Experimental Design and Statistical Rationale

A total of 69 fractionated samples were analysed, including two biological replicates for the ES (49 fractions from 2 biological replicates) and one biological replicate for the L3 somatic samples (20 fractions). Each biological replicate from the ES samples was fractionated by SDS-PAGE or Offgel to maximise sample separation and peptide discovery and analysed by mass spectrometry (25 from SDS fractions and 24 from Offgel fractions). L3 somatic samples were fractionated by SDS-PAGE. Adult somatic data was obtained from previous studies [11]. Different search engines were employed to increase the confidence of identified PSMs and maximize peptide identification [32], including Mascot, X!Tandem and Comet as described in section “Protein identification”. For peptide and protein identification, a False Discovery Rate (FDR) of 1% was applied.

Ethics

Ethical approval for hamster animal experimentation to obtain N. americanus adult worms was obtained according to the Brazilian Guidelines on Animal Work, and approved by the local Animal Ethics Committee (CEUA) of the Federal University of Minas Gerais (Protocol# 51/2013). Ethical approval for human experimental infection with N. americanus and subsequent culturing of L3 was obtained from the James Cook University Human Research Ethics Committee (ID# H5936).

Parasite material

Adult N. americanus were manually isolated from the intestines of experimentally infected golden hamsters (Mesocricetus auratus) upon euthanasia. For the isolation and purification of ES products, the worms were washed 3 times in phosphate-buffered saline (PBS) before being cultured overnight in a humidified incubator at 37°C, 5% CO₂, in RPMI 1640 (100U/ml penicillin, 100 μg/ml streptomycin sulphate, 0.25 μg/ml amphotericin B). The supernatant was collected the following day and debris was removed by centrifuging the concentrated samples at 1,500 g for 3 minutes in a benchtop microfuge. ES products were concentrated with a 3 kDa cut-off Centricon filter membrane (Merck Millipore) and samples were stored at -80°C until use. For somatic adult extracts, data was used from Tang et al [11]. In brief, whole worms were ground under liquid nitrogen and solubilized using lysis buffer (1.0% (v/v) Triton X-100 in 40 mM Tris, 0.1% (w/v) SDS, pH 7.4). The extract was filtered through 20μm filter before fractionation.

For isolation of N. americanus L3, stool samples were collected from infected human volunteers and cultured as follows. Reverse osmosis water was added to the stool sample until a thick paste was formed. This paste was then distributed onto moistened Filter Paper (VWR, Standard Grade, 110 mm) in Petri dishes (Sarstedt, 150 mm) and placed in a 25°C incubator for 8 days. Following incubation, the edges of each plate were gently rinsed with RO water to obtain clean L3 preparations. Somatic extracts were prepared by adding 100 μl lysis buffer (3 M Urea, 0.2% SDS, 1% Triton X, 50 mM Tris-HCl) to approximately 6,000 larvae before repeated vortexing and sonication (4°C, probe sonicator, pulse setting) to digest the larvae. The extract was passed through a 0.45 μm filter (Millipore) before in-gel fractionation.

SDS-PAGE fractionation and in-gel trypsin digestion

A total of 30 μg of N. americanus adult ES products was buffer exchanged into 50 mM NH₄HCO₃, freeze-dried and resuspended in Laemmli buffer. The sample was boiled at 95°C for 5 minutes and electrophoresed on a 12% SDS-PAGE gel for 40 minutes at 150V. The gel was stained with Coomassie Blue and 20 pieces (approximately 1 mm thick) were cut and placed into Eppendorf tubes. For the in-gel digestion, slices were de-stained and freeze-dried before incubating them at 65°C for 1 h in reduction buffer (20 mM dithiothreitol (DTT), Sigma, 50 mM NH₄HCO₃). Samples were then alkylated in 50 mM iodoacetamide (IAM, Sigma), 50 mM NH₄HCO₃ for 40 minutes at 37°C, washed three times with 25 mM NH₄HCO₃ and digested with 20 μg/ml of trypsin (Sigma) by incubating them for 16 h at 37°C with gentle agitation. Digestion was stopped and peptides were released from the slices by adding 0.1% TFA, 70% acetonitrile. This step was repeated 3 times with pooling of the corresponding supernatants to maximise peptide recovery for each sample. Finally, each sample was desalted with a ZipTip (Merck Millipore) and stored at -80°C until use.

In-solution trypsin digestion and off-gel fractionation

A total of 70 μg of each N. americanus extract (L3 somatic and adult ES products) was buffer exchanged into 50 mM NH₄HCO₃ before adding DTT to 20 mM and incubating for 10 minutes at 65°C. Alkylation was carried out by adding IAM to 55 mM and incubating for 45 minutes at room temperature in the dark. Samples were digested with 2 μg of trypsin by incubating for 16 h at 37°C with gentle agitation. Following trypsin digestion, peptides were fractionated using a 3100 OFFGEL Fractionator (Agilent Technologies) according to the manufacturer’s protocol with a 24-well format as described previously [19]. In brief, Immobiline DryStrip pH 3–10 (24 cm) gel strips were rehydrated in rehydration buffer in the assembled loading frame. Digested peptides were diluted in dilution buffer to a volume of 3.6 ml and loaded equally across the 24 well cassette. The sample was run at a current of 50 μA until 50 kilovolt hours had elapsed. Upon completion of the fractionation, samples were collected, desalted with ZipTip and stored at -80°C until use.

