Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform

Abstract

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.

Babesia bovisRad51 ortholog influences switching ofvesgenes but is not essential for segmental gene conversion in antigenic variation

“…conversion. Yet, in B. bovis orthologs may be identified only for RPA, Rad51, and Rad54, and many similar examples of "missing" proteins hold [13,31], suggesting the merging of functions. In this study, we wished to understand the contributions of BbRad51 to SGC because proteins of this family are considered essential to HR and gene conversion in other systems, including other apicomplexans [29,40].…”

“…DNA damage. Previously, we knocked out the Bbrad51 gene, resulting in a parasite phenotype of MMS-sensitivity and apparent loss of the ability to integrate exogenous DNA by HR, but not significant difference in rates of growth [31]. Cells that suffer environmental insult may up-regulate DNA repair proteins, including Rad51 [32].…”

“…Structures of these plasmids are shown in S1 Figure, and sequences are provided in S2_File and S3_File. Reporter parasites were exposed to 250 M MMS, a level that reproducibly inhibits growth by approximately 10% after 48 hours in wild-type B. bovis CE11 parasites [31], presumably sufficient to induce Bbrad51 expression in response but low enough not to significantly reduce viability. Under these conditions, no evidence of an alteration in BbRad51 expression was observed after either 3 hours ( Figure 1A A second measure of phenotype commonly used in studies of DNA repair is sensitivity to -irradiation.…”

“…Although overall DNA repair following damage from ionizing radiation was not measurably impaired by the loss of BbRad51, sensitivity to MMS and loss of ability to integrate selectable plasmids via long sequence tracts suggested that BbRad51 plays a role in aspects of HR [31], and perhaps in repair of stalled replication forks [36].…”

“…Three immediate subclones of CE11 wild-type and three independent knockout clonal lines were studied; their origin is described in [31]. Total RNAs were collected from each clonal line at one month and five months post-cloning.…”