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Title: Identification of interchangeable cross-species function of elongation factor-1 alpha promoters in babesia bigemina and babesia bovis

Author
item Suarez, Carlos
item Knowles Jr, Donald
item SILA, MARTA - Washington State University

Submitted to: Parasites & Vectors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/25/2016
Publication Date: 11/11/2016
Citation: Suarez, C.E., Knowles, D.P., Sila, M. 2016. Identification of interchangeable cross-species function of elongation factor-1 alpha promoters in babesia bigemina and babesia bovis. Parasites & Vectors. doi:10.1186/s13071-016-1859-9.

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 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 combination with transfection technology, B. bigemina gene regulatory regions need to be identified. In this study we identify and characterize a bidirectional promoter of B. bigemina. The data demonstrate the existence of two distinct promoters with homologous and heterologous promoter function in B. bigemina and Babesia bovis which is described for the first time in Babesia species. The data presented in this study is of significance for the future development of interspecies stable transfection systems for B. bigemina and for B. bovis.

Technical Abstract: Background: 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 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 combination with transfection technology, B. bigemina gene regulatory regions need to be identified. Effective promoters, required to regulate expression of transgenes, such as the elongation factor 1-alpha (ef-1a), have been identified in a related B. bovis 1.4 kb genomic region separating two identical elongation factor 1-alpha (ef-1a) genes, and in other apicomplexans such as Plasmodium falciparum. Methods: The B. bigemina ef-1a locus was defined by searching a partial genome library of B. bigemina (Sanger Institute). Locus structure was confirmed by sequencing PCR amplification products. Presence of an intron in the 5’ untranslated region (UTR) was determined by RACE analysis performed on total RNA extracted from in vitro cultured B. bigemina and sequencing. Promoter activity was determined by measurement of luciferase expression at several time points after electroporation (1.25 kV, 200 O, 25 µF in a BioRad electroporator), efficiency of transfections and normalization of data was determined by quantitative PCR methods and the computation of the percentage of parasitized erythrocytes. Results: The ef-1a locus, contains two identical head to head ef-1a genes separated by a 1,425 Kb intergenic (IG) region. The organization deduced for the ef-1a locus of B. bigemina is similar to the ef-1a genes in B. bovis and Plasmodium falciparum. Sequence comparisons among the B. bovis and B. bigemina ef-1a loci shows high homology in the ef-1a open reading frames (ORFs), but sequences in their IG regions differ significantly. Regardless of this, several structural similarities occur among the B. bovis and B. bigemina IG regions, including an almost identical length (1424 vs. 1425 bp), the presence of inverted repeats in each of the 5’ and 3’ ends, and introns present in each 5’ untranslated region of both ef-1a genes. Significant sequence divergence in the regions upstream of the inverted repeats (IR) on each side of the B. bigemina IG region suggest independent regulation mechanisms for controlling expression of each of the two ef-1a genes. Plasmid constructs containing the 5’ and 3’ halves of the IG regions controlling the expression of the luciferase gene containing a 3’ region of a B. bigemina rap-1a gene, were generated for the testing of luciferase activity in transiently transfected parasites. Both halves of the ef-1a IG region tested showed the ability to promote high level production of luciferase. Interestingly, both B. bigemina ef-1 a promoters are also active in transiently transfected B. bovis and conversely, a B. bovis ef-1a promoter is active in transiently transfected B. bigemina. Conclusions: Collectively these data demonstrate the existence of two distinct promoters with homologous and heterologous promoter function in B. bigemina and Babesia bovis which is described for the first time in Babesia species. The data presented in this study is of significance for the future development of interspecies stable transfection systems for B. bigemina and for B. bovis.