Mass spectrometry

The extracts were analyzed by LC-MS/MS on a Shimadzu Prominence Nano HPLC (Japan) coupled to a Triple Time of Flight (TOF) 5600+ mass spectrometer (SCIEX, Canada) equipped with a nano electrospray ion source. Fifteen (15) μl of each extract was injected onto a 50 mm x 300 μm C18 trap column (Agilent Technologies, Australia) at 60 μl/min. The samples were de-salted on the trap column for 6 minutes using 0.1% formic acid (aq) at 60 μl/min. The trap column was then placed in-line with the analytical nano HPLC column, a 150 mm x 100 μm 300SBC18, 3.5 μm (Agilent Technologies, Australia) for mass spectrometry analysis. For peptide elution and analysis the nano-HPLC pump was initially held at 2% solvent B for 6 minutes followed by a linear gradient of 2–40% solvent B over 80 minutes at 500 nl/minute flow rate and then a steeper gradient from 40% to 80% solvent B in 10 minutes was applied. Solvent B was held at 80% for 5 minutes for washing the column and returned to 2% solvent B for equilibration prior to the next sample injection. Solvent A consisted of 0.1% formic acid (aq) and solvent B contained 90/10 acetonitrile/ 0.1% formic acid (aq). The ionspray voltage was set to 2200V, declustering potential 100V, curtain gas flow 25, nebulizer gas 1 12 and interface heater at 150°C. The mass spectrometer acquired 250 ms full scan TOF-MS data followed by 20 by 250 ms full scan product ion data in an Information Dependent Acquisition mode. Full scan TOF-MS data was acquired over the mass range 300–1600 and for product ion ms/ms 80–1600. Ions observed in the TOF-MS scan exceeding a threshold of 150 counts and a charge state of +2 to +5 were set to trigger the acquisition of product ion, MS/MS spectra of the resultant 20 most intense ions. The data was acquired and processed using Analyst TF 1.6.1 (SCIEX, Canada).

Proteogenomics

The mass spectrometry raw data was searched against the N. americanus protein database [11] using SequestHT algorithm in Proteome Discoverer 2.1 (Thermo Scientific, Bremen, Germany). Trypsin was used as the protease, allowing a maximum of two missed cleavages. Carbamidomethylation of cysteine was specified as a fixed modification, and oxidation of methionine was included as variable modifications. The minimum peptide length was specified as 6 amino acids. The mass error of parent ions was set to 10 ppm, and for fragment ions it was set to 0.05 Da. Protein inference was based on the rule of parsimony and required one or more unique peptides. FDR of 1% at peptide and protein levels was applied. The unassigned spectra from the protein database search were further searched against the six-frame translated genome database of N. americanus [11]. The genome sequences were downloaded from WormBase ParaSite release 9 (http://parasite.wormbase.org/Necator_americanus_prjna72135/Info/Index) in FASTA format and translated in all six frames using in-house python scripts. All the sequences greater than 10 amino acids between any two stop codons were added to the database. The search was again performed using SequestHT with precursor mass tolerance of 50 ppm and fragment ion tolerance of 1 Da. Carbamidomethylation of cysteine was specified as a fixed modification, and oxidation of methionine was included as variable modifications. Results were obtained at 1% FDR for both protein and peptide level.

The identified peptides in the search against the six-frame translated genome were mapped back to the protein database using standalone BLAST. Any sequences that mapped 100% to the protein database were discarded. The filtered peptides were mapped to the N. americanus genome using the standalone tblastn program [11]. Peptide identifications that unambiguously mapped to a single region in the genome, also known as Genome Search Specific Peptides (GSSPs), were considered to perform proteogenomics-based annotation of novel coding regions. The known gene annotation GFF3 was downloaded from WormBase ParaSite for N. americanus [11]. Using in-house scripts, we categorized the GSSPs into various categories: intergenic, intronic, exon-extension, alternative frame, N-terminal extensions or repeat regions with respect to the known regions from GFF3. Additionally, for all the intergenic peptides, we determined if there was a potential open reading frame (ORF) within the stretch of amino acids. Each of the spectra was further manually validated. All newly identified ORFs were blasted against a custom database containing the proteomes of A. caninum, A. ceylaninum and A. duodenale in order to annotate them. Interpro IDs were used to identify functional domains and SMART (http://smart.embl-heidelberg.de/) domain prediction software was used independently to determine which sequences contained a functional domain.

