Primer sequences used in experiments.

Tetraspanins (TSPANs) are a family of highly conserved proteins present in a wide variety of eukaryotes. Although protein-protein interactions of TSPANs have been well established in eukaryotes including parasitic protists, the role they play in parasitism and pathogenesis remains largely unknown. In this study, we characterized three representative members of TSPANs, TSPAN4, TSPAN12, and TSPAN13 from the human intestinal protozoan Entamoeba histolytica. Co-immunoprecipitation assays demonstrated that TSPAN4, TSPAN12 and TSPAN13 are reciprocally pulled down together with several other TSPAN-interacting proteins including TSPAN binding protein of 55kDa (TBP55) and interaptin. Blue native-PAGE analysis showed that these TSPANs form several complexes of 120–250 kDa. Repression of tspan12 and tspan13 gene expression led to decreased secretion of cysteine proteases, while repression of tspan4 led to a four-fold increase in the activity of cysteine proteases in crude extracellular vesicles (EVs) fraction. Meanwhile, strains overexpressing HA-tagged TSPAN12 and TSPAN13 demonstrated reduced adhesion to collagen. Altogether, this study reveals that the TSPANs, especially TSPAN12 and TSPAN13, are engaged with complex protein-protein interactions and are involved in the pathogenicity-related biological functions such as protease secretion and adhesion, offering insights into the potential regulatory mechanisms of tetraspanins in protozoan parasites.