapeutic targets in parasitic nematodes.

Proteolytic degradation of hemoglobin in the intestine of the human hookworm Necator americanus.

Blood-feeding parasites use mechanistically distinct proteases to digest hemoglobin (Hb), often as multienzyme cooperative cascades. We investigated the roles played by 3 distinct proteases from adults of the human hookworm Necator americanus. The aspartic protease Na-APR-1 and the cysteine protease Na-CP-3 were expressed in catalytically active form in yeast, and the metalloprotease Na-MEP-1 was expressed in catalytically active form in baculovirus. Antibodies to all 3 proteases were used to immunolocalize each native enzyme to the intestine of adult N. americanus. Recombinant Na-APR-1 cleaved intact human Hb. In contrast, Na-CP-3 and Na-MEP-1 could not cleave Hb but instead cleaved globin fragments that had been hydrolyzed by Na-APR-1, implying an ordered process of hemoglobinolysis. Seventy-four cleavage sites within Hb alpha- and beta-chains were characterized after digestion with all 3 proteases. All of the proteases demonstrated promiscuous subsite specificities within Hb; noteworthy preferences included aromatic and hydrophobic P1 residues and hydrophobic P1' residues for Na-APR-1 and hydrophobic P1 residues for Na-MEP-1. We conclude that Hb digestion in N. americanus involves a network of distinct proteases, some of which act in an ordered fashion, providing a potential mechanism by which some of these hemoglobinases exert their efficacy as recombinant vaccines against hookworm infection.

Improved insights into the transcriptomes of the human hookworm Necator americanus--fundamental and biotechnological implications.

Hookworms of humans are blood-feeding parasitic nematodes of major socio-economic significance in a wide range of countries. They cause a neglected tropical disease (NTD) called "hookworm disease" (=necatoriasis and/or ancylostomiasis). Necator americanus is the most widely distributed hookworm of humans and is a leading cause of iron deficiency anaemia, which can cause physical and mental retardation and deaths in children as well as adverse maternal-foetal outcomes. Currently, there is a significant focus on the development of new approaches for the prevention and control of hookworms in humans. Technological advances are underpinning the discovery of drug and vaccine targets through insights into the molecular biology and genomics of these parasites and their relationship with the human host. In spite of the widespread socio-economic impacts of human necatoriasis, molecular datasets for N. americanus are scant, limiting progress in molecular research. The present article explores all currently available EST datasets for adult and larval stages of N. americanus using a semi-automated bioinformatic pipeline. In the current repertoire of molecules now available, some have been or are being considered as candidate vaccines against N. americanus. Among others, the most abundant sets of molecules relate to the pathogenesis-related protein (PRP) superfamily, comprising various members, such as the Ancylostoma-secreted or activation-associated proteins (ASPs) and the kunitz-type proteins, both of which are inferred to play key roles in the interplay between N. americanus and the human host. Understanding the molecular biology of these and other novel molecules discovered could have important implications for finding new ways of disrupting the pathways that they are involved in, and should facilitate the identification of new drug and vaccine targets. Also, the bioinformatic prediction of the essentiality of genes and gene products as well as molecular network connectivity of nematode-specific genes, together with sequencing by 454 technology, are likely to assist in the genomic discovery efforts in the very near future, to also underpin fundamental, molecular research of hookworms.

A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy.

The gastrointestinal tracts of multi-cellular blood-feeding parasites are targets for vaccines and drugs. Recently, recombinant vaccines that interrupt the digestion of blood in the hookworm gut have shown efficacy, so we explored the intestinal transcriptomes of the human and canine hookworms, Necator americanus and Ancylostoma caninum, respectively. We used Laser Microdissection Microscopy to dissect gut tissue from the parasites, extracted the RNA and generated cDNA libraries. A total of 480 expressed sequence tags were sequenced from each library and assembled into contigs, accounting for 268 N. americanus genes and 276 A. caninum genes. Only 17% of N. americanus and 36% of A. caninum contigs were assigned Gene Ontology classifications. Twenty-six (9.8%) N. americanus and 18 (6.5%) A. caninum contigs did not have homologues in any databases including dbEST-of these novel clones, seven N. americanus and three A. caninum contigs had Open Reading Frames with predicted secretory signal peptides. The most abundant transcripts corresponded to mRNAs encoding cholesterol-and fatty acid-binding proteins, C-type lectins, Activation-Associated Secretory Proteins, and proteases of different mechanistic classes, particularly astacin-like metallopeptidases. Expressed sequence tags corresponding to known and potential recombinant vaccines were identified and these included homologues of proteases, anti-clotting factors, defensins and integral membrane proteins involved in cell adhesion.

Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase.

A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the endoplasmic reticulum (ER) resident, homologous chaperone protein, protein disulfide isomerase (PDI) was able to increase the secretion of (Na-ASP1) protein in high copy clones. The effect of PDI levels on secretion of Na-ASP1 protein was examined in clones with varying copy number of PDI gene. Increase in secreted Na-ASP1 secretion is correlated well with the PDI copy number. Increasing levels of PDI also increased overall Na-ASP1 protein production in all the clones. Nevertheless, there was still accumulation of intracellular Na-ASP1 protein in P. pastoris clones over-expressing Na-ASP1 and PDI proteins.

Metabolism of lipid peroxidation products by the gastro-intestinal nematodes Necator americanus, Ancylostoma ceylanicum and Heligmosomoides polygyrus.

Somatic extracts of the three parasitic nematodes Necator americanus, Ancylostoma ceylanicum and Heligmosomoides polygyrus were able to detoxify a model hydroperoxide and a putative natural peroxide by glutathione-dependent peroxidase activity while cytotoxic carbonyls could be metabolized by NADPH-linked reduction activities. Unlike cestodes and digeneans, the nematodes in this study could not enzymatically conjugate carbonyls with glutathione. The results indicate that the three nematodes can protect themselves against possible host-immune initiated lipid peroxidation of their membranes at the level of the hydroperoxide and at the level of cytotoxic carbonyl, although other protective enzymatic mechanisms are also likely to exist (superoxide dismutase and catalase).