Genome annotation

The genome annotation from N. americanus [11] was updated using the MAKER pipeline v2.31.8 [33]. The genome assembly (GenBank assembly accession: GCA_000507365.1) was softmasked for repetitive elements with RepeatMasker v4.0.6 using a species-specific repeat library created by RepeatModeler v1.0.8, RepBase repeat libraries [34] and a list of known transposable elements provided by MAKER [33]. From the NCBI Sequence Read Archive, N. americanus RNA-Seq data [11] (Adult: SRR609895, SRR831085, SRR892200; L3: SRR609894, SRR831091, SRR89220) were obtained. After adapter and quality trimming using Trimmomatic v0.36 [35], RNA-Seq reads were aligned to the genome using HISAT2 v2.0.5 [36] with the—dta option and subsequently assembled using StringTie v1.2.4 [37]. The resulting alignment information and transcript assembly were used by BRAKER [38] and MAKER pipelines, respectively, as extrinsic evidence data. In addition, protein sequences from SwissProt UniRef100 [39] and WormBase ParaSite WS258 [40] (Ancylostoma ceylanicum PRJNA231479, Brugia malayi PRJNA10729, Caenorhabditis elegans PRJNA13758, Onchocerca volvulus PRJEB513, Pristionchus pacificus PRJNA12644, Trichinella spiralis PRJNA12603 and Strongyloides ratti PRJEB125) were provided to MAKER as protein homology evidence. Following the developer’s recommendation [41], the protein-coding gene models of Tang et al. [11] were passed to MAKER as pred_gff to update the models by adding new 3’ and 5’ exons, additional UTRs, and merging split models. This method, however, cannot change internal exons nor create new annotations where evidence suggests a gene but no corresponding model is previously present. To address this shortcoming, additional ab initio gene predictions were generated using BRAKER v2.0.1 [38] and passed to MAKER so that the intron-exon model that best matched the evidence could be included in the final annotation set. Within the BRAKER pipeline, the gene prediction tools GeneMark [42] and AUGUSTUS [43] were trained utilizing the N. americanus RNA-Seq alignment and protein homology information from C. elegans. The GFFPs identified in the present study were used to confirm the validity of proposed annotation changes and resolve competing gene predictions through manual curation. Gene models with no evidence support were not included in the final annotation build to reduce false positives in the existing annotations. However, ab initio gene predictions that encoded Pfam domains as detected by InterProScan v5.19 [44] were rescued to enhance overall accuracy by balancing sensitivity and specificity [33, 45]. Our approach, while being conservative, relied on the quality and completeness of previously published genomes, and may have missed some of species-specific gene models. The completeness of the annotated gene set was assessed using BUSCO v3.0 with Eukaryota-specific single copy orthologs (OrthoDB v9) [46].

Protein identification

Mascot version 2.5 (Matrix Science), X!Tandem (The Global Proteome Machine Organisation) version Jackhammer and Comet v2014.02 rev.2 were used to analyze data from the mass spectrometer. Searches were carried out against a database comprised of either the updated genome annotation provided with this study (15,728 sequences), or the N. americanus genome [11] (19,153 sequences), both appended to the common repository of adventitious proteins (cRAP; http://www.thegpm.org/crap/; 116 sequences) database (to detect potential contamination) and their corresponding decoy sequences (total of 38,538 sequences when using the N. americanus previous published genome version [11] and 31,688 when using the updated genome annotation). The following parameters were used: enzyme, trypsin; variable modifications, oxidation of methionine, carbamidomethylation of cysteine, deamidation of asparagine and glutamine; maximum missed cleavages, 2; precursor ion mass tolerance, 50 ppm; fragment ion tolerance 0.1 Da; charge states, 2+, 3+ 4+. A FDR of 0.1% was applied, and a filter of greater than 2 significant unique sequences was used to further improve the robustness of data. The mass spectrometry data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD010669. A False Discovery Rate (FDR) of 1% at peptide and protein levels was applied in all searches.

Bioinformatic Analysis of Proteomic Sequence Data

Gene ontology (GO) annotations were assigned using the program Blast2GO and Pfam analysis was performed using HMMER [47]. Pfam domains were detected at the P<0.001 threshold for the HMMER software. Putative signal peptides were predicted with SignalP v4.1 and transmembrane domains with TMHMM v2.0 [48, 49]. REViGO, an online tool, was used to summarise and plot GO terms [50]. UpSetR was used to group proteins based on whether they had a GO term with one, two or all of the GO categories (biological process, molecular function or cellular process) [51].

Similarity analysis

A similarity analysis was carried out based on the Parkinson and Blaxter method using an in-house script [26, 52]. Given the difficulty of working with N. americanus specifically (in terms of accessibility to samples and establishment of life cycle in other hosts), other hookworms are frequently used to model this human parasite. Data from the secreted proteome of adult N. americanus (described herein) was compared to the published secreted proteomes from related adult nematode species including H. polygyrus, A. caninum and Nippostrongylus brasiliensis [15, 19, 53].

Protein family similarity visualization

H. polygyrus, A. caninum and N. brasiliensis SCP/TAPS and protease protein sequences were obtained from their respective published secreted proteomes [15, 19, 53]. These sequences were aligned with adult N. americanus homologous protein sequences using BLAST. Significant sequence alignments were visualized using Circos [54].

Phylogenetic analyses

SCP/TAPS proteins were identified from published secretomes of 6 species of parasitic helminths including A. caninum, Ascaris suum, H. polygyrus, N. brasiliensis, Trichuris muris and Toxocara canis. SCP/TAPS proteins were identified from N. americanus ES products using the proteome data generated in this study. Sequences were sorted into single and double SCP/TAPS domain proteins for individual phylogenetic analysis due to distinct differences described previously [55]. A list of the proteins and their respective sequences used for this analysis can be found in S1 Table. A multiple sequence alignment was carried out using the alignment program MUSCLE. Outliers with poor alignment (long unaligned regions) were detected and filtered out using ODseek. PhyML, a phylogeny software, was used for a maximum-likelihood phylogenetic analyses with bootstrapping of SCP/TAPS amino acid sequences. The tree was visualized with The Interactive Tree of Life (iTOF) online phylogeny tool (https://itol.embl.de/) [56].

