Babesia bovis establishes persistent infections of long duration in cattle, despite the development of effective anti-disease immunity. One mechanism used by the parasite to achieve persistence is rapid antigenic variation of the VESA1 cytoadhesion ligand through segmental gene conversion (SGC), a phenomenon thought to be a form of homologous recombination (HR). To begin investigation of the enzymatic basis for SGC we initially identified and knocked out the Bb rad51 gene encoding the B . bovis Rad51 ortholog. BbRad51 was found to be non-essential for in vitro growth of asexual-stage parasites. However, its loss resulted in hypersensitivity to methylmethane sulfonate (MMS) and an apparent defect in HR. This defect rendered attempts to complement the knockout phenotype by reinsertion of the Bb rad51 gene into the genome unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3, into which the complete Bb rad51 locus was inserted, for expression of BbRad51 under regulation by autologous elements. Maintenance of BbACc3 makes use of centromeric sequences from chromosome 3 and telomeric ends from chromosome 1 of the B . bovis C9.1 line. A selection cassette employing human dihydrofolate reductase enables recovery of transformants by selection with pyrimethamine. We demonstrate that the BbACc3 platform is stably maintained once established, assembles nucleosomes to form native chromatin, and expands in telomere length over time. Significantly, the MMS-sensitivity phenotype observed in the absence of Bb rad51 was successfully complemented at essentially normal levels. We provide cautionary evidence, however, that in HR-competent parasites BbACc3 can recombine with native chromosomes, potentially resulting in crossover. We propose that, under certain circumstances this platform can provide a useful alternative for the genetic manipulation of this group of parasites, particularly when regulated gene expression under the control of autologous elements may be important.
The tick-borne apicomplexan parasite, Babesia bovis, a highly persistent bovine pathogen, expresses VESA1 proteins on the infected erythrocyte surface to mediate cytoadhesion. The cytoadhesion ligand, VESA1, which protects the parasite from splenic passage, is itself protected from a host immune response by rapid antigenic variation. B. bovis relies upon segmental gene conversion (SGC) as a major mechanism to vary VESA1 structure. Gene conversion has been considered a form of homologous recombination (HR), a process for which Rad51 proteins are considered pivotal components. This could make BbRad51 a choice target for development of inhibitors that both interfere with parasite genome integrity and disrupt HR-dependent antigenic variation. Previously, we knocked out the Bbrad51 gene from the B. bovis haploid genome, resulting in a phenotype of sensitivity to methylmethane sulfonate (MMS) and apparent loss of HR-dependent integration of exogenous DNA. In a further characterization of BbRad51, we demonstrate here that ΔBbrad51 parasites are not more sensitive than wild-type to DNA damage induced by γ-irradiation, and repair their genome with similar kinetics. To assess the need for BbRad51 in SGC, RT-PCR was used to observe alterations to a highly variant region of ves1α transcripts over time. Mapping of these amplicons to the genome revealed a significant reduction of in situ transcriptional switching (isTS) among ves loci, but not cessation. By combining existing pipelines for analysis of the amplicons, we demonstrate that SGC continues unabated in ΔBbrad51 parasites, albeit at an overall reduced rate, and a reduction in SGC tract lengths was observed. By contrast, no differences were observed in the lengths of homologous sequences at which recombination occurred. These results indicate that, whereas BbRad51 is not essential to babesial antigenic variation, it influences epigenetic control of ves loci, and its absence significantly reduces successful variation. These results necessitate a
24 Babesia bovis establishes persistent infections of long duration in cattle, despite the development 25 of effective anti-disease immunity. One mechanism used by the parasite to achieve persistence is 26 rapid antigenic variation of the VESA1 cytoadhesion ligand through segmental gene conversion 27 (SGC), a phenomenon thought to be a form of homologous recombination (HR). To begin 28 investigation of the enzymatic basis for SGC we initially identified and knocked out the Bbrad51 29 gene encoding the B. bovis Rad51 ortholog. BbRad51 was found to be non-essential for in vitro 30 growth of asexual-stage parasites. However, its loss resulted in hypersensitivity to 31 methylmethane sulfonate (MMS) and an apparent defect in HR. This defect rendered attempts to 32 complement the knockout phenotype by reinsertion of the Bbrad51 gene into the genome 33 unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3, 34 into which the complete Bbrad51 locus was inserted, for expression of BbRad51 under 35 regulation by autologous elements. Maintenance of BbACc3 makes use of centromeric 36 sequences from chromosome 3 and telomeric ends from chromosome 1 of the B. bovis C9.1 line. 37 A selection cassette employing human dihydrofolate reductase enables recovery of transformants 38 by selection with pyrimethamine. We demonstrate that the BbACc3 platform is stably 39 maintained once established, assembles nucleosomes to form native chromatin, and expands in 40 telomere length over time. Significantly, the MMS-sensitivity phenotype observed in the absence 41 of Bbrad51 was successfully complemented at essentially normal levels. We provide cautionary 42 evidence, however, that in HR-competent parasites BbACc3 can recombine with native 43 chromosomes, potentially resulting in crossover. We propose that, under certain circumstances 44 this platform can provide a useful alternative for the genetic manipulation of this group of 45 parasites, particularly when regulated gene expression under the control of autologous elements 46 may be important. 47 48 49 Keywords 50 artificial chromosome; Babesia bovis; chromatin; gene complementation; homologous 51 recombination; Rad51 52 53 Introduction 54 Babesiosis is a tick-borne disease caused by apicomplexan parasites of the genus Babesia. 55 Humans are not the natural host for any babesial parasite but may be an incidental host, 56 acquiring zoonotic infections with a variety of different species. Babesia microti is the most 57 common species of Babesia to infect humans, although in western Europe infections commonly 58 occur with Babesia divergens. In the U.S. infections have been observed with Babesia duncani 59 and B. divergens-like organisms, as well as the unspeciated WA1 and MO1 isolates (reviewed in 60 [1]). Many individuals may carry asymptomatic infections [2], including as a result of inadequate 61 drug treatment of acute parasitemia [3, 4], posing a serious risk to the blood supply [5]. In cattle 62 babesiosis may be caused by at least five different ...
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