Geno- and phenotypic characteristics of a transfected Babesia bovis 6-Cys-E knockout clonal line
© The Author(s). 2017
Received: 25 October 2016
Accepted: 19 April 2017
Published: 2 May 2017
Babesia bovis is an intra-erythrocytic tick-transmitted apicomplexan protozoan parasite. It has a complex lifestyle including asexual replication in the mammalian host and sexual replication occurring in the midgut of host tick vector, typically, Rhipicephalus microplus. Previous evidence showed that certain B. bovis genes, including members of 6-Cys gene family, are differentially expressed during tick and mammalian stages of the parasite’s life cycle. Moreover, the 6-Cys E gene is differentially expressed in the T3Bo strain of B. bovis tick stages, and anti 6-Cys E antibodies were shown to be able to inhibit in vitro growth of the phenotypically distinct B. bovis Mo7clonal line.
In this study, the 6-Cys E gene of B. bovis T3Bo strain was disrupted by transfection using a plasmid containing 6-Cys gene E 5′ and 3′ regions to guide homologous recombination, and the egfp-bsd fusion gene under control of a ef-1α promoter, yielding a B. bovis clonal line designated 6-Cys EKO-cln. Full genome sequencing of 6-Cys EKO-cln parasites was performed and in vitro inhibition assays using anti 6-Cys E antibodies.
Full genome sequencing of 6-Cys EKO-cln B. bovis demonstrated single insertion of egfp-bsd gene that disrupts the integrity of 6-Cys gene E. Undistinguishable growth rate of 6-Cys EKO-cln line compared to wild-type 6-Cys E intact T3Bo B. bovis strain in in vitro cultures indicates that expression of gene 6-Cys E is not essential for blood stage replication in this strain. In vitro inhibition assays confirmed the ability of anti-6 Cys E antibodies to inhibit the growth of the wild-type Mo7 and T3Bo B. bovis parasites, but no significant inhibition was found for 6-Cys EKO-cln line parasites.
Overall, the data suggest that the anti-6 Cys E antibody neutralising effect on the wild type strains is likely due to mechanical hindrance, or cross-reactivity, rather than due to functional requirements of 6-Cys gene E product for survival and development of the erythrocyte stages. Further investigation is underway to determine if the 6-Cys E protein is required for replication and sexual stage development of B. bovis during tick stages.
KeywordsB. bovis transfection 6-Cys E gene Cell sorting Transfected clonal line
Babesia bovis is an apicomplexan tick-borne parasite, mainly transmitted by Riphicephalus microplus, responsible for acute disease in bovines. The disease causes important economic loses in endemic areas worldwide, and improved methods of control are needed. A common method used to prevent acute babesiosis in endemic areas, such as Australia, Argentina and Mexico, is the use of B. bovis attenuated vaccines, which have multiple limitations . Subunit vaccines able to elicit sterile immunity or prevent acute babesiosis would be ideal prevention tools, but they remain unavailable. Additional tools for enhanced control include vaccines designed to block transmission of the parasites by the tick vector. Development of such vaccines requires the identification of relevant antigens expressed in the tick stages of the parasites. Recent work resulted in the identification of candidate antigens that can be used for the developing of Babesia transmission blocking vaccines , including the members of the 6-Cys gene family. The apicomplexan 6-Cys gene family was originally identified in the Babesia-related Plasmodium parasites [3–5]. Members of the 6-Cys family are defined by a unique arrangement of 6-Cysteine residues, although other alternative arrangements involving 4, 5 and seven cysteine residues are also possible . The members of the 6-Cys family contain signal peptides and are thus likely expressed on the surface of the parasites . Interestingly, Plasmodium 6-Cys proteins such as pfs230 and s48/45 are expressed in sexual stages of the parasite, occurring exclusively in the midgut of the mosquito vector, whereas other Plasmodium 6-Cys proteins are differentially expressed in the sporozoite and or merozoite stages . Plasmodium 6-Cys proteins are known to play important roles in the sexual stage forms and are required in oocyte formation, and are strong candidates for the development of transmission-blocking vaccines [6, 7]. In addition, some Plasmodium 6-Cys proteins such as 6-Cys protein P12 and P41 are also known to be expressed in erythrocyte stages of the parasite . Similar to Plasmodium parasites, B. bovis encodes for 6-Cys proteins . The B. bovis 6-Cys gene family was originally described as containing six genes termed 6-Cys A-F, but, further searches of the genome revealed the presence of four additional genes (6-Cys G-J), in this family . Currently, the roles of the 6-Cys gene family members in survival during the tick and mammalian stage of the life-cycle are unknown. The gene 6-Cys E was found transcribed only in tick stages, but not in the blood stages of parasites of the T3Bo strain . In addition, previous studies performed on the biologically cloned B. bovis strain Mo7 suggested that the 6-Cys E protein might be a suitable candidate for a subunit vaccine because it was found to be expressed on the surface of the cloned B. bovis Mo7 strain, while developing in the blood stage,  and contained neutralization sensitive epitopes. We were interested in further analysing the biological significance of this previous finding and into determining whether expression of 6-Cys E is essential for the survival of the blood stage of the life-cycle of B. bovis. Importantly, if 6-Cys E mutants are viable and able to develop in in vitro cultures, it would provide an essential tool for further testing of the possible functional role of the 6-Cys E gene in erythrocyte and tick stages of the life cycle of B. bovis.
