A comparative 'omics' approach for prediction of candidate Strongyloides stercoralis diagnostic coproantigens

Abstract

Human infection with the intestinal nematode Strongyloides stercoralis is persistent unless effectively treated, and potentially fatal in immunosuppressed individuals. Epidemiological data are lacking, partially due to inadequate diagnosis. A rapid antigen detection test is a priority for population surveillance, validating cure after treatment, and for screening prior to immunosuppression. We used a targeted analysis of open access 'omics' data sets and used online predictors to identify S. stercoralis proteins that are predicted to be present in infected stool, Strongyloides-specific, and antigenic. Transcriptomic data from gut and non-gut dwelling life cycle stages of S. stercoralis revealed 328 proteins that are differentially expressed. Strongyloides ratti proteomic data for excreted and secreted (E/S) proteins were matched to S. stercoralis, giving 1,057 orthologues. Five parasitism-associated protein families (SCP/TAPS, prolyl oligopeptidase, transthyretin-like, aspartic peptidase, acetylcholinesterase) were compared phylogenetically between S. stercoralis and outgroups, and proteins with least homology to the outgroups were selected. Proteins that overlapped between the transcriptomic and proteomic datasets were analysed by multiple sequence alignment, epitope prediction and 3D structure modelling to reveal S. stercoralis candidate peptide/protein coproantigens. We describe 22 candidates from seven genes, across all five protein families for further investigation as potential S. stercoralis diagnostic coproantigens, identified using open access data and freely-available protein analysis tools. This powerful approach can be applied to many parasitic infections with 'omic' data to accelerate development of specific diagnostic assays for laboratory or point-of-care field application.

Fig 1. Schematic of the method for identifying coproantigens of Strongyloides stercoralis using ‘omic’ data and computational analyses.

Filled shapes represent datasets, colours represent analyses against three characteristics of a candidate coproantigen. E/S, excreted and secreted; MSA, multiple sequence alignment; Ss, Strongyloides stercoralis; BLAST, basic local alignment search tool; DE, differentially expressed.

Fig 2. Strongyloides stercoralis life stages.

Asterisks indicate life stages for which transcriptomic data were obtained by Stoltzfus et al. (2012). We grouped these transcriptomic data by presence in the host gut (boxed asterisks), and outside the host gut. P Females, parasitic females; PP, post parasitic; FL, Free-living; PFL, post free-living; L1, stage 1 larva; L3, stage 3 larva; iL3, infectious third stage larva; L3+, tissue-migrating larva; L3a, autoinfective L3. Figure modified from Stoltzfus et al. (2012) [37] under a CC BY license. Accession numbers are given for NCBI SRA for the triplicate reads in Table 1.

Fig 3. Strongyloides stercoralis proteins differentially expressed (DE) between gut-dwelling and non-gut-dwelling life stages and their overlap with other analyses.

Panel A; DE proteins grouped by sequence similarity with protein families identified. Colours refer to features or families; the 5 priority protein families analysed in depth (SCP/TAPS; TTL; AChE; aspartic peptidases, and POP) are labelled with an asterisk and boxed text; pale grey indicates proteins not identified by any method, dark grey indicates disordered proteins. Panel B, overlap, in numbers of proteins, between the DE proteins and other analyses. In both panels, labels indicate: a, the 328 S. stercoralis DE proteins; b, proteins orthologous to S. ratti E/S proteins; c, proteins containing epitopes predicted by BcePred (left in Panel A) and BepiPred (right in Panel A); d, the 203 proteins that were DE in gut-dwelling life stages. Gene accession numbers and all protein family names for the DE proteins can be found in S2 and S4 Files for E/S orthologues. These results originate from methods in Fig 1, boxes C-H and M).

Fig 4. Phylogenetic alignment of Strongyloides stercoralis high priority family proteins with other S. stercoralis proteins and outgroups.

Proteins of the five priority families of S. stercoralis (dark green), from the differentially expressed dataset, in phylogenetic alignment with BLAST hits from S. stercoralis itself (black) and outgroups (other colours). Colours reaching the outer edge represent Strongyloides species, whereas shorter colour bands are non-Strongyloides outgroups. (A) SCP/TAPS, (B) transthyretin-like (TTL), (C) acetylcholinesterase (AChE), (D) aspartic peptidase, (E) prolyl oligopeptidase (POP). Arrows indicate clusters of S. stercoralis proteins containing species-specific regions; derived from both gut- and non-gut-dwelling differential expression data, augmenting detection of lack of species specificity. These results originate from methods in Fig 1, box K.

Fig 5. A transthyretin-like (TTL) protein of Strongyloides stercoralis in rotated view showing surface-exposed epitope regions and N-linked glycosylation sites.

Accession number SSTP_0000700800. Blue, BepiPred predicted epitope aa’s 124–143; pink, bcepred predicted epitope aa’s 99–123; green, predicted N-linked glycosylation site at N165 in the motif NVS. The bcepred epitope (pink) extends to residue 141 but has been shortened to show the BepiPred epitope (blue), which overlaps with it. These results originate from methods shown in Fig 1, boxes M-P.

Fig 6. Strongyloides stercoralis acetylcholinesterase in rotated views showing surface-exposed epitope regions and N-linked glycosylation sites.

Accession number SSTP_0000509400. Predicted features indicated in colour: blue, BepiPred 2.0 predicted epitope at residues 85–103; and yellow at 390–411; green, predicted N-linked glycosylation sites at N38 (NVT), N89 (NFS) and N319 (NLT). The site at N89 is within the blue epitope sequence. Both predicted epitope regions (blue and yellow) were selected as candidate coproantigens in this study. These results originate from methods shown in Fig 1, boxes M-P.