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The microbial parasite  Blastocystis  colonizes the large intestines of numerous animal species and increasing evidence has linked  Blastocystis  infection to enteric diseases with signs and symptoms including abdominal pain, constipation, diarrhea, nausea, vomiting, and flatulence. It has also recently been reported to be an important member of the host intestinal microbiota. Despite significant advances in our understanding of  Blastocystis  cell biology and host-parasite interactions, a genetic modification tool is absent. In this study, we successfully established a robust gene delivery protocol for  Blastocystis  subtype 7 (ST7) and ectopic protein expression was further tested using a high sensitivity nano-luciferase (Nluc) reporter system, with promoter regions from several genes. Among them, a strong promoter encompassing a region upstream of the legumain 5′ UTR was identified. Using this promoter combined with the legumain 3′ UTR, which contains a conserved, precise polyadenylation signal, a robust transient transfection technique was established for the first time in  Blastocystis . This system was validated by ectopic expression of proteins harbouring specific localization signals. The establishment of a robust, reproducible gene modification system for  Blastocystis  is a significant advance for  Blastocystis  research both  in vitro  and  in vivo . This technique will spearhead further research to understand the parasite’s biology, its role in health and disease, along with novel ways to combat the parasite.

Successful Genetic Transfection of the Colonic Protistan Parasite Blastocystis for Reliable Expression of Ectopic Genes