BACKGROUND: Venom allergen-like proteins (VALs) are abundant in the excretory-secretory products (ESPs) of numerous parasitic helminths and have been extensively studied for over 30 years because of their potential to interact with host systems. Despite substantial research, however, the precise functions of these proteins remain largely unresolved. Schistosomes, parasites of the circulatory system, are no exception, with 29 SmVAL genes identified in the genome of Schistosoma mansoni to date. The eggs of these parasites, as primary pathogenic agents, interact directly with host tissues and release excretory-secretory products that aid their egress from the host. Although SmVALs have been detected in the egg secretome in the past, direct evidence of their secretion and functional interaction with host molecules has never been demonstrated. These findings fuel the ongoing debate as to whether egg-expressed SmVALs interact with the mammalian host or are rather miracidial proteins synthesized within the egg during larval development. RESULTS: Based on complete revision of the SmVAL family and an associated robust transcriptomic meta-analysis of gene expression across the life cycle, we show that many of SmVAL genes, including 6 newly identified genes, are expressed in the infective larvae-producing stages (eggs and sporocysts). Following localization of two "egg-specific" SmVAL9 and SmVAL29 did not prove active secretion of these molecules into surrounding tissues but were aligned with miracidial structures interfacing with the molluscan host, specifically the larval surface and penetration glands. Finally, we show the complete lack of interactions between candidate SmVAL proteins and an array of 755 human cell receptors via a state-of-the-art SAVEXIS screen. CONCLUSIONS: Overall, we conclude that these "egg" SmVALs are not involved in direct host‒parasite interactions in the mammalian host and are rather proteins employed during intermediate host invasion. Our study revisits and updates the SmVAL gene family, highlighting the limitations of in silico protein function predictions while emphasizing the need for up-to-date datasets and tools together with experimental validation in host-parasite interactions. By uncovering the diversity, expression patterns, and interaction dynamics of SmVALs, we open new avenues for understanding host manipulation and reevaluating orthologous proteins in other helminths.

Department of Biology University of York York UK

Department of Ecology Center of Infectious Animal Diseases Faculty of Environmental Sciences Czech University of Life Sciences Prague Czechia

Department of Parasitology Faculty of Science Charles University Prague Czechia

Department of Zoology and Fisheries Center of Infectious Animal Diseases Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Czechia

Hull York Medical School University of York York UK

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Prague Czechia

York Biomedical Research Institute University of York York UK

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World Health Organisation. Schistosomiasis. 2023. In: https://www.who.int/news-room/fact-sheets/detail/schistosomiasis.

Schwartz C, Fallon PG. Schistosoma “Eggs-iting” the host: Granuloma formation and egg excretion. Front Immunol. 2018. 10.3389/fimmu.2018.02492. PubMed PMC

Ashton PD, Harrop R, Shah B, Wilson R, a,. The schistosome egg: development and secretions. Parasitology. 2001;122:329–38. PubMed

Carson JP, Robinson MW, Hsieh MH, Cody J, Le L, You H, McManus DP, Gobert GN. A comparative proteomics analysis of the egg secretions of three major schistosome species. Mol Biochem Parasitol. 2020. 10.1016/j.molbiopara.2020.111322. PubMed PMC

Cass CCL, Johnson JR, Califf LL, Xu T, Hernandez HJ, Stadecker MJ, Yates JR, Williams DL. Proteomic analysis of Schistosoma mansoni egg secretions. Mol Biochem Parasitol. 2007;155:84–93. PubMed PMC

Mathieson W, Wilson RA. A comparative proteomic study of the undeveloped and developed Schistosoma mansoni egg and its contents: The miracidium, hatch fluid and secretions. Int J Parasitol. 2010;40:617–28. PubMed

Colley DG, Secor WE. Immunology of human schistosomiasis. Parasite Immunol. 2014;36:347–57. PubMed PMC

Costain AH, MacDonald AS, Smits HH. Schistosome Egg Migration: Mechanisms, Pathogenesis and Host Immune Responses. Front Immunol. 2018. 10.3389/fimmu.2018.03042. PubMed PMC

Everts B, Hussaarts L, Driessen NN, et al. Schistosome-derived omega-1 drives Th2 polarization by suppressing protein synthesis following internalization by the mannose receptor. J Exp Med. 2012;209:1753–67. PubMed PMC

