Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase

Abstract

Theileria equi, an intraerythrocytic protozoan parasite, causes equine piroplasmosis, a disease which negatively impacts the global horse industry. Genetic manipulation is one of the research tools under development as a control method for protozoan parasites, but this technique needs to be established for T. equi. Herein, we report on the first development of a stable transgenic T. equi line expressing enhanced green fluorescent protein/blasticidin S deaminase (eGFP/BSD). To express the exogenous fusion gene in T. equi, regulatory regions of the elongation factor-1 alpha (ef-1α) gene were identified in T. equi. An eGFP/BSD-expression cassette containing the ef-1α gene promoter and terminator regions was constructed and integrated into the T. equi genome. On day 9 post-transfection, blasticidin-resistant T. equi emerged. In the clonal line of T. equi obtained by limiting dilution, integration of the eGFP/BSD-expression cassette was confirmed in the designated B-locus of the ef-1α gene via PCR and Southern blot analyses. Parasitaemia dynamics between the transgenic and parental T. equi lines were comparable in vitro. The eGFP/BSD-expressing transgenic T. equi and the methodology used to generate it offer new opportunities for better understanding of T. equi biology, with the add-on possibility of discovering effective control methods against equine piroplasmosis.

Figure 1

Construction of the enhanced green fluorescent protein/blasticidin S deaminase (eGFP/BSD)-expression cassette targeting the elongation factor-1α-B gene locus in T. equi genome. (a) Visualisation of the amplicons obtained from 5′-RACE PCR (5′) and 3′-RACE PCR (3′). M, 100 base-pair DNA ladder marker. (b) Structure of head-to-head orientation of two copies of elongation factor-1α (ef-1α) genes in the T. equi (USDA strain) genome. The ef-1α gene loci are flanked by ribonucleoside-diphosphate reductase (rdrs) and glutamyl-tRNA synthetase (gluRS) genes. Regions containing the terminator (ter), a part of the intergenic region (IG), homologous flanking fragment A (flank A), and homologous flanking fragment B (flank B) that were selected for plasmid construction are highlighted in grey, orange, blue, and yellow, respectively. (c) Magnified structure of the IG containing inverted region-A (IR-A), non-inverted region, and inverted region-B (IR-B). (d) The eGFP/BSD-expression cassette. The expression cassette included green fluorescent protein (gfp) and blasticidin S deaminase (bsd) genes. (e) Genomic integration of the eGFP/BSD-expression cassette into the ef-1α-B gene locus of T. equi.

Figure 2

Transfection and isolation of the enhanced green fluorescent protein/blasticidin S deaminase (eGFP/BSD)-expressing transgenic T. equi parasite. (a) Selecting the T. equi transgenic line using blasticidin pressure. Blasticidin treatment was initiated on day 1 post-transfection. Blasticidin-resistant T. equi emerged on day 9 post-transfection. (b) Visualisation of T. equi at one year after the isolation of clonal line. A green fluorescent signal was emitted in both the merozoite (ring, paired, and Maltese cross forms) and trophozoite (ring form) stages of the transfected parasites.

Figure 3

Parasitaemia curves of the transgenic T. equi line expressing enhanced green fluorescent protein/blasticidin S deaminase (eGFP/BSD) and the parental line. (a) Transgenic T. equi was resistant to blasticidin but the parental line was not when cultured in medium containing 70-µM blasticidin. (b) The parasitaemia dynamics were comparable between the transgenic and parent lines when cultured in normal medium.

Figure 4

Graphical illustration of genomic integration of the enhanced green fluorescent protein/blasticidin S deaminase (eGFP/BSD)-expression cassette in transgenic T. equi. Genomic DNA extracted from the transgenic T. equi line (TL) and parental line (PL) was subjected to PCR assays. (a) A PCR assay (P1) confirmed the integration of the eGFP/BSD-expression cassette into one of the two elongation factor-1α (ef-1α) gene loci. (b) Two PCR assays (P2 and P3) confirmed that integration of the eGFP/BSD-expression cassette had occurred in the ef-1α-B gene locus. (c) Five PCR assays (P4–P8) confirmed the integration and correct orientation of the eGFP/BSD-expression cassette components in the ef-1α-B gene locus of the transgenic T. equi. A 1000 base-pair DNA ladder marker was used in all panels.

Figure 5

Graphical illustration of Southern blot and Western blot analyses. Genomic DNA extracted from the transgenic T. equi line (TL) and parental line (PL) was subjected to Southern blotting analyses after digestion with EcoRV. (a) A probe targeting the green fluorescent protein gene (GFP-probe) detected a single DNA fragment (4.9-kbp) in the genomic DNA (gDNA) obtained from the transgenic TL, but no such fragment was detectable in the PL. (b) A probe targeting the promoter region of the ef-1α gene (Prom-probe) detected a single 4.97-kbp fragment in the gDNA from the transgenic TL, whereas a 2.99-kbp fragment was detected in the PL. (c) Graphical illustration of ef-1α gene loci in the genome of the transgenic TL and EcoRV recognition sites. (d) Graphical illustration of ef-1α gene loci in the PL genome and EcoRV recognition sites. (e) In Western blot, an anti-GFP rabbit polyclonal antibody recognized a 40-kilodalton protein, corresponding to the size of eGFP/BSD, in lysate of TL but not in the lysates of PL and uninfected erythrocytes (UE).