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


 BackgroundBabesia species, the agentic pathogens of human and animal babesiosis, are spread worldwide. Over the last decade, genetic manipulation approaches have been applied with many protozoan parasites, including Plasmodium falciparum, Trypanosoma cruzi, Cryptosporidium parvum, Theileria annulata, Theileria parva, Babesia bovis, Babesia bigemina, Babesia ovata, Babesia gibsoni, and Babesia ovis. For Babesia sp. Xinjiang (Bxj), which is the causative pathogen of ovine babesiosis mainly in China, the efficiency of these techniques remain unclear. MethodsFirst, a plasmid bearing the elongation factor-1 alpha promoter, as well as the firefly luciferase reporter gene and rap stop region were transfected into Bxj by electroporation and nucleoporation to determine the most suitable transfection solution. Then, six program setting were evaluated to confirm the best for Bxj transient transfection and a series of different amounts of plasmid DNA were applied to generate relatively high luminescence values. Finally, the activities of four promoters derived from Bxj were evaluated using the developed transient transfection system. ConclusionsIn this study, a transient transfection system for Bxj was optimized. These findings provided critical information for Bxj genetic manipulation, as an essential tool to identify virulence factors and to further elucidate the basic biology of pathogens.


Abstract
Background Babesia species, the agentic pathogens of human and animal babesiosis, are spread worldwide. Over the last decade, genetic manipulation approaches have been applied with many protozoan parasites, including Plasmodium falciparum, Trypanosoma cruzi, Cryptosporidium parvum, Theileria annulata, Theileria parva, Babesia bovis, Babesia bigemina, Babesia ovata, Babesia gibsoni, and Babesia ovis. For Babesia sp. Xinjiang (Bxj), which is the causative pathogen of ovine babesiosis mainly in China, the e ciency of these techniques remain unclear.

Methods
First, a plasmid bearing the elongation factor-1 alpha promoter, as well as the re y luciferase reporter gene and rap stop region were transfected into Bxj by electroporation and nucleoporation to determine the most suitable transfection solution. Then, six program setting were evaluated to con rm the best for Bxj transient transfection and a series of different amounts of plasmid DNA were applied to generate relatively high luminescence values. Finally, the activities of four promoters derived from Bxj were evaluated using the developed transient transfection system.

Conclusions
In this study, a transient transfection system for Bxj was optimized. These ndings provided critical information for Bxj genetic manipulation, as an essential tool to identify virulence factors and to further elucidate the basic biology of pathogens. Background Babesiosis, caused by apicomplexan intraerythrocytic protozoan pathogens of the genus Babesia infective to humans, as well as domestic and wild animals, is a re-emerging tick-borne disease in tropical and subtropical regions worldwide [1]. Since the rst documented Babesia species was reported in 1888, more than 100 others have been identi ed globally, although few (Babesia ovis, Babesia motasi, and Babesia crassa) have been con rmed as causative agents of ovine babesiosis. This group of Babesia spp. has received much attention since B. ovis is the main etiological agent of ovine babesiosis, while B. motasi and B. crassa have been implicated in human cases of babesiosis throughout Asia [2][3][4][5][6]. Additionally, a novel Babesia species, named Babesia sp. Xinjiang (Bxj), experimentally transmitted by Hyalomma anatolicum anatolicum (Koch, 1844), was rst isolated from a sheep bitten by Rhipicephalus sanguineus and H. anatolicum anatolicum [7]. Epidemiological investigations revealed that the seropositivity rate of Bxj ranged from 0 to 68.13% in 22 provinces across China [8]. Ovine babesiosis is an important socioeconomic factor, particularly in animal mortality, costs of livestock treatment, and productivity loss [9].
The di culty in controlling ovine babesiosis is due to the lack of effective vaccines, limited choices of therapeutic drugs, and the emergence of several drug-resistant Babesia strains. Understanding the basic biology of Babesia species is pivotal to the development of effective therapeutic drugs and preventive vaccines to control this disease [10]. 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. In this study, a suitable transfection system for Bxj merozoites was developed using electroporation and nucleofactor technologies.

Methods
In vitro culture of Babesia sp. Xinjiang A monoclonal line (G5) of Bxj was cultured in vitro as reported previously [22]. Brie y, when parasitemia reached 20%-30%, 4 mL of a suspension of infected red blood cells (RBCs) were cultured in a T75 ask supplemented with 5% fresh sheep erythrocytes and 20% fetal bovine serum (Gibco, Carlsbad, CA, USA) in Roswell Park Memorial Institute 1640 medium (Lonza Biologics, Portsmouth, NH, USA) at 37°C under an atmosphere of 5% CO 2 /95% air.

Plasmid constructs
Schematic diagrams of the transient transfection plasmids used in this study are presented in Fig. 1a and 1b. Five distinct plasmids were generated, named as phosphate-buffered saline (PBS)-LuRaA, PBS-LuRaB, PBS-LuRaAc, and PBS-LuRaRa. Initially, the plasmid PBS-LuRa was constructed by amplifying the re y luciferase gene and Bxj rhoptry-associated protein-1 3'-asking region (rap3') by PCR from the plasmid pENT12luc and genomic DNA of Bxj, respectively, using speci c primer pairs ( Table 1). The PCR products were inserted into the Xho I restriction site of the plasmid pBluescript SK(+) (provided by Carlos E. Suarez) using the MultiS One Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, China). Then, the promotors of Bxj ef1α IG-A, ef-1α IG-B, actin 5'-untranslated region (UTR), and rap 5'-UTR were ampli ed by PCR and cloned into the Sal I restriction site of PBS-LuRa, respectively. A free-promoter plasmid (PBS-LuRa) was used as a negative control. The constructed plasmids were con rmed by sequencing and puri ed using the QIAGEN Plasmid Maxi Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions.