Knuhr K, Langhans K, Nyenhuis S, Viertmann K, Overgaard Kildemoes AM, Doenhoff MJ, Haas H, Schramm G. Schistosoma mansoni egg-released IPSE/alpha-1 dampens inflammatory cytokine responses viabasophil interleukin (IL)-4 and IL-13. Front Immunol. 2018. 10.3389/fimmu.2018.02293. PubMed PMC

Farias LP, Rodrigues D, Cunna V, Rofatto HK, Faquim-Mauro EL, Leite LCC. Schistosoma mansoni venom allergen like proteins present differential allergic responses in a murine model of airway inflammation. PLoS Negl Trop Dis. 2012. 10.1371/journal.pntd.0001510. PubMed PMC

Wilbers RHP, Schneiter R, Holterman MHM, Drurey C, Smant G, Asojo OA, Maizels RM, Lozano-Torres JL. Secreted venom allergen-like proteins of helminths: Conserved modulators of host responses in animals and plants. PLoS Pathog. 2018. 10.1371/journal.ppat.1007300. PubMed PMC

Chalmers IW, Hoffmann KF. Platyhelminth Venom Allergen-Like (VAL) proteins: Revealing structural diversity, class-specific features and biological associations across the phylum. Parasitology. 2012;139:1231–45. PubMed PMC

Chalmers IW, McArdle AJ, Coulson RMR, Wagner MA, Schmid R, Hirai H, Hoffmann KF. Developmentally regulated expression, alternative splicing and distinct sub-groupings in members of the Schistosoma mansoni venom allergen-like (SmVAL) gene family. BMC Genomics. 2008. 10.1186/1471-2164-9-89. PubMed PMC

Hunt VL, Tsai IJ, Coghlan A, et al. The genomic basis of parasitism in the Strongyloides clade of nematodes. Nat Genet. 2016;48:299–307. PubMed PMC

Buddenborg SK, Lu Z, Sankaranarayan G, Doyle SR, Berriman M. The stage- and sex-specific transcriptome of the human parasite Schistosoma mansoni. Sci Data. 2023. 10.1038/s41597-023-02674-2. PubMed PMC

Fernandes RS, Barbosa TC, Barbosa MMF, Miyasato PA, Nakano E, Leite LCC, Farias LP. Stage and tissue expression patterns of Schistosoma mansoni venom allergen-like proteins SmVAL 4, 13, 16 and 24. Parasit Vectors. 2017. 10.1186/s13071-017-2144-2. PubMed PMC

Perally S, Geyer KK, Farani PSG, et al. Schistosoma mansoni venom allergen-like protein 6 (SmVAL6) maintains tegumental barrier function. Int J Parasitol. 2021;51:251–61. PubMed PMC

Rofatto HK, Parker-Manuel SJ, Barbosa TC, Tararam CA, Alan Wilson R, Leite LCC, Farias LP. Tissue expression patterns of Schistosoma mansoni Venom Allergen-Like proteins 6 and 7. Int J Parasitol. 2012;42:613–20. PubMed

Attenborough T, Rawlinson KA, Soria CLD, Ambridge K, Sankaranarayanan G, Graham J, Cotton JA, Doyle SR, Rinaldi G, Berriman M. A single-cell atlas of the miracidium larva of the human blood fluke Schistosoma mansoni: cell types, developmental pathways and tissue architecture. 2024. 10.7554/eLife.95628.2. PubMed PMC

Peterková K, Konečný L, Macháček T, Jedličková L, Winkelmann F, Sombetzki M, Dvořák J. Winners vs. losers: Schistosoma mansoni intestinal and liver eggs exhibit striking differences in gene expression and immunogenicity. PLoS Pathog. 2024. 10.1371/journal.ppat.1012268. PubMed PMC

Yoshino TP, Brown M, Wu XJ, Jackson CJ, Ocadiz-Ruiz R, Chalmers IW, Kolb M, Hokke CH, Hoffmann KF. Excreted/secreted Schistosoma mansoni venom allergen-like 9 (SmVAL9) modulates host extracellular matrix remodelling gene expression. Int J Parasitol. 2014;44:551–63. PubMed PMC