Recombinant protein expression and purification

We selected four SCP/TAPS protein family members representing double- (NAME_01818; NAME_01068; NAME_1070) and a single-domain (NAME_10402) protein from distinct phylogenetic clades for immunogenicity studies. We had proteomic evidence for secretion of the three double-domain proteins but there was no evidence of secretion of NAME_10402 in adult worm ES products, despite its upregulation at the mRNA level in the adult worm [11]. Complete open reading frames (without their signal peptides) were codon optimised for expression in the yeast Pichia pastoris, commercially synthesized and cloned into the pPICZαA expression vector (ThermoFisher) by Integrated DNA Technologies (Singapore). Sequences were cloned into the EcoRI and XbaI sites of pPICZαA such that they were in frame with the vector’s alpha-factor secretion signal (to facilitate secretion of the expressed protein into culture media) and C-terminal 6xHis tag to facilitate purification by Immobilised Metal Affinity Chromatography. Recombinant plasmids (40 μg) were linearized by digestion with SacI, purified by phenol:chloroform extraction and ethanol precipitation and resuspended in 20 μl of H₂O. Linearized vector was transformed into electrocompetent P. pastoris X-33 cells by electroporation (2 ms in 2 mm cuvettes using a BioRad Gene Pulser Xcell—2 kV, 25 μF capacitance, 200 Ohms resistance, square wave electroporation), plated onto yeast extract peptone dextrose agar plates supplemented with 2,000 μg/mL zeocin, covered in foil and incubated overnight for 3 days at 28°C. Integration of the expression cassette into the yeast chromosome was confirmed by colony PCR. Ten positive colonies were each seeded into 2 mL of buffered glycerol-complex (BMGY) media in 50 mL tubes, incubated at 30°C overnight with shaking at 250 rpm and the resultant cells centrifuged (2,500 g for 5 mins at 10°C), resuspended in 3 mL of buffered minimal methanol-complex medium (BMMY) to induce expression and similarly incubated for 96 hours. Cultures were supplemented with 0.5% methanol every 24 h to maintain promoter activity and 20 μL samples of culture media were taken every day and analysed for protein expression by Western blotting with an anti-His-HRP monoclonal antibody (ThermoFisher) using standard methods. A 10 μL sample of the highest expressing culture of each SCP/TAPS domain-containing protein was used to inoculate 25 mL of BMGY in a 250 mL conical flask, which was cultured overnight with shaking (250 rpm) at 30°C. The 25 mL culture was then used to inoculate 250 mL of BMGY in a 2.5 L conical flask, which was cultured overnight with shaking (250 rpm) at 30°C. To induce protein expression, the cells were pelleted at 2,500 g for 30 mins at 10°C, and then resuspended in 2 L of BMMY and split between four 2.5 L conical flasks, which were incubated with shaking (250 rpm) at 30°C for 96 hours. Methanol was added to a final concentration of 0.5% (2.5 mL/flask) every 24 hours to maintain promoter activity. Cells were pelleted at 2,500 g for 30 mins at 10°C. The supernatant was decanted, filtered through a 0.45 μm filter and applied to a pre-packed 5.0 mL His-Trap Excel IMAC column (GE Healthcare), that was charged with 100 mM nickel sulfate and equilibrated with 10 column volumes of binding buffer (50 mM Na₂HPO₄/NaH₂PO₄, pH 8, 300 mM NaCl, 10 mM imidazole), at a flow rate of 1.0 mL/min. Bound proteins were gradient-purified by washing and eluting with binding buffer containing an increasing concentration of imidazole (40–500 mM). Fractions taken throughout the purification process were analyzed by 12% SDS-PAGE and eluate fractions that contained protein were pooled, the concentration adjusted to 1.0 mg/mL using a 3 kDa cut-off Centricon filter (Merck Millipore) and verified using gel densitometry in comparison with BSA standards.

