Hamster blood for donor RBC was collected from hamsters into EDTA K2 (solution/RBCs = 1:9; 10% EDTA-2K), and the animals were anesthetized with isoflurane for retro-orbital venipuncture. All blood samples were centrifuged at 500×g for 10 min, followed by removing the plasma and buffy coat through three washes of the cells by 5 volumes of PSG, with careful removal of the supernatant and buffy coat at each wash. Next, the washed RBC was stored at 4°C for a maximum of 2 weeks in an equal volume of PSG plus extra glucose (20 g glucose/L) with a final concentration of 200 μg/ml streptomycin and 200 U/ml penicillin.

Babesia duncani strain WA1 (ATCC PRA-302™) was obtained from the ATCC and maintained in our laboratory (State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, China). Babesia duncani was cultured in vitro (Abraham et al., 2018; McCormack et al., 2019) in the presence of HL-1 supplemented with 1 mg/ml AlbuMAX I (Gibco Life Technologies, Shanghai, China), 200 μM L-glutamine (Sigma, Shanghai, China), 2% antibiotic/antimycotic 100× (Corning, Shanghai, China), and 20% FBS at 37°C in a microaerophilous stationary phase (5% CO₂, 2% O₂, 93% N₂).

According to previous reports, a putative glutamyl tRNA synthase gene was located next to the ef-1α B gene, and a gene homologous to the putative ribonucleotide reductase R2 subunit was located downstream of the ef-1α A gene (Suarez et al., 2006). To confirm that ef-1α is a double-copy gene, we designed primers on two adjacent genes (Table 1). The primer of ef-locus-1 is located in the ef-1α gene, and the primers of ef-locus-2 and ef-locus-3 are located in the ribonucleotide reductase R2 subunit gene and the glutamyl tRNA synthase gene upstream of ef-1a A and downstream of ef-1a B, respectively. Using these three primers for PCR amplification and sequencing, we confirmed that B. duncani has two copies of ef-1α, but failed to obtain the sequence between the two copies. To obtain the sequence between the two copies, four more primers (ef-locus-4, ef-locus-5, ef-locus-6, and ef-locus-7) with a higher annealing temperature of 72°C were designed at the ef-1α location, and using these four primers, the intergenic (IG) region was successfully amplified and sequenced. For the analysis of the ef-1α locus of the B. duncani WA1 strain, we obtained two overlapping fragments by PCR amplification (ef-locus-8, ef-locus-9) of B. duncani genomic DNA extracted from in-vitro-cultured parasites using a genomic DNA purification kit (Tiangen, Beijing, China).

Disrupting the TPX-1 gene requires the accurate genomic location of the TPX-1 gene, so two primers (tpx-locus-f and tpx-locus-r) were designed to amplify the TPX-1 gene. We obtained one fragment by PCR amplification of B. duncani genomic DNA, and the PCR product was sequenced directly.

Babesia duncani was cultured in 96-well plates with various concentrations of WR99210 (20, 10, 5, 2.5, 1.25, 0.6125, 0.306, 0.15, 0.075, and 0 nM). Parasite growth was determined by the SYBR Green assay as previously reported (Abraham et al., 2018). The normalized luciferase activities were plotted using GraphPad Prism 6.

The plasmid schematic diagrams used in this study are shown in Figure 1. For pBS-EMR, the ef-1α B promoter and rap-1 terminator were amplified from the B. duncani genome, the mCherry fragment was amplified from the plasmid of pBS-PAC-mCherry, and these fragments were cloned separately into the pBluescript (pBS) backbone plasmid using the ClonExpress MultiS One Step Cloning Kit (Vazyme, China). The plasmid of pBS-EHEG was modified on the basis of plasmid pBS-EMR, replacing the mCherry gene and 3′UTR of rap-1 with the hDHFR-eGFP and 3′UTR of ef-1αB by the ClonExpress MultiS One Step Cloning Kit (Vazyme, China). The plasmid of pBS-DHFR-EGFP-TPX-1 KO was cloned using the same method. Briefly, on the basis of the plasmid pBS-EHEG, the 5′UTR of the TPX-1 gene was inserted into the upstream region of the ef-1α B promoter and the 3′UTR of ef-1αB was replaced with the 3′UTR of TPX-1. All plasmids were sequenced to confirm the accuracy of the sequence. All primers used in this study are shown in Table 1.

