Date Published: August 6, 2019
Publisher: Public Library of Science
Author(s): Erin A. Mack, Yu-Ping Xiao, David R. Allred, Ulrike Gertrud Munderloh.
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 Bbrad51 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 Bbrad51 gene into the genome unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3, into which the complete Bbrad51 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 Bbrad51 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.
Babesiosis is a tick-borne disease caused by apicomplexan parasites of the genus Babesia. Humans are not the natural host for any babesial parasite but may be an incidental host, acquiring zoonotic infections with a variety of different species. Babesia microti is the most common species of Babesia to infect humans, although in western Europe infections commonly occur with Babesia divergens. In the U.S. infections have been observed with Babesia duncani and B. divergens-like organisms, as well as the unspeciated WA1 and MO1 isolates (reviewed in ). Many individuals may carry asymptomatic infections , including as a result of inadequate drug treatment of acute parasitemia [3, 4], posing a serious risk to the blood supply . In cattle babesiosis may be caused by at least five different species, with Babesia bovis generally considered the most virulent. B. bovis shares many parallels with the human malarial parasite, Plasmodium falciparum, including immune evasion via cytoadhesion and antigenic variation, and the capacity for development of a lethal cerebral disease .
With increasing human population density and environmental encroachment, as well as climate change, many zoonotic diseases are emerging and/or expanding in range, including babesiosis [47–49]. It is thus important that we understand well the biology of these parasites, and prepare to defend against their expansion. Key to understanding the biology of parasites at the molecular level is the ability to manipulate them genetically, something which is not yet well developed for babesial parasites. We have been especially interested in the mechanisms of immune evasion employed by B. bovis as a potential “Achilles heel” of this parasite which, if compromised, could lead to its control during infection . At least two mechanisms are used to evade an ongoing immune response: cytoadhesion in the deep vasculature and rapid antigenic variation of the cytoadhesion ligand . Previously, we have shown that antigenic variation relies in large part upon segmental gene conversion (SGC) for modification of the expressed member of the ves gene family . With SGC assumed to be a form of homologous recombination, a process thought to be dependent upon the activities of Rad51 and related proteins [18–21, 51], we knocked out the Bbrad51 gene to begin an assessment of its contribution to this phenomenon. Experience in other systems lends credence to this idea. For example, in Trypanosoma spp., which also uses SGC to form mosaic isoforms of VSG proteins [16, 17], knockout of TbRad51 or TbRad51-3 expression reduced the overall rate of antigenic variation dramatically [52, 53]. Here, we demonstrate that a lack of BbRad51 led to hypersensitivity to MMS, an alkylating agent that methylates adenosine bases . The methyl adducts can result in stalled replication forks and/or single-stranded DNA breaks, both of which can advance to double-stranded DNA breaks, and lead to MMS-hypersensitivity in organisms lacking Rad51 [36–38].