Enzyme-Linked Immunosorbent Assay using human sera

The diagnostic potential of each SCP/TAPS protein was assessed by indirect IgG ELISA using a cohort of hookworm-positive (grouped according to World Health Organisation stratification: heavy infection—≥4,000 eggs per gram of faeces (epg), n = 10; moderate infection—2,000–3,999 epg, n = 11; or light infection—≤1,999 epg, n = 44), hookworm-negative (n = 7) and hookworm-negative/Ascaris-positive (n = 12) serum samples from a hookworm-endemic region in Minas Gerais, Brazil. In 2005, 1,494 individuals between the ages 4 and 66 years (inclusive) were enrolled (with informed consent) into a cross-sectional study in an N. americanus-endemic area of Northeastern Minas Gerais state in Brazil, using protocols approved by the George Washington University Institutional Review Board (117040 and 060605), the Ethics Committee of Instituto René Rachou and the National Ethics Committee of Brazil (CONEP; protocol numbers 04/2008 and 12/2006). Venous blood (15 mL) was collected from individuals determined to be positive for N. americanus. Microlon high-binding 96-well microtiter ELISA plates (Greiner) were incubated overnight at 4°C with each antigen (2 μg/ml in 0.1 M Na₂CO₃/NaHCO₃, pH 9.6). Somatic extract of N. americanus L3 was similarly used as a positive control antigen. After washing with PBST (PBS + 0.05% Tween), the plates were blocked for 2 hours at RT with 100 μl of PBST/5% BSA. One hundred microliters of sera (1:200 in PBST/1% BSA) were added to the wells and incubated overnight at 4°C, then the plates were washed with PBST and 100 μl of goat anti-human IgG-HRP (Sigma) (1:5000 in PBST) was added to the plates. Plates were incubated for 2 hours at RT and then washed with PBST. Plates were developed by adding 100 μl of 3,3’,5,5’-tetramethylbenzidine and incubating for 30 minutes at RT in the dark. One hundred microliters of 1M HCl was added to stop the colorimetric reaction, which was read at a wavelength of 450 nm on a Polarstar Omega microplate reader (BMG Labtech). Assays were performed in triplicate and blank-corrected values were plotted using Graphpad Prism 7. The same software was also used to generate single and multi-antigen Receiver-Operating Characteristic (ROC) curves. Reactivity cutoffs were determined as the average plus 1SD of the values of the hookworm-negative group and the frequency of recognition of each antigen was determined using this cutoff.

Proteogenomic analysis and genome annotation

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD010669. To access the data, please use the below login details.

Username: reviewer49665@ebi.ac.uk

Password: fybW0mgJ

Proteomes from two life stages (infective L3 somatic extract and adult ES products) of N. americanus were analysed by mass spectrometry in a 5600 ABSciex Triple Tof to perform a proteogenomic analysis of the hookworm genome. We identified 198 adult ES proteins and 590 L3 somatic proteins. In an earlier study we identified 458 proteins from a somatic extract of adult N. americanus [11], and those sequences were used herein to facilitate the annotation process. After excluding peptides that mapped accurately to the existing protein database entry, a total of 218 novel peptides were identified that did not match any protein sequences of N. americanus contained in the annotations of Tang et al [11]. Of these 218 novel peptides, 83 were found exclusively in adult somatic extracts, 50 exclusively in L3, and 67 exclusively in adult ES, while 10 peptides were found in both the adult somatic extract and ES products, and 8 were found in both adult and larval somatic extracts. No common peptides were identified from adult ES products and larval somatic extract.

Newly identified peptides could be grouped into the following six categories: (1) peptides mapping to intergenic regions; (2) peptides mapping to introns; (3) peptides mapping to alternative reading frames; (4) peptides extending gene boundaries (exon extensions); (5) peptides mapping to N-terminal extensions; and (6) peptides mapping to repeat regions (not gene) regions (Fig 1). Of the identified peptides, more than half belonged to group 1, 18% to group 2, 15% to group 3 and a small number to groups 4, 5 and 6 (Fig 1). The translated protein sequences with their respective peptides, six-frame translated genome ID and ORF length are provided in S2 Table. Group 1 peptides were further analyzed for an ORF including whether or not they contained a methionine; see S3 Table. From this data, one-third of group 1 peptides were found to overlap with a viable ORF, with the shortest having just 21 amino acids and the longest 381 amino acids. In addition, in order to further annotate these newly identified ORFs, we blasted the sequences against a custom database containing the proteomes of A. caninum, A. ceylaninum and A. duodenale. A total of 54 proteins were significantly similar to proteins from other ancylostomatids, while 7 had no known homologs (S4 Table). Furthermore, only 41 proteins contained a valid Pfam domain (S5 Table).

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Fig 1. Depiction of the proteogenomic process as well as the types and numbers of peptide corrections identified.

https://doi.org/10.1371/journal.pntd.0008237.g001

These results highlighted the need for improving the previously published gene models, so we updated the genome annotation of N. americanus using a new RNA-Seq based gene calling pipeline as outlined in the Materials and Methods (Table 1). The total number of predicted N. americanus genes decreased by 3,425, with substantial increases in both the number of exons and introns. The total coding sequence (CDS) length increased by 2.22 Mb with the mean gene length (including introns and UTRs) nearly doubling from 4.3 kb to 8.1 kb. Furthermore, the percentage of the genome covered by genes increased by 18.3%, and the percentage of detected BUSCOs among the predicted genes increased from 95.7% to 97.4% (with 31% reduction in the number of fragmented BUSCOs). While these improvements are attributable in most part to the use of more sophisticated genome annotation methods utilizing RNA-Seq data and the inclusion of more extensive, up-to-date homologous protein databases, the peptide sequences generated in this study contributed directly to the refinement of 14 gene models. The newly annotated, improved gene models were used in subsequent proteomic analysis of ES products, and are publicly available on Nematode.net [57, 58].

