Identification and characterization of EhSWP8, a conserved heparin-dependent adhesin from the shrimp microsporidia Ecytonucleospora hepatopenaei.

Ecytonucleospora hepatopenaei (EHP), the causative agent of shrimp hepatopancreatic microsporidiosis (HPM), induces growth retardation, heightened secondary infection susceptibility, and substantial global shrimp aquaculture economic losses. The lack of mechanistic insights into EHP pathogenesis, particularly key virulence factors, has impeded diagnostics and therapeutics development. Here, a novel EHP spore wall protein, EhSWP8, was characterized by bioinformatics profiling, prokaryotic expression/antibody production, indirect immunofluorescence assay (IFA), and immunoelectron microscopy (IEM). EhSWP8 (326 aa, 36.8 kDa) contained 34 predicted phosphorylation sites, two N-glycosylation sites, and a heparin-binding motif (HBM, residues 50-55, YKKMKQ) characterized by an overall positively charged surface and high relative solvent accessibility (RSA > 0.5), suggesting that the motif is surface-exposed and structurally competent to mediate molecular interactions. EhSWP8 shared the closest phylogenetic affinity with Enterocytozoon bieneusi hypothetical protein EDQ31248.1, and its identity was 100% across five geographic EHP strains. Recombinant EhSWP8 expressed in Escherichia coli Rosetta was confirmed by SDS-PAGE. Western blot verified the polyclonal antibody specifically against native EhSWP8 in purified EHP spores. IFA/IEM revealed predominant exospore localization of EhSWP8. Re-analysis of three public omics datasets further showed that EhSWP8 is the fifth most abundant EHP transcript during host infection (median ranks #4-#8 of 2536 transcripts), that the EhSWP8 protein increases 41.6-fold with infection burden. Crucially, a heparin magnetic-bead assay provided functional evidence that native, surface-exposed EhSWP8 mediates heparin-dependent adhesion of EHP spores, with free-heparin competition and antibody blocking reducing binding by 70.6% and 39.4%, respectively. These findings establish EhSWP8 as a conserved, heparin-dependent adhesin, positioning it as both a novel surveillance target and a foundation for mechanistic insights into EHP pathogenesis.