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

Abstract

Babesia bigemina and Babesia bovis, are the two major causes of bovine babesiosis, a global neglected disease in need of improved methods of control. Here, we describe a shared method for the stable transfection of these two parasites using electroporation and blasticidin/blasticidin deaminase as a selectable marker. Stably transfected B. bigemina and B. bovis were obtained using a common transfection plasmid targeting the enhanced green fluorescent protein-BSD (egfp-bsd) fusion gene into the elongation factor-1α (ef-1α) locus of B. bigemina and B. bovis under the control of the B. bigemina ef-1α promoter. Sequencing, Southern blotting, immunoblotting and immunofluorescence analysis of parasite-infected red blood cells, demonstrated that the egfp-bsd gene was expressed and stably integrated solely into the ef-1α locus of both, B. bigemina and B. bovis. Interestingly, heterologous B. bigemina ef-1α sequences were able to drive integration into the B. bovis genome by homologous recombination, and the stably integrated B. bigemina ef-1α-A promoter is fully functional in B. bovis. Collectively, the data provides a new tool for genetic analysis of these parasites, and suggests that the development of vaccine platform delivery systems based on transfected B. bovis and B. bigemina parasites using homologous and heterologous promoters is feasible.

Figure 1

Experimental strategy used for the stable transfection of B. bigemina and B. bovis cell lines. (a) Schematic representation of the target ef-1α locus of B. bigemina in B. bigemina parasites (bigemina-big-ef-egfp-bsd). (b) Schematic representation of the stable transfection plasmid pbig-ef-egfp-bsd used for electroporation. (c) Schematic representation of the target ef-1α locus of B. bigemina in B. bovis parasites (bovis-big-ef-egfp-bsd). BgIII: restriction enzyme.

Figure 2

Analysis of B. bigemina and B. bovis parasites by fluorescence. (a) Bigemina-big-ef-egfp-bsd and bovis-big-ef-egfp-bsd transfected parasites observed under bright field, green laser (eGFP), blue laser (DAPI) and merge images. (b) B. bigemina and B. bovis wild-type parasites observed under bright field, green laser (eGFP), blue laser (DAPI) and merge images. Amplification 630x.

Figure 3

In vitro growth curve of B. bigemina and B. bovis parasites up to 72 hr. (a) B. bigemina growth curve. (b) B. bovis growth curve. Tbg + bsd: bigemina-big-ef-egfp-bsd parasites in the presence of blasticidin; Tbg-bsd: bigemina-big-ef-egfp-bsd parasites in the absence of blasticidin; wt Bbg + bsd: B. bigemina wild-type parasites in the presence of blasticidin; wt Bbg-bsd: B. bigemina wild-type parasites in the absence of blasticidin; Tbo + bsd: bovis-big-ef-egfp-bsd parasites in the presence of blasticidin; Tbo-bsd: bovis-big-ef-egfp-bsd parasites in the absence of blasticidin; wt Bbo + bsd: B. bovis wild-type parasites in the presence of blasticidin; wt Bbo-bsd: B. bovis wild-type parasites in the absence of blasticidin. Normalized PPE values (Y axis) obtained from Babesia spp. in the presence or absence of blasticidin (X axis). Media was used as negative controls for no inhibition. Error bars indicate standard deviations for each sample tested from triplicate culture. Data were compared using ANOVA analysis. *Represents P < 0.05 indicating a statistically significant difference between groups.

Figure 4

Southern blot analysis using dig-labeled probes against: (a) B. bigemina rap-1a; (b) B. bovis msa-1; (c) egfp and (d) ef-1α. 1: wild-type B. bigemina gDNA digested with BgIII; 2: wild-type B. bigemina gDNA undigested; 3: bigemina-big-ef-egfp-bsd digested with BgIII; 4: bigemina-big-ef-egfp-bsd undigested; DM: Dig labeled DNA marker II; Pr-: pBS promoterless control plasmid; C-: pbig-ef-egfp-bsd plasmid control; 5: wild-type B. bovis gDNA digested with BgIII; 6: wild-type B. bovis gDNA undigested; 7: bovis-big-ef-egfp-bsd digested with BgIII; 8: bovis-big-ef-egfp-bsd undigested. Vertical black stripes on the blot indicate cropping of the blot. A full image of the original blot can be seen in Supplementary Info section.

Figure 5

PCR integration analysis in B. bigemina using two different set of primers. (a) egfp-Fwd and ef1α-Rev. (b) egfp-Fwd and egfp-Rev. Line 1: wild-type B. bigemina gDNA; Line 2: bigemina-big-ef-egfp-bsd; C-: pbig-ef-egfp-bsd plasmid control; MW: molecular size ladder in bp, 1 Kb Plus DNA ladder.

Figure 6

PCR integration analysis in B. bovis culture using two different sets of primers. (a) egfp-Fwd and Bbov-UpS-efB-Rev. (b) rap-1a-Fwd and rap-1a-Rev. Line 1: bovis-big-ef-egfp-bsd gDNA; line 2: wild-type gDNA B. bovis; MW: molecular size ladder in bp, 1 Kb Plus DNA ladder. (c) Alignments among the regions of insertion of the transfected genes into bigemina-big-ef-egfp-bsd and bovis-big-ef-egfp-bsd parasites. Vertical black stripes on the blot indicate where the image was cropped. Full image of the original agar electrophoresis gel can be seen in Supplementary Info section.

Figure 7

Immunoblot analysis. (1) wild-type B. bigemina. (2) bigemina-big-ef-egfp-bsd. (3) wild-type B. bovis. (4) bovis-big-ef-egfp-bsd. (5) Uninfected bovine RBC. Samples were incubated with antibodies: (a) pre-immune mouse serum; (b) anti-B. bigemina rap-1 MAb. (c) anti-B. bovis rap-1 MAb. (d) anti-GFP MAb. (M) molecular size ladder in kDa indicated by arrows. Vertical black stripes on the blot indicate cropping of the blot. A full image of the original blot can be seen in Supplementary Info section.