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Table 1. Comparison of original and updated genome annotations.

https://doi.org/10.1371/journal.pntd.0008237.t001

Analysis of the ES products from N. americanus adult worms

A comprehensive analysis of the ES products from adult worms was carried out using in-gel and off-gel fractionation and the tryptic peptides were analyzed using LC-MS/MS. Mascot, X!Tandem and Comet searches were carried out against a database including the predicted proteins from the annotated N. americanus genome available in this study and the cRAP sequences available at http://www.thegpm.org/crap/. Identified contaminant and host proteins (M. auratus) are detailed in S6 Table. A total of 186 and 141 proteins were identified using Mascot and X!Tandem/Comet, respectively. The two search methods combined found a total of 198 proteins, S7 Table). These 198 proteins were obtained using the updated genome annotation generated as part of this study. In comparison, using the first version of the annotated genome sequence we identified 203 proteins using the same analytical software [11]. Using the exponential modified protein abundance index (emPAI) and the newly annotated genome, the most abundant proteins in the ES products of N. americanus adult worms were ranked, and the top 10 are shown in Table 2. The conserved Pfam domains of the 198 ES proteins identified were analyzed. The most abundant protein family in the ES products was the SCP/TAPS family with 54/198 proteins containing a single or double cysteine-rich secretory protein family (CAP) domain (PF00188) (Fig 2B). The top 10 most abundant protein families are displayed in Fig 2B. Many of these proteins were described by Blast2GO as ‘SCP-partial’ or ‘SCP-like’, but for a more standardized annotation in our analysis we have grouped them all as ‘SCP/TAPS’. The second most frequently represented group (with 42 proteins) were proteins with one or more domains of unknown function (DUF). Other abundant families included Ancylostoma secreted protein related (ASPR) proteins and metalloproteases with 35 and 9 of each identified respectively (S8 Table). Despite some published reports classifying ASPRs as SCP/TAPS proteins, they are a diverse set of secreted cysteine rich proteins based on Pfam annotation and therefore we have grouped them separately [28]. Of the 198 identified ES proteins, 48% contained a predicted signal peptide. Supporting the accuracy of the new gene model, 96 of the identified adult ES proteins were predicted to contain a signal peptide compared to just 75 using the previous genome annotation.

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Fig 2.

(A) UpSetR plot displaying the number of each gene ontology (GO) term categories (biological process, molecular function and/or cellular component) available (Blast2GO) for adult Necator americanus excretory/secretory (ES) proteins. Proteins with no available GO terms are broken down in a pie chart into ‘SCP/TAPS’ proteins ‘other’. (B) Top 10 most abundant protein families in the ES products of adult N. americanus. (C) Biological processes of adult N. americanus ES proteins ranked by nodescore (Blast2GO) and plotted using REViGO. Semantically similar GO terms plot close together, increasing heatmap score signifies increasing nodescore from Blast2GO, while circle size denotes the frequency of the GO term from the underlying database. (D) Molecular functions of adult N. americanus ES proteins ranked by nodescore (Blast2GO) plotted using REViGO.

https://doi.org/10.1371/journal.pntd.0008237.g002

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Table 2. Top 10 most abundant proteins in the excretory/secretory proteins of N. americanus.

https://doi.org/10.1371/journal.pntd.0008237.t002

In total, 30 GO terms were identified (following the removal of parent child redundancy) belonging to one of the three GO database categories: biological processes, molecular function or cellular component (Fig 2A, for raw data see S9 Table). Blast2GO returned biological process GO terms for 87/198 proteins, molecular function GO terms for 99/198 proteins and cellular component GO terms for 83/198 proteins (Fig 2A). The most prominent biological process term was proteolysis (Fig 2D), with 15% (29 proteins) of total ES products being involved in a proteolytic process. The most prominent single molecular function term was “hydrolase activity” followed by “peptidase activity” and “metal ion binding” (Fig 2C). Interestingly, 50/198 proteins did not return any GO terms (Fig 2A). Of these, SCP/TAPS proteins made up 64% with no known biological process, molecular function, or cellular component. This highlights a significant knowledge gap surrounding SCP/TAPS proteins produced by helminths.

Similarity analysis of the ES products from different gastrointestinal nematode species

A similarity analysis of ES proteomic data from N. americanus and three of the most commonly used animal models for human hookworms, A. caninum, H. polygyrus and N. brasiliensis was carried out (Fig 3). A total of 15, 10 and 1 N. americanus ES proteins had unique homology to ES proteins from A. caninum, H. polygyrus and N. brasiliensis respectively. This included one SCP/TAPS protein (NAME_13724) which was similar only to an H. polygyrus protein, while 3 of the proteins were similar only to A. caninum ES proteins with domains of unknown function (NAME_07734, NAME_09181, NAME_09182). Seven proteins shared homology with only A. caninum and H. polygyrus proteins, 11 shared homology with only A. caninum and N. brasiliensis proteins and 7 shared homology with only H. polygyrus and N. brasiliensis proteins. One hundred and twenty-two N. americanus proteins shared different degrees of homology with proteins from A. caninum, H. polygyrus and N. brasiliensis.

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Fig 3. Excretory/secretory (ES) products from Ancylostoma caninum, Heligmosomoides polygyrus, and Nippostrongylus brasiliensis were compared with the ES proteome of Necator americanus and displayed in a Simitri plot.

