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Tetraspanins (TSPANs) are a family of proteins highly conserved in all 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. Author summary: Tetraspanins (TSPANs) are involved in critical roles such as cellular communication, adhesion, and immune responses. However, their function in parasitic protozoa, particularly in relation to disease-causing mechanisms, remains poorly understood. Our study explores the role of three tetraspanins (TSPAN4, TSPAN12, and TSPAN13) in Entamoeba histolytica, an intestinal parasite which causes amebiasis, and a significant health problem affecting millions of people worldwide. Our findings reveal that these amebic TSPANs form various protein complexes similar to what is observed in other organisms. We also found that amebic TSPANs influence crucial processes related to the parasite's pathogenicity, such as adhesion to collagen and secretion of cysteine proteases (CPs). Gene silencing of TSPAN12 and TSPAN13 caused reduction in CP secretion, while overexpression resulted in decreased adhesion. Additionally, TSPAN4 appears to play a distinct role in exosome-mediated pathways. Our findings provide insights into a potential alternative mechanism of CP secretion via exosomes, in which amebic TSPANs may play a role. [ABSTRACT FROM AUTHOR]

Tetraspanins are membrane proteins involved in intra- and/or intercellular signaling, and membrane protein complex formation. In some organisms, their role is associated with virulence and pathogenesis. Here, we investigate known and potential tetraspanins in the human intestinal protozoan parasite Entamoeba histolytica. We conducted sequence similarity searches against the proteome data of E. histolytica and newly identified nine uncharacterized proteins as potential tetraspanins in E. histolytica. We found three subgroups within known and potential tetraspanins, as well as subgroup-associated features in both their amino acid and nucleotide sequences. We also examined the subcellular localization of a few representative tetraspanins that might be potentially related to pathogenicity. The results in this study could be useful resources for further understanding and downstream analyses of tetraspanins in Entamoeba. [ABSTRACT FROM AUTHOR]