Farias LP, Chalmers IW, Perally S, et al. Schistosoma mansoni venom allergen-like proteins: phylogenetic relationships, stage-specific transcription and tissue localization as predictors of immunological cross-reactivity. Int J Parasitol. 2019;49:593–9. PubMed PMC

Wu XJ, Sabat G, Brown JF, Zhang M, Taft A, Peterson N, Harms A, Yoshino TP. Proteomic analysis of Schistosoma mansoni proteins released during in vitro miracidium-to-sporocyst transformation. Mol Biochem Parasitol. 2009;164:32–44. PubMed PMC

Howe KL, Bolt BJ, Shafie M, Kersey P, Berriman M. WormBase ParaSite − a comprehensive resource for helminth genomics. Mol Biochem Parasitol. 2017;215:2–10. PubMed PMC

Buddenborg SK, Tracey A, Berger DJ, et al. Assembled chromosomes of the blood fluke Schistosoma mansoni provide insight into the evolution of its ZW sex-determination system. 2021. 10.1101/2021.08.13.456314.

Prawer YDJ, Stroehlein AJ, Young ND, Kapoor S, Hall RS, Ghazali R, Batterham P, Gasser RB, Perry T, Anstead CA. Major SCP/TAPS protein expansion in Lucilia cuprina is associated with novel tandem array organisation and domain architecture. Parasit Vectors. 2020. 10.1186/s13071-020-04476-6. PubMed PMC

Käll L, Krogh A, Sonnhammer ELL. Advantages of combined transmembrane topology and signal peptide prediction-the Phobius web server. Nucleic Acids Res. 2007. 10.1093/nar/gkm256. PubMed PMC

Teufel F, Almagro Armenteros JJ, Johansen AR, Gíslason MH, Pihl SI, Tsirigos KD, Winther O, Brunak S, von Heijne G, Nielsen H. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol. 2022;40:1023–5. PubMed PMC

HMMER: biosequence analysis using profile hidden Markov models. http://hmmer.org.

Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol Biol Evol. 2013;30:772–80. PubMed PMC

Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25:1972–3. PubMed PMC

Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Von Haeseler A, Lanfear R, Teeling E. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol Biol Evol. 2020;37:1530–4. PubMed PMC

FigTree. http://tree.bio.ed.ac.uk/software/figtree/.

Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. Jalview Version 2-A multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25:1189–91. PubMed PMC

Wangwiwatsin A, Protasio AV, Wilson S, Owusu C, Holroyd NE, Sanders MJ, Keane J, Doenhoff MJ, Rinaldi G, Berriman M. Transcriptome of the parasitic flatworm schistosoma mansoni during intra-mammalian development. PLoS Negl Trop Dis. 2020;14:1–25. PubMed PMC

Smid M, Coebergh van den Braak RRJ, van de Werken HJG, et al. Gene length corrected trimmed mean of M-values (GeTMM) processing of RNA-seq data performs similarly in intersample analyses while improving intrasample comparisons. BMC Bioinformatics. 2018. 10.1186/s12859-018-2246-7. PubMed PMC

Jedličková L, Dvořák J, Hrachovinová I, Ulrychová L, Kašný M, Mikeš L. A novel Kunitz protein with proposed dual function from Eudiplozoon nipponicum (Monogenea) impairs haemostasis and action of complement in vitro. Int J Parasitol. 2019;49:337–46. PubMed

Crosnier C, Hokke CH, Protasio AV, et al. Screening of a Library of Recombinant Schistosoma mansoni Proteins With Sera From Murine and Human Controlled Infections Identifies Early Serological Markers. In: Journal of Infectious Diseases. Oxford University Press, 2022. pp 1435–1446. PubMed PMC

Sun Y, Gallagher-Jones M, Barker C, Wright GJ. A benchmarked protein microarray-based platform for the identification of novel low-affinity extracellular protein interactions. Anal Biochem. 2012;424:45–53. PubMed PMC

Shilts J, Severin Y, Galaway F, Müller-Sienerth N, Chong ZS, Pritchard S, Teichmann S, Vento-Tormo R, Snijder B, Wright GJ. A physical wiring diagram for the human immune system. Nature. 2022;608:397–404. PubMed PMC