The focus of the present study was to determine whether expression of the 6-Cys gene E is needed for the replication of the blood stage of the parasite in vitro. Here, we describe the production of a B. bovis 6-Cys E knockout (KO) clonal line derived from the T3Bo strain of B. bovis using transfection methods [9–13] that can be applied for future gene functional analysis. The B. bovis T3Bo line was selected for this study because recent unpublished evidence suggests that the phenotypically distinct Mo7 strain, which was used in previous studies, is not likely transmissible by ticks, and therefore not suited to perform further tick transmission studies required to prove the role of 6-Cys proteins in transmission.
We also compared the ability of anti-6-Cys E antibodies to inhibit the in vitro growth of KO and wild-type B. bovis parasites. Collectively, the results indicated that the gene 6-Cys E is not essential for the development of erythrocyte stages of B. bovis. Although the results are not supportive of using the 6-Cys E as a blood stage vaccine component, they suggest that this protein could still be a candidate for developing transmission blocking vaccines against B. bovis.
The Texas (Tx) T3Bo  and Mo7 strains of B. bovis were grown in long-term microaerophilic stationary phase culture as previously described [10, 11]. Both strains, Mo7 and T3Bo, of B. bovis [10, 12] are maintained as a cryopreserved stabilate in liquid nitrogen when not in use .
Construction of the transfection plasmid p6-Cys-EKO
Transfection of B. bovis
The plasmid p6-Cys-EKO was electroporated into B. bovis Tx T3Bo strain infected erythrocytes as previously described [15, 16]. Plasmid pBluescript was used in identical transfections as a negative control. The transfected parasites were transferred to an incubator at 37 °C, selected with blasticidin as described previously , and monitored on a regular basis for the presence of transfected parasites with a fluorescence microscope at 60× amplification.
Cloning transfected parasites
Cloning was performed using a fluorescence activated cell sorter with a Clonecyte attachment (FACS Vantage, Becton Dickenson, Immunocytometry Systems, San Jose, CA) as previously described . The cells were sorted depending on the fluorescence expressed from transfected parasites with the egfp gene. Single transfected parasites were sorted into three 96 well plates. The cultures were maintained for 3 weeks as described  with a daily change of medium. PCR analysis was performed on 1 μl samples obtained from each well to identify parasites using the rap-1 primer BoFN primer (5′-TCA ACA AGG TAC TCT ATA TGG CTA CC-3′) and BoRN primer (5′-CTA CCG AGC AGA ACC TTC TTC ACC AT-3′) . PCR positive wells were further analysed by PCR to detect a disrupted 6-Cys gene E using a forward primer that anneals in the intergenic region upstream of 6-Cys gene E IGE for (5′-GCT AAG CAC CTA TTT AGC GTA AC-3′) and a reverse primer EF-Pr-R-6-HIII [5′-GAC CAT AAG CTT AGT AAA CGA TAG AAC AGA CTA AG-3′] .
Phenotypic and genotypic analysis of transfected clones
Multiple approaches were used to define the phenotypic and genotypic characteristics and to confirm the integrity and stability of the transfected clones, as described below.
Emission of fluorescence by transfected parasites was verified using a Zeiss Fluorescence microscope. Wells containing clones of parasites with uniform bright fluorescence (N = 62 clones) were selected for further analysis.
