Abstract
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.
Highlights
Blastocystis is a common enteric microbial eukaryote belonging to the Stramenopiles, infecting more than 1 billion humans along with a large variety of non-human hosts, such as farm animals, rodents, birds, reptiles and others[1,2]
Genetic modifications in which specific parasite molecules or organelles are rendered visible via reporter systems or epitope tags may lead to a better understanding of the cell biology of this poorly understood but widespread parasite
A genetic tool for manipulating gene expression in Blastocystis is hitherto lacking, transient or stable transfection approaches have been successfully achieved in several parasitic protists, including trypanosomatid parasites Trypanosoma brucei[34,35], T. cruzi[36] and Leishmania spp.[36]; apicomplexan parasites Plasmodium spp.[37], Cryptosporidium spp38. and Toxoplasma gondii[39]; parasitic excavates such as Trichomonas vaginalis[40] and Giardia intestinalis[41,42] and parasitic amoeba Entamoeba histolytica[43]
Summary
Received: 30 October 2018 Accepted: 17 January 2019 Published: xx xx xxxx of Ectopic Genes. 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. 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. Genetic modifications in which specific parasite molecules or organelles are rendered visible via reporter systems or epitope tags may lead to a better understanding of the cell biology of this poorly understood but widespread parasite
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