Babesia bovis Rad51 ortholog influences switching of ves genes but is not essential for segmental gene conversion in antigenic variation

Abstract

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 makes BbRad51 a choice target for development of inhibitors that could 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 a failure to upregulate the Bbrad51 gene in response to DNA damage. Moreover, we demonstrate that {Delta}Bbrad51 parasites are not more sensitive than wild-type to DNA damage induced by {gamma}-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 ves1 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 {Delta}Bbrad51 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 reconsideration of the likely enzymatic mechanism(s) underlying SGC and suggest the existence of additional targets for development of small molecule inhibitors. Author summaryB. bovis establishes highly persistent infections in cattle, in part by using cytoadhesion to avoid passage through the spleen. While protective, a host antibody response targeting the cytoadhesion ligand is quickly rendered ineffective by antigenic variation. In B. bovis, antigenic variation relies heavily upon segmental gene conversion (SGC), presumed to be a form of homologous recombination (HR), to generate variants. As Rad51 is generally considered essential to HR, we investigated its contribution to SGC. While diminishing the parasites capacity for HR-dependent integration of exogenous DNA, the loss of BbRad51 did not affect the parasites sensitivity to ionizing radiation, overall genome stability, or competence for SGC. Instead, loss of BbRad51 diminished the extent of in situ transcriptional switching (isTS) among ves gene loci, the accumulation of SGC recombinants, and the mean lengths of SGC sequence tracts. Given the overall reductions in VESA1 variability, compromise of the parasites capacity for in vivo persistence is predicted.