SCP/TAPS are represented by a turquoise triangle, proteases by an orange square and any other protein by a grey circle. Points on the diagram triangle represent sequences which only had similarity to the labelled species. Points along the edges of the triangle are sequences which had similarity to two of the three species (given at the respective ends of the edge). Any sequence in the middle area of the triangle represents a sequence with similarity to all three compared species.

https://doi.org/10.1371/journal.pntd.0008237.g003

From the 54 SCP/TAPS proteins found in N. americanus ES products, one (NAME_13724) shared homology with a protein found in only H. polygyrus, while another SCP/TAPS protein (NAME_15177) shared homology with proteins from both A. caninum and N. brasiliensis. Fifty-one (51) of 54 SCP/TAPS proteins were similar to all compared species, leaving a single SCP/TAPS protein (NAME_11218) which did not have homology to any SCP/TAPS protein from the compared species. Twenty-five N. americanus ES proteins did not have homology to any proteins in the secretome of A. caninum, H. polygyrus or N. brasiliensis. Notable proteins among these 25 were NAME_01848 (aspartyl protease) and NAME_05081 (zinc metalloprotease).

Of the 26 proteases in the N. americanus ES products, 22 had homologs in all compared species, while one serine protease (NAME_06735) only had homologs in H. polygyrus and A. caninum and one metalloprotease (NAME_00535, peptidase family M1) only had homologs in the ES products of A. caninum, and N. brasiliensis. One zinc metalloprotease (NAME_05081) and one serine protease (NAME_01250) were only found in the ES products of N. americanus and therefore did not have any similarity to ES products from the other species.

Homology analysis of SCP/TAPS and proteases in the ES products of N. americanus

Since SCP/TAPS proteins and proteases from N. americanus numerically dominate the ES protein dataset and likely play key roles in infection, migration and parasite establishment, we performed an in-depth analysis of these families of proteins between the human and three model gastrointestinal nematode species. Adult N. americanus ES SCP/TAPS protein sequences were aligned with homologs from H. polygyrus, A. caninum and N. brasiliensis ES SCP/TAPS protein sequences using BLAST. Sequences were visualized using Circos (Fig 4). SCP/TAPS from N. americanus are more similar to A. caninum than H. polygyrus or N. brasiliensis, as denoted by thicker, darker ribbons (Fig 4). The sequences, their homologs and the corresponding blast scores are detailed in S10 Table. As with the similarity analysis, NAME_11218 had no significant alignment to any of the compared species.

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Fig 4. SCP/TAPS proteins in the excretory/secretory products (ES) of Necator americanus are most closely related to SCP/TAPS proteins in the ES of Ancylostoma caninum.

Protein names are displayed in a circle with N. americanus (purple), A. caninum (blue), Heligmosomoides polygyrus (pink) and Nippostrongylus brasiliensis (green). Ribbon thickness is relative to the maximum score obtained in the BLAST search while darker ribbons denote higher sequence percent identity. The corresponding bars provide relative sequence length of each protein.

https://doi.org/10.1371/journal.pntd.0008237.g004

A similar analysis was performed for the proteases from each of the aforementioned species (Fig 5). These proteases have been grouped together into their respective mechanistic classes: aspartyl (ASP), cysteine (CYS), metallo (MET) or serine (SER) proteases. In general, all three comparator species had high protease sequence homology to metalloproteases from N. americanus. Aspartyl protease sequences were more similar between H. polygyrus, N. brasiliensis and N. americanus than sequences from A. caninum. It is interesting to note that N. americanus contained more aspartyl proteases than the other nematode species analyzed (N. americanus– 8; A. caninum– 4; H. polygyrus– 6; N. brasiliensis– 6). Serine proteases had the lowest sequence homology across all of the compared species and protease subclasses.

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Fig 5. Proteases in the excretory/secretory (ES) products of Necator americanus are most closely related to proteases in the ES of Ancylostoma caninum.

Protein names are displayed in a circle with N. americanus (purple), A. caninum (blue), Heligmosomoides polygyrus (pink) and Nippostrongylus brasiliensis (green). Ribbon thickness is relative to the maximum score obtained in the BLAST search while darker ribbons denote higher sequence percent identity. The corresponding bars provide relative sequence length of each protein. Respective protease mechanistic classes: aspartic (ASP), cysteine (CYS), metallo- (MET) or serine (SER).

https://doi.org/10.1371/journal.pntd.0008237.g005

Phylogenetic analysis of SCP/TAPS proteins

SCP/TAPS sequences of 6 parasitic nematodes were obtained from published secretomes and compared to N. americanus SCP/TAPS identified in this study. Sequences were grouped by whether they had a single or double domain and then aligned using MUSCLE and PhyML. From the 7 total species, 232 SCP/TAPS were reported with 134 single-domain proteins and 98 double-domain. For the single SCP/TAPS-domain proteins an unrooted tree was generated. The analysis identified seven main clades (Fig 6). Three of these clades consisted entirely of sequences from N. americanus and A. caninum, while sub-clades in 3 of the other main clades followed a similar trend. A majority of T. muris single-domain sequences formed a sub-clade with A. suum and T. canis, grouping together the non-clade V helminths. An unrooted tree was also generated for double domain sequences. The analysis identified five main clades. N. americanus SCP/TAPS clustered almost exclusively with sequences from A. caninum again, representing one main clade and several sub-clades (Fig 7). H. polygyrus and N. brasiliensis also formed a number of distinct sub-clades. T. canis, the only non-clade V helminth, was reported to only produce one double-domain SCP/TAPS protein in its ES products which was not closely related to any of the compared sequences. Interestingly, no double-domain SCP/TAPS were reported for A. suum or T. muris. Another trend across both single and double domain trees was the diversity of evolution between SCP/TAPS within a single species. For example, the single-domain tree included a sub-clade of 7 N. americanus-only sequences while other N. americanus sequences had more similarity to mouse hookworm sequences.

