Identification of villin-interacting virulence factors of Blastocystis hominis using yeast two-hybrid screening

Blastocystis hominis (B. hominis), one of the most common intestinal protozoa infecting humans worldwide, is associated with gastrointestinal disorders, yet it remains unclear whether it secretes virulence factors that interact with villin, a key cytoskeletal protein essential for brush border homeostasis and epithelial barrier function. In this study, we constructed a high-quality normalized full-length B. hominis cDNA library (capacity 5.2 × 10⁶ CFU; recombination rate 95.8%) and performed yeast two-hybrid screening using villin as bait. Thirty initial positive clones were obtained, and sequencing combined with BLAST identified 24 unique prey proteins. Among these, 11 were in-frame (ORF-true), 8 proteins were selected for pairwise verification, and 6 proteins were ultimately confirmed to specifically interact with villin, including pyridine nucleotide transhydrogenase (XP_014525634), an ATP/GTP-binding protein-like molecule (OAO14457), and four hypothetical proteins (XP_012896330, XP_012895057, XP_012893676, XP_012894607). GO and KEGG analyses performed on the 24 prey proteins indicated enrichment in cellular metabolic processes and membrane-associated pathways. These findings identify multiple B. hominis proteins capable of interacting with villin, provide new insights into parasite-induced epithelial damage, and offer potential biomarkers and candidate targets for drug and vaccine development against blastocystosis.