Novel insights into the somatic proteome of Strongyloides stercoralis infective third-stage larvae

Abstract

Background: Strongyloidiasis is a neglected tropical disease affecting an estimated 600 million people, particularly in resource-limited settings. The infection can persist lifelong due to unusual auto-infective cycle of Strongyloides stercoralis. The lack of a diagnostic gold standard and limited knowledge of the mechanisms underpinning this chronic infection are key issues in disease management. To date, only a few proteomics studies have been conducted to elucidate the molecular mechanisms associated with Strongyloides parasitism or to highlight novel immunological markers, with the result that our knowledge of S. stercoralis proteome remains limited. This study aims at expanding the characterization of S. stercoralis infective larvae (iL3) in order to further explore the mechanisms of parasitism and to highlight possible novel targets for serodiagnosis.

Methods: iL3 obtained from an infected subject were analysed by high-throughput tandem mass spectrometry. To achieve a more comprehensive characterization of the iL3 proteome we analysed the experimental dataset using an automatic search strategy combined with manual annotation, which included gene ontology (GO) analysis, InterPro annotation, assessment of the homology with Homo sapiens and other pathogens of clinical importance and B-cell epitope prediction.

Results: Our pipeline identified 430 S. stercoralis proteins, 187 (43%) of which were uncharacterized. Oxidoreductases and peptidases were amongst the most represented protein categories, as highlighted by molecular function GO analyses, while membrane and mitochondrial proteins were the most represented cellular component GO categories. A high proportion of proteins bearing the CAP, SCP or thioredoxin domain or belonging to cysteine-rich secretory, transthyretin-like or peptidase protein families were also identified. Additionally, we highlighted nine proteins displaying low homology with H. sapiens or other related pathogens and bearing amino acid sequences with immunogenic properties.

Conclusions: Our comprehensive description and annotation of the S. stercoralis iL3 proteome contribute to expanding the 'omics characterization of this parasite and provide experimental evidence on the most represented proteins associated with S. stercoralis parasitism, as inferred from genomic and transcriptomic data. Moreover, novel candidate immunogenic proteins to be evaluated as novel serological diagnostic markers are highlighted.

Keywords: Annotation; B-cell epitope prediction; Proteomics; Serodiagnosis; Strongyloides stercoralis; iL3.

Fig. 1

Study flowchart. Pipeline followed in the present study. GO, Gene ontology; iL3, infective filariform larvae; LC-MS/MS, Liquid chromatography-tandem mass spectrometry. Other pathogens include: Ancylostoma duodenale; Ancylostoma ceylanicum; Necator americanus; Ascaris lumbricoides; Trichuris trichiura; Toxocara canis; Loa loa; Mansonella perstans; Mansonella ozzardi; Wuchereria bancrofti; Onchocerca volvulus; Brugia malayi; Brugia timori; Dirofilaria immitis; Dirofilaria repens; Trichinella spiralis; Taenia saginata; Taenia solium; Echinococcus granulosus; Hymenolepis nana; Schistosoma mansoni; Schistosoma haematobium; Schistosoma japonicum; Fasciola hepatica; Plasmodium falciparum; Plasmodium vivax; Plasmodium ovale; Plasmodium malariae; Plasmodium knowlesi

Fig. 2

Gene ontology results. Frequency of the GO terms for the three categories cellular component (A), molecular function (B) and biological process (C) across identified proteins. ER, Endoplasmic reticulum

Fig. 3

InterPro annotation results. The top 10 most frequent InterPro terms for the categories domain and family represented among all identified proteins (n = 430), as established by InterPro annotation. For each term, the number of uncharacterized and characterized proteins is represented in different color shades. The complete annotation is reported in Additional file 1: Table S1

Fig. 4

B-cell epitope prediction results. The results for the protein A0A0K0ECK4—galectin are reported as an example. a FASTA sequence showing the results obtained with each tool (Chou & Fasman Beta-Turn Prediction; Emini Surface Accessibility Prediction; Kolaskar & Tongaonkar Antigenicity; Parker Hydrophilicity Prediction, BepiPred2.0; all available via http://tools.iedb.org/bcell/). All residues having a score above their threshold are highlighted in grey. The purple squares indicate the sequences highlighted as being potentially immunogenic as reported in the Methods section. b Protein structures as predicted by AlphaFold showing the model confidence. c Mapping of the potentially immunogenic epitopes on the protein structure. The same images for all other selected proteins are reported in Additional file 4: Figures S1–S8