Fig 1.
Depiction of the proteogenomic process as well as the types and numbers of peptide corrections identified.
Table 1.
Comparison of original and updated genome annotations.
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.
Table 2.
Top 10 most abundant proteins in the excretory/secretory proteins of N. americanus.
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.
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.
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).
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.
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.
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.
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.