title: Transfection of Babesia bovis by Double Selection with WR99210 and Blasticidin-S and Its Application for Functional Analysis of Thioredoxin Peroxidase-1.
abstract: Genetic manipulation is an essential technique to analyze gene function; however, limited methods are available for Babesia bovis, a causative pathogen of the globally important cattle disease, bovine babesiosis. To date, two stable transfection systems have been developed for B. bovis, using selectable markers blasticidin-S deaminase (bsd) or human dihydrofolate reductase (hdhfr). In this work, we combine these two selectable markers in a sequential transfection system. Specifically, a parent transgenic B. bovis line which episomally expresses green fluorescent protein (GFP) and human dihydrofolate reductase (hDHFR), was transfected with a plasmid encoding a fusion protein consisting of red fluorescent protein (RFP) and blasticidin-S deaminase (BSD). Selection with WR99210 and blasticidin-S resulted in the emergence of parasites double positive for GFP and RFP. We then applied this method to complement gene function in a parasite line in which thioredoxin peroxidase-1 (Bbtpx-1) gene was knocked out using hDHFR as a selectable marker. A plasmid was constructed harboring both RFP-BSD and Bbtpx-1 expression cassettes, and transfected into a Bbtpx-1 knockout (KO) parasite. Transfectants were independently obtained by two transfection methods, episomal transfection and genome integration. Complementation of Bbtpx-1 resulted in full recovery of resistance to nitrosative stress, via the nitric oxide donor sodium nitroprusside, which was impaired in the Bbtpx-1 KO parasites. In conclusion, we developed a sequential transfection method in B. bovis and subsequently applied this technique in a gene complementation study. This method will enable broader genetic manipulation of Babesia toward enhancing our understanding of the biology of this parasite.
authors.affiliation: Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto, Nagasaki, Japan; National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Asada
authors.affiliation: Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto, Nagasaki, Japan.
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Yahata
authors.affiliation: Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto, Nagasaki, Japan; National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Japan.
authors.forename: Hassan
authors.initials: H
authors.lastname: Hakimi
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Japan.
authors.forename: Naoaki
authors.initials: N
authors.lastname: Yokoyama
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Igarashi
authors.affiliation: Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto, Nagasaki, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Kaneko
authors.affiliation: Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, United States of America; Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Pullman, Washington, United States of America.
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Suarez
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Japan.
authors.forename: Shin-ichiro
authors.initials: S
authors.lastname: Kawazu
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D016793:Babesia bovis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D001404:Babesiosis / Q000469:parasitology*; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology*; D015870:Gene Expression; D055786:Gene Knockout Techniques; D023061:Gene Order; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D056426:Genetic Loci; D009705:Nucleosides / Q000494:pharmacology; D054464:Peroxiredoxins / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D015203:Reproducibility of Results; D013312:Stress, Physiological; D014162:Transfection; D014227:Triazines / Q000494:pharmacology
chemical_list: D009705:Nucleosides; D014227:Triazines; C006201:BRL 6231; C004500:blasticidin S; D054464:Peroxiredoxins