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Fig 6. Phylogenetic relationships of single-domain SCP/TAPS proteins determined with MUSCLE alignment software.

PhyML was used for a maximum-likelihood phylogenetic analysis with bootstrapping and results were visualized with The Interactive Tree of Life (iTOF) online phylogeny tool. Necator americanus sequences are highlighted in purple with comparator species each denoted by a different color (see key). Black solid circles on branches denote bootstrap values with greater than or equal to 70% support.

https://doi.org/10.1371/journal.pntd.0008237.g006

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Fig 7. Phylogenetic relationships of double-domain SCP/TAPS proteins determined with MUSCLE alignment software.

PhyML was used for a maximum-likelihood phylogenetic analysis with bootstrapping and results were visualized with The Interactive Tree of Life (iTOF) online phylogeny tool. Necator americanus sequences are highlighted in purple with comparator species each denoted by a different color (see key). Black solid circles on branches denote bootstrap values with greater than or equal to 70% support.

https://doi.org/10.1371/journal.pntd.0008237.g007

Diagnostic potential of SCP/TAPS proteins

Using the recombinant N. americanus SCP/TAPS proteins expressed in this study (and N. americanus L3 extract as a comparator), we analyzed antibody responses in the serum of N. americanus-infected individuals (Fig 8A–8E) to each antigen and the AUC generated from the ROC curves (S1 Fig) were used to determine the sensitivity and specificity of each antibody response, and the predictive value of infection (Table 3). Antibodies to all antigens were significantly reactive in subjects with a heavy hookworm infection (≥4,000 epg) compared to hookworm-negative controls. Further anti-01068 and anti-01070 IgG responses were also significant in subjects with a light hookworm load (≤1,999 epg). For the L3 extract and three of the antigens, IgG reactivity in the hookworm-negative/Ascaris-positive group was higher (not significantly so) compared to the hookworm-negative group with no Ascaris infection. The two Ascaris SCP/TAPS proteins share between 16–31% identity with the respective N. americanus sequences, so overall the similarity is relatively low. The most likely explanation for the small number of Ascaris-positive/hookworm-negative individuals is prior or active low intensity infection of these few subjects with hookworm. Hookworm is co-endemic with Ascaris in this region of Brazil, and although the subjects were negative for hookworm eggs in the feces, the low sensitivity of the Kato-Katz test means that they might have had low intensity hookworm infection. Of the recombinant antigens tested, the highest positive predictive value of infection (PPV) in hookworm-positive vs hookworm-negative subjects was generated by the antibody response to 01070 (AUC = 0.77). Hookworm-positive subjects with antibody levels against a particular antigen above the reactivity cut-off were recorded as positive for recognition of that antigen and, because the recognition pattern of each antigen among the infected population was different, we investigated whether a combination of antigens would increase the frequency of recognition and/or PPV among hookworm-positive subjects compared to any one antigen alone. A minimum combination of three SCP/TAPS proteins (01070 + 01068 + 10402) increased the FoR to 82% of hookworm-positive subjects, compared to an FoR of 60% and 66% for 01070 and the L3 extract, respectively (Fig 8F and Table 3). Further, this antigen combination gave a PPV of 0.83, which was comparable to 0.86 obtained for the L3 extract.

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Fig 8. Serological diagnosis of hookworm infection by detection of serum antibodies against hookworm L3 extract and recombinant SCP/TAPS proteins.

Scatter plots showing the (A) anti-N. americanus L3 extract IgG response, (B) anti-01818 IgG response, (C) anti-01068 IgG response, (D) anti-10402 IgG response (E) anti-01070 IgG response. Hookworm-positive subjects were characterized (WHO stratification) as either having a heavy (≥4,000 epg), moderate (2,000–3,999 epg) or light (≤1,999 epg) infection. “HW -ve” = hookworm egg negative subjects from an endemic area, “HW -ve/ASC +ve” = hookworm egg negative, Ascaris egg positive subjects from an endemic area. Differences in responses between groups were analyzed by Student’s t test. *P≤0.05, **P≤0.01. (F) Schematic showing frequency of recognition of N. americanus L3 extract and recombinant SCP/TAPS proteins by hookworm-positive subjects. The recognition cutoff was defined as the average response of the HW -ve group + 1SD. ^#represents the minimal antigen combination (01070, 01818 and 10402) which gives the highest frequency of recognition. AUC = area under receiver operator curves generated for each antigen and the antigen combination, which determines the positive predictive value of infection.

https://doi.org/10.1371/journal.pntd.0008237.g008

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Table 3. Frequency of Recognition and Area Under the Curve values for N. americanus L3 extract and recombinant SCP/TAPS proteins in the diagnosis of hookworm infection.

https://doi.org/10.1371/journal.pntd.0008237.t003