Stable transformation of Babesia bigemina and Babesia bovis using a single transfection plasmid

Authors: SILVA, MARTA - Washington State University; Knowles Jr, Donald; MAZUZ, MONICA - Kimron Veterinary Institute; COOKE, BRIAN - Monash University; Suarez, Carlos

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/26/2018
Publication Date: 4/17/2018
Citation: Silva, M.G., Knowles, D.P., Mazuz, M.L., Cooke, B.M., Suarez, C.E. 2018. Stable transformation of Babesia bigemina and Babesia bovis using a single transfection plasmid. Scientific Reports. 8:6096. doi:10.1038/s41598-018-23010-4.
DOI: https://doi.org/10.1038/s41598-018-23010-4

Interpretive Summary: Tick borne Babesia bigemina is responsible for acute and potentially lethal hemolytic disease in cattle. The development of genetic manipulation tools necessary to the better understanding of parasite biology and novel vaccines is currently limited by the lack of a complete parasite genome and experimental tools such as transfection. A necessary tool is a robust transfection system for genetic analysis and genome modification of B. bigemina. In this study we describe stable transfection in B. bigemina using a plasmid containing B. bigemina gene expression control and integration sequences. In addition, the transfection construct was able to integrate and function in Babesia bovis despite sequence discrepancies among both parasites. The data presented in this study is of significance for the future development of novel transfected vaccines for B. bigemina and B. bovis, and vaccine delivery systems based on transfected Babesia parasites.

Technical Abstract: Bovine babesiosis caused by Babesia bovis and B. bigemina is a severe, often fatal, tick-borne disease of cattle for which improved methods for control are urgently needed. One of our long term goals is to develop effective vaccines based on genetically modfied B. bigemina and B. bovis parasites. Although methods to transfect and transform B. bovis have been developed, stable transfection methods for B. bigemina remain unavailable. Here, we have developed a stable transfection system for B. bigemina, and B. bovis using a previously described transient transfection system and B. bigemina control of gene expression sequences. Blasticidin demonstrated a potent inhibitory activity on the growth of B. bigemina in vitro with an IC50 of 3 'g/ml thus the blasticidin/ blasticidin deaminase (BSD) system was adopted as the drug-selectable marker for the B. bigemina stable transfection system.We generated a transfection plasmid construct designed to integrate an enhanced green fluorescent protein-BSD (egfp-bsd) fusion gene into the elongation factor-1a (ef-1'' locus of B. bigemina under the control of the B. bigemina ef-1''' promoter, and transfected it into B. bigemina (Puerto Rico strain) and B. bovis (T3Bo strain) parasites by electroporation of parasite-infected red blood cells (iRBC), followed by selection for blasticidin resistance. Both B. bigemina and B. bovis, blasticidin-resistant parasites emerged in in vitro cultures following addition of blasticidin. PCR, sequencing, Southern blotting, immunoblotting and immunofluorescence analysis of iRBC, demonstrated that the egfp-bsd gene was expressed and stably integrated solely into the ef-1' locus of B. bigemina and B. bovis blasticidin-resistant parasites. Interestingly, heterologous B. bigemina ef-1' sequences were able to integrate into the B. bovis genome by homologous recombination despite sequence discrepancies in the targeted ef-1' genes among both parasites. In addition, we have shown that the B. bigemina ef-1'-A promoter was fully functional in B. bovis. These findings suggests that future development of vaccine platform delivery systems based on transfected B. bovis and B. bigemina parasites using homologous and heterologous promoters is feasible.