To prepare B. duncani-infected RBCs (iRBCs) for transfection, cell pellets were washed twice with PBS and once with cytomix buffer (120 mM KCl, 0.15 mM CaCl₂, 10 mM K₂HPO₄, 10 mM KH₂PO₄, 25 mM HEPES, pH 7.6, 2 mM EGTA, and 5 mM MgCl₂). Electroporation was performed in BTX using 0.2 cm cuvettes containing filter-sterilized cytomix buffer at a final volume of 200 l, and parameters for transfection were 1,200 V, 25 μF, and 2 times, with 50 μg plasmid in 100 μl iRBC and 100 μl cytomix buffer.

After transfection, the mixtures were transferred into 24-well culture plates containing 5% fresh RBCs and 20% FBS, followed by incubation for 24 h and the addition of 5 nM WR99210 (MCE, Shanghai, China). To obtain a clonal strain, 1 μl iRBC was collected at 10% parasitemia and diluted to 6 infected RBCs/ml with completed medium containing 5% of fresh RBCs. To make the clonal line, 0.6 iRBC was added into each well with a total volume of 100 μl in a 96-well culture plate, replacing 70 μl culture medium every 3 days until the parasitemia reached 1%. After 12 days of culture, parasites could usually be observed, and the culture was transferred to a 48-well plate for further analysis.

To select the recombinant monoclones, blood samples of recombinant parasites were collected in the 96-well plate, and RBCs were lysed using 0.1% saponin in phosphate buffer saline (PBS), followed by boiling the sample in boiling water for 10 min and using it directly for PCR. Parasite genomic DNAs were isolated with DNeasy blood kits and used for PCR amplification.

Three pairs of primer (Table 1) (pcr-1-hDHFR; pcr-2-EGFP; pcr-1-ef; pcr-2-ef; pcr-3-ef) were designed to confirm the integration of hDHFR-eGFP into the ef-1αB locus. The position of the primer and the size of the product are shown in Figure 5. The same method (three pairs of primer: pcr-1-hDHFR; pcr-2-EGFP; pcr-1-tpx; pcr-2-tpx; pcr-3-tpx) was used to identify the TPX-1 KO strain. All primers are shown in Table 1.

For live-cell imaging, parasite-infected blood was washed twice with PBS, followed by staining the cells with 1 μg/ml Hoechst 33342 (Sigma, Shanghai, China) in PBS. All images were captured and processed using identical settings in the OLYMPUS FRAME_BX63 scanning confocal microscope with a ×100 numerical-aperture (NA) oil objective.

To quantify the proportion of GFP-positive parasites, TPX-1 KO parasites were washed with PBS and analyzed with a flow cytometer. The cell nuclei were first stained with 2 μg/ml Hoechst 33342 (Sigma, Shanghai, China) and 2 μg/ml PI (propidium iodide) in PBS, with PI being used to count the dead cells. After a single wash, 100,000 cells were counted on the CytoFLEX LX flow cytometer, followed by selecting the cell populations without red fluorescent signal for data analysis using CytExpert 2.4, with gating for nuclear stain Hoechst 33342 and green fluorescence.

To collect parasites, RBCs were lysed using 0.1% saponin in PBS to wash and remove the hemoglobin. Total proteins extracted from parasite pellets were separated on 12.5% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (GE, Shanghai, China), followed by incubation with blocking buffer (TBST with 5% skimmed milk) at room temperature for 1 h and then at 4°C overnight with anti-GFP (rabbit; 1:5,000; Proteintech, Shanghai, China). Next, the PVDF membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit for 2 h at room temperature, followed by three washes with blocking buffer to enhance chemiluminescence (Sahi et al., 2009) detection, cutting the membrane between 30 and 45 kDa, and incubation with anti-GAPDH (rabbit; 1:2,000; Proteintech, Shanghai, China). The antibody of GAPDH was used as the internal control.