Crosnier C, Bustamante LY, Bartholdson SJ, et al. Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum. Nature. 2011;480:534–7. PubMed PMC

Takaki KK, Rinaldi G, Berriman M, Pagán AJ, Ramakrishnan L. Schistosoma mansoni Eggs Modulate the Timing of Granuloma Formation to Promote Transmission. Cell Host Microbe. 2021;29:58-67.e5. PubMed PMC

Collins JJ, King RS, Cogswell A, Williams DL, Newmark PA. An atlas for schistosoma mansoni organs and life-cycle stages using cell type-specific markers and confocal microscopy. PLoS Negl Trop Dis. 2011. 10.1371/journal.pntd.0001009. PubMed PMC

Haeberlein S, Angrisano A, Quack T, Lu Z, Kellershohn J, Blohm A, Grevelding CG, Hahnel SR. Identification of a new panel of reference genes to study pairing-dependent gene expression in Schistosoma mansoni. Int J Parasitol. 2019;49:615–24. PubMed

Lu Z, Sessler F, Holroyd N, Hahnel S, Quack T, Berriman M, Grevelding CG. Data Descriptor: A gene expression atlas of adult Schistosoma mansoni and their gonads. Sci Data. 2017. 10.1038/sdata.2017.118. PubMed PMC

Linder E. The Schistosome Egg in Transit. Ann Clin Pathol. 2017;5:1110.

Curwen RS, Ashton PD, Sundaralingam S, Wilson RA. Identification of Novel Proteases and Immunomodulators in the Secretions of Schistosome Cercariae That Facilitate Host Entry. Mol Cell Proteomics. 2006;5:835–44. PubMed

Fernandes RS, Fernandes LGV, de Godoy AS, Miyasato PA, Nakano E, Farias LP, Nascimento ALTO, Leite LCC. Schistosoma mansoni venom allergen-like protein 18 (SmVAL18) is a plasminogen-binding protein secreted during the early stages of mammalian-host infection. Mol Biochem Parasitol. 2018;221:23–31. PubMed

Hansell E, Braschi S, Medzihradszky KF, Sajid M, Debnath M, Ingram J, Lim KC, McKerrow JH. Proteomic analysis of skin invasion by blood fluke larvae. PLoS Negl Trop Dis. 2008. 10.1371/journal.pntd.0000262. PubMed PMC

Dolečková K, Albrecht T, Mikeš L, Horák P. Cathepsins B1 and B2 in the neuropathogenic schistosome Trichobilharzia regenti: Distinct gene expression profiles and presumptive roles throughout the life cycle. Parasitol Res. 2010;107:751–5. PubMed

Parker-Manuel SJ, Ivens AC, Dillon GP, Wilson RA. Gene expression patterns in larval Schistosoma mansoni associated with infection of the mammalian host. PLoS Negl Trop Dis. 2011. 10.1371/journal.pntd.0001274. PubMed PMC

Ruth Lawson J, Wilson RA. The survival of the cercariae of Schistosoma mansoni in relation to water temperature and glycogen utilization. Parasitology. 1980;81:337–48. PubMed

Darwiche R, Mène-Saffrané L, Gfeller D, Asojo OA, Schneiter R. The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein. J Biol Chem. 2017;292:8304–14. PubMed PMC

Kelleher A, Darwiche R, Rezende WC, Farias LP, Leite LCC, Schneiter R, Asojo OA. Erratum : Schistosoma mansoni venom allergen-like protein 4 (SmVAL4) is a novel lipid-binding SCP, TAPS protein that lacks the prototypical CAP motifs. Corrigendum (Acta Crystallographica Section D: Biological Crystallography D70, 2014 2186–2196). Acta Crystallogr D Biol Crystallogr. 2015;71:1022. PubMed PMC

Ma D, Xu X, An S, et al. A novel family of RGD-containing disintegrins (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets αIIbβ3 or αVβ3 and inhibits platelet aggregation and angiogenesis. Thromb Haemost. 2011;105:1032–45. PubMed PMC

Xu E, Francischetti IMB, Lai R, Ribeiro JMC, Andersen JF. Structure of protein having inhibitory disintegrin and leukotriene scavenging functions contained in single domain. J Biol Chem. 2012;287:10967–76. PubMed PMC