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

Abstract

BackgroundTick-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. Effective promoters, required to regulate expression of transgenes, such as the elongation factor-1 alpha (ef-1α), have been identified in other apicomplexans such as Babesia bovis and Plasmodium falciparum. MethodsThe B. bigemina ef-1a locus was defined by searching a partial genome library of B. bigemina (Sanger Institute). Presence of an intron in the 5’ untranslated region was determined by 5’ Rapid Amplification of cDNA Ends (RACE) analysis. Promoter activity was determined by measurement of luciferase expression at several time points after electroporation, efficiency of transfections and normalization of data was determined by quantitative PCR and by the percentage of parasitized erythrocytes.ResultsThe ef-1α locus contains two identical head to head ef-1α genes separated by a 1.425 kb intergenic (IG) region. Significant sequence divergence in the regions upstream of the inverted repeats on each side of the B. bigemina IG region suggest independent regulation mechanisms for controlling expression of each of the two ef-1α 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-1α IG region tested showed the ability to promote high level production of luciferase. Moreover, both B. bigemina ef-1α promoters are also active in transiently transfected B. bovis and conversely, a B. bovis ef-1α promoter is active in transiently transfected B. bigemina. ConclusionsCollectively these data demonstrate the existence of two distinct promoters with homologous and heterologous promoter function in B. bigemina and B. bovis which is described for the first time in Babesia species. This study is of significance for development of interspecies stable transfection systems for B. bigemina and for B. bovis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1859-9) contains supplementary material, which is available to authorized users.

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

“…Figure 2 ) provide hints on the mechanisms of homologous recombination operating in the parasite. Additionally, and consistent with previous findings 10 , the ef-1α promoter of B . bigemina was able to generate expression levels of the egfp-bsd gene that are sufficient to sustain growth of transfected parasites at high levels of blasticidin.…”

“…bigemina and B . bovis cultures were grown in 96-well plates in bovine red blood cells (RBC) at 5% or 10% hematocrit, respectively, using HL-1 culture media at pH 7.2, and were incubated at 37 °C in an atmosphere of 5% CO 2 and 90% N 2 10 .…”

“…The plasmid containing the ef-1α-A promoter and luciferase gene along with the 3′ rap-1a stop region used in the B . bigemina transient transfection 10 was digested with Eco RI restriction enzyme to remove the luciferase gene and the resulting linearized vector now containing just the B . bigemina ef-1a-A promoter and B .…”

“…bigemina genome ( http://www.sanger.ac.uk/resources/downloads/protozoa/babesia-bigemina.html ) and the previous characterization of the B . bigemina ef-1α locus 10 , indicate that the structure of this locus is essentially identical in both B . bigemina and B .…”

Establishment of a transient transfection system for Babesia sp. Xinjiang with the use of homologous promoters

“…Genetic manipulation tools are considered as effective measures to identify virulence factors of particular agents, understand host-parasite interactions, and elucidate the mechanisms underlying drug resistance [11]. Such techniques have been described in several protozoan parasites, including Plasmodium falciparum, Toxoplasma gondii, Cryptosporidium parvum, Theileria annulata, Babesia bovis, Babeisa bigemina, Babeisa ovata, Babesia gibsoni, and Babesia ovis [12][13][14][15][16][17][18][19][20][21]. Mapping and sequencing of the Bxj genome were completed by our laboratory (Vector and Vector-Borne Diseases Laboratory, Lanzhou Veterinary Research Institute) in 2006.…”