The promoter of the ef-1α gene is most commonly used in various apicomplexan protozoa (Adamson et al., 2001; Suarez and McElwain, 2009; Asada et al., 2012; De Goeyse et al., 2015; Hakimi et al., 2016; Liu et al., 2018), and the ef-1α gene has two copies in the genome, providing a suitable site for the stable transfection of the gene. However, due to the lack of genomic data, the ef-1 gene promoter sequence remains unknown. By comparing multiple sequences of Babesia and designing specific PCR primers, we successfully obtained the locus of the ef-1α gene. For the analysis of the ef-1α locus of the B. duncani WA1 strain, two overlapping fragments were obtained by PCR amplification of B. duncani genomic DNA (Figure 2). Similar to B. bovis (Suarez et al., 2006), the ef-1a locus in B. duncani contains two identical ef-1α genes (denoted as ef-1αA and ef-1αB in Figure 2). Both ef-1α ORFs are 1,347 bp, arranged in a head-to-head orientation and separated by a 1,302-bp IG region. The sequence information of ef-1α obtained in this study was submitted to GenBank with the accession number OL804102.

The success of an efficient transfection method requires effective promoters and a suitable strategy for DNA transfection (Suarez and McElwain, 2010). To establish the B. duncani transfection method, a plasmid (pBS-EMR) expressing mCherry was constructed, using 658-bp 5′UTR of ef-1α B as the promoter and 300-bp 3′UTR of rap-1 as the terminator. Based on the B. bovis transfection method (Suarez and McElwain, 2008), we have made some modifications to achieve higher transfection efficiency. Electroporation was performed in BTX with 0.2 cm cuvettes containing filter-sterilized cytomix buffer at a final volume of 200 μl, using the parameters of 1,200 V, 25 μF, and 2 times for transfection, with 50 μg plasmid in 100 μl iRBC and 100 μl cytomix buffer. At 24 h post-transfection, the parasites expressing mCherry were observed by live fluorescence microscopy (Figure 3), proving the feasibility of this electroporation method.

To examine the inhibition of WR99210 on B. duncani, the parasites were cultured with different concentrations of WR99210 (0 to 20 nM). The experiment was performed in triplicate wells, and parasitemia was calculated on day 3 after adding the drug into the culture. The IC50 of WR99210 was 1.01 nM, and 5 nM WR99210 could inhibit 80% of the growth of B. duncani (Figure 4), indicating that 5 nM WR99210 could be used to select genetically modified parasites.

At 12 days post-selection by 5 nM WR99210, eGFP-expressing parasites appeared in cultures transfected with linearized plasmids (Figure 5A). The integration of hDHFR-eGFP into the ef-1α B locus was initially evaluated by PCR. After confirming the integration event, the transfected B. duncani clone line was obtained by limiting dilution. PCR1, PCR2, and PCR3 primer pairs could successfully amplify 1,031, 1,273, and 2,066 bp fragments in the clone line, respectively, but not in the wild type (WT) of B. duncani (Figure 5B). hDHFR-eGFP expression was detected by live fluorescence microscopy (Figure 5C), and fluorescence could be observed in the cytoplasm of different forms of parasites. Additionally, the growth curves showed no significant difference between the WT and eGFP parasites (Figure S1A). These results suggested that eGFP can be stably expressed in B. duncani, with no side effect on the growth of the parasites.

The gene manipulation ability of this method for B. duncani was tested by disrupting the TPX-1 gene through homologous recombination established in this study. The 780-bp 5′UTR and 780-bp 3′UTR of the TPX-1 gene were used as the homology arms, and hDHFR was used as the drug selection marker (Figure 6A). After transfecting the parasites with circular plasmids, the daily PPE (Figure 6B) and the proportion of eGFP expression were calculated by flow cytometry (Figures 6C, D). Approximately 0.75% of parasites could express the fluorescent protein at 24 h post-transfection. After 13 days of drug screening, 96.3% of parasites could stably express eGFP and show green fluorescence (Figures 6C, D). Resistant parasites were observed by Giemsa staining on day 7, and parasites with green fluorescence could be easily observed by flow cytometry (Figure 6D). To obtain the clonal line, transfected B. duncani was subjected to limiting dilution. After 12 days of culture, 11 clone lines were obtained, which were identified by PCR, indicating that the TPX-1 gene was successfully knocked out (with only 3 clone lines shown in the results) (Figure 6E). The PCR1, PCR2, and PCR3 primer pairs could successfully amplify 1.6, 2.0, and 2.1 kbp DNA fragments in the KO clone line, respectively, but not in B. duncani WT (Figure 6E). Western blot results also confirmed the expression of the eGFP protein, in contrast to no signal for WT (Figure 6F). Meanwhile, no obvious difference was observed in the growth curves between the WT and TPX-1 KO parasites (Figure S1B).