PCR and DNA sequencing
Primers sequence used in PCR and sequencing
Rev: 5′-ctacgaggatcctccttt gtgaggttcacg-3′
Rev: 5′-ctacgaggatcctccttt gtgaggttcacg-3′
Southern blot analysis
The patterns of insertion of the EKO plasmid into the B. bovis T3Bo gDNA were analysed using hybridization of undigested and BglII digested gDNA, using DIG-labeled 6-Cys E 3′ end, Egfp-bsd, Ef-1α and ampicillin probes in Southern blots. The probes were prepared as previously described . The gDNA used in this analysis included. DNA extracted from the non-transfected parental T3Bo strain, 6-Cys EKO, and 6-Cys EKO-cln.
Clonal line genome sequencing
Approximately 7 μg of gDNA were extracted from in vitro cultured B. bovis using the DNEasy kit (Qiagen, Hilden, Germany) with special care being taken to avoid shearing of large DNA fragments. The SMRT Bell Template Prep Kit V1.0 was used for library preparation, and DNA was size-selected at > 15 kb using the Blue Pippon (Sage Biosciences, Edmonton, Canada), the resulting library had an average fragment size of ~ 21 kb. Sequencing was performed on the Pacific Biosciences RSII sequencer using MagBead Loading and P6/C chemistry. Sequences were assembled using the Hierarchal Genome Assembly Process 2 (HGAP2), and the resulting assembly was further analysed using CLC Genomics Workbench. Comparison of the re-sequencing data from the transfected clone to that of the T2Bo reference genome was performed using BLAT 2.0 using default settings .
Phenotypic analysis of mutant strain and clone
To identify the possible impact of the gene 6-Cys E mutation on the growth of the B. bovis parasite, we compared the ability of the 6-Cys EKO strain, 6-Cys EKO-cln and wild-type T3Bo strains to grow in in vitro culture. Cultures of each strain were initiated at 0.5% parasitemia in triplicate wells in the presence and absence of blasticidin . Culture medium was replaced and the parasitemia calculated daily for 8 days. Statistical analysis was performed with the Student t-test, and the probability value of less than 5% (P < 0.05) was considered significant.
In vitro neutralisation assay
Inhibition of B. bovis merozoite invasion of erythrocytes was performed on the Mo7, T3Bo, EKO, and EKO cln, strains as previously described by Hines et al. . Briefly, B. bovis merozoites were separated from erythrocytes by centrifugation, and approximately 5 × 105 viable merozoites, as determined using 6-carboxyl fluorescein diacetate , were used in each antibody neutralisation reaction: rabbit antisera specific for B. bovis 6-Cys E-peptides 1 and 2 , or pre-immune rabbit sera. Mouse polyclonal sera against a non-Babesia sp. protein (sera specific for Operon-associated protein (OpAG) 2 encoded protein of A. marginal)  was used as negative control, and a previously described B. bovis neutralising monoclonal mouse anti-MSA-1 antibody (BABB35A4) was used as positive control [22, 24]. All sera were heat-inactivated for 30 min at 56 °C, diluted 1:1 in culture medium and incubated with the merozoites for 30 min at 4 °C. Mouse antibodies were used at a concentration of 1.5 μg/ ml. After the incubation period, an equal volume of 5% (v/v) bovine erythrocytes in culture medium was added to each well. The plates were incubated at 37 °C in a 5% CO2 atmosphere. Parasites were grown in micro-aerophilous stationary phase culture as previously described . Percentages of parasitized erythrocytes (PPE) were determined every 24 h up to 72 h by counting parasites in smears stained with Diff-Quik on an optical microscopic. The assay was performed in triplicate in 96-well plates. The results were analyzed by the one-way ANOVA with 95% confidence level.