Optimization of parasite transfection
When parasitemia of Bxj-infected RBCs reached 25%-30%, the cultures were centrifuged at 800 × g for 10 min. Then, the supernatant was discarded and pellets of Bxj-infected RBCs were washed twice in PBS and once in cytomix buffer (120 mM KCl, 0.15 mM CaCl 2 , 10 mM K 2 HPO 4 /KH 2 PO 4 , pH 7.6, 25 mM HEPES. pH7.6, 2 mM EGTA, 5 mM MgCl 2 , nal pH 7.6) [14]. Parasite transfections were performed with the use of a Gene Pulser Xcell™ Electroporation System (Bio-Rad Laboratories, Hercules, CA, USA) and 0.2-cm cuvettes containing 50 μL of sterilized cytomix buffer or 20 μg of plasmids and 1 × 10 8 infected RBCs to a nal volume of 100 μL. Initially, two types of nucleofection buffer (human T cell Nucleofector solution and Basic Parasite Nucleofector® Solution) were used to screen the optimization buffer with the program settings U-033, V-024, X-001, D-023, T-023, and T-020. Cytomix buffer was used for electrotransfection at 1200 V and 25 μF. Subsequently, optimized parameters for Bxj transfection were employed to determine the amounts of plasmid, which varied from 5 to 150 μg. After transfection, the mixtures were transferred into the wells of a 24-well culture plate containing 7.5% fresh sheep RBCs and

Results And Discussion
Optimization of Bxj merozoite transfection parameters Experimental conditions for transit transfection of Bxj merozoites were determined using a Gene Pulser Xcell™ Electroporation System and a Nucleofector™ II/2b Transfection Device (Lonza). To con rm the suitability of the transient transfection solution, luciferase activity and PPEs were determined at 24, 48, and 72 h, when 20 µg of plasmid DNA with each buffer (human T cell nucleofector solution and Basic parasite nucleofector solution) were transfected with the V-024 program setting, and while the cytomix buffer was electrotransfected with 20 μg of PBS-LuRaB at 1200 V and 25 μF (Fig. 1c). As compared to the other two solutions, the luciferase activity of the transfection system with human T cell nucleofector solution was relatively high ( *** p < 0.001), indicating that this solution was suitable for Bxj transient transfection. With the exception of the cytomix buffer, there was no decrease in relative luciferase activity in the human T cell nucleofector solution or basic parasite nucleofector solution over a period of 72 h. In previous reports, there was a downward trend in relative luciferase activity during transfection of B. bovis, B. ovis, and Babesia microti. One possible explanation for this exceptional situation was that human T cell nucleofector solution and basic parasite nucleofector solution were more capable of conveying protective effects to the ovine erythrocyte membrane.
In terms of the impact of each transfection buffer to PPEs, a signi cant high gures using basic parasite nucleofector solution can be obtained at 72 h post-transfection compared to other transfection solutions (Fig. 1d), while the highest level of relative luminescence units observed in human T cell nucleofector solution group instead of cytomix and basic parasite nucleofector solution groups (Fig. 1c). These data provide additional evidence that human T cell nucleofector solution was more effective and suitable for transient transfection of Bxj.
Furthermore, as compared to the other ve program settings (U-033, X-001, D-023, T-023, and T-020), luciferase activity was signi cantly greater with V-024 ( **** p < 0.0001). Relative luciferase activities at 24, 48, and 72 h post-transfection are presented in Fig. 2e. Subsequently, human T cell Nucleofector solution and V-024 program were used as transient transfection parameters to determine the amount of plasmid DNA. With an increase in the amount of plasmid DNA, relative luminescence units tended to increase, while almost similar values were observed with 20, 50, and 100 μg (p > 0.05 ) (Fig. 1f). As compared to the use of 10 μg, 20 μg generated a relatively high relative light unit value ( * p < 0.05). Taken together, these results, including relative light units and PPEs, showed that human T-cell solution, the program V-024, and 20 μg of plasmid DNA were the most favorable conditions for transient transfection in subsequent studies.

Evaluation of promoter activity
To determine promoter activity, the optimal transfection conditions mentioned above, including the transfection program, buffer, and amount of plasmid DNA, were employed to transfect Bxj merozoites with ve types of plasmids (PBS-LuRaA, PBS-LuRaB, PBS-LuRaAc, PBS-LuRaRa and PBS-LuRa). The time courses of luciferase activities in the transfected Bxj culture are presented in Fig. 2a. Although four of the promoters tested in this study were able to promote luciferase expression, a signi cantly higher level of luciferase activity was observed with transfection of the Bxj merozoites with either plasmid PBS-LuRaAc or PBS-LuRaB after electroporation. According to values of luciferase activity post-transfection, the extent of promoter activity can be classi ed as strong (actin 5' UTR), medium (ef1α IG-B), or low (ef1α IG-A and rap 5'-UTR). The PPEs of each group at 24, 48, and 72 h post-transfection were determined by calculating the proportion of infected RBCs out of 2000 cells under a light microscope. There were no obvious difference in PPE values among the four groups at 24, 48, and 72 h post-transfection (p > 0.05) (Fig. 2b).

Conclusions
In conclusion, the conditions for transient transfection of Bxj, including the transfection solution, program settings, and quality of plasmid DNA, were optimized. In addition, promoter activity was systematically evaluated. This study is the rst effort to create a platform for transient transfection of Bxj and provides basic information for the development of a stable transfection method for Bxj and to further elucidate the biology of this parasite, as well as the parasite-vector and parasite-host interactions.