Results and discussion
Transfection and biological cloning of transfected B. bovis parasites
Genotypic analysis of transfected B. bovis parasites
To further confirm specific disruption of the targeted 6-Cys E locus, we also performed comparative PCR among the T3Bo, 6-Cys EKO, and 6-Cys EKO-cln using primers designed for the amplification of rap-1 (control non-targeted gene), full-size 6-Cys gene E, and egfp-bsd. Additionally, to demonstrate specific integration at the attempted 6-Cys E locus, we also performed a PCR reaction using forward primers IGE (representing sequences in the intergenic region located 5′ of the E gene, and not represented in the transfection plasmid p6-Cys-EKO) and PRO (representing sequences in the ef-1B promoter) as the reverse primer (Table 1 and Fig. 3b). All products derived from these PCR reactions, shown in Fig. 3b were fully sequenced. The control rap-1 gene was identically amplified from gDNA extracted from all three strains. However, gDNA from strains 6-Cys EKO and 6-Cys EKO-cln, but not from the T3Bo strain, generated products when the egfp-bsd and IGE-PRO primers were employed, confirming the presence of the egfp-bsd gene and integration of transfected genes into the 6-Cys gene E locus exclusively in the transfected parasites. Furthermore, sequence analysis of PCR amplicons indicates the integration of the structure of the locus by sequences present in the transfected plasmid, which successfully integrated into the genome by homologous recombination. Interestingly, primers designed for the amplification of the full size 6-Cys E orf only generated PCR products of identical size and composition in the T3Bo and 6-Cys EKO lines, and no products were visible upon amplification of the 6-Cys EKO-cln line, consistent with disruption of the 6-Cys gene E in these parasites (Fig. 3b). Taken together, these data also suggest that the transfected line 6-Cys EKO contains a mixed population of parasites, either with or without disrupted 6-Cys gene E, and that, in contrast, the 6-Cys EKO-cln line contains a single insertion of the egfp-bsd gene disrupting the integrity of the 6-Cys gene E of B. bovis.
Collectively, this Southern blot and PCR data support stable insertion of the intended egfp-bsd gene disrupting the integrity of the 6-Cys gene E orf in in the parasite line 6-Cys EKO-cln, as operated by homologous recombination. Full genome sequencing of the 6-Cys EKO-cln line again confirmed the gene 6-Cys E interruption and is consistent with the PCR and Southern blot data. Sequence analysis of chromosome number 2, where the 6-Cys gene E locus is located indicates that the originally 3878 bp targeted area became enlarged to 7117 bp as a result of the integration of the egfp-bsd gene, its 3′ rap and the ef-1α B promoter regions included in the transfection, interrupting the 6-Cys gene E, exactly as described in Figs. 1 and 3a. The pattern of insertion and the conservation of restriction sites derived from the plasmid vector are shown in Additional file 1: Figure S1. The sequence of the interrupted 6-Cys gene E region in the 6-Cys E-KO-cln parasites was deposited in GenBank (Accession no. KX247383). Analysis of the full genome of the 6-Cys EKO-cln gDNA indicated that no other insertions derived from the transfection changes occurred in the genome of parasites of the 6-Cys EKO-cln line. Comparison of the original reference B. bovis T2Bo genome sequence  to the re-sequenced 6-Cys EKO-cln revealed random SNP and short insertion/deletion mutations but did not result in gross chromosomal re-arrangements. However, such minor differences among these genomes are expected as they result from comparing sequences derived from a clonal parasite line with a parasite line composed of multiple parasite subpopulations.
Phenotypic characterization of B. bovis transfected parasites
In the current study, we were able to produce the clonal line 6-Cys EKO cln, and demonstrate that this clonal line contain a disruption of the B. bovis 6-Cys gene E. The egfp-bsd fusion gene insertion in the clonal line 6-Cys EKO cln seem to be limited to the intended 6-Cys gene E, and transfection does not appear to have altered the genome of 6-Cys EKO cln at sites other than that the targeted 6-Cys E gene, nor causing any gross chromosomal re-arrangements. The 6-Cys EKO cln mutant line has neither obvious morphological nor significant phenotypical differences compared to the parental non-transfected strain T3Bo parasites, and it can grow at identical rates regardless of the presence or absence of the selection agent blasticidin. Moreover, 6-Cys EKO cln can resist the blocking inhibitory effects of antibodies against 6-Cys E. Taken together, the data indicate a negligible role of the 6-Cys E protein for the development of B. bovis parasite in in vitro cultures. We plan next to compare transmission fitness of the 6-Cys EKO cln and 6-Cys intact parasites to start assessing the possible role of the 6-Cys E in the transmission of B. bovis.
We are thankful to Paul lacy, Jacob Laughery and Grace Chung for excellent technical support.
This work was supported by scholarship from Egyptian government, the Ministry of Higher Education and Scientific Research, and the United States Department of Agriculture Research Service Current Research Information System Project No. 2090-32000-039-00D.
Availability of data and materials
All data generated or analyzed in this study are included and its Additional file 1: Figure S1 and Additional file 2: Table S1. Sequences were submitted to GenBank database under accession numbers KX247384 and KX247383.
Project designing and manuscript preparations CES and HFA; sample collection HFA; data acquisition: HFA, WCD, DAS, MGS, CES and DRH. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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