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

Abstract

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 ...

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

“…(i) In the first strategy, plasmid pBb rad51 wt_comp (S5 Fig) was engineered to replace all sequences associated with the knockout plasmid by double crossover homologous recombination. The goal was to replace them with the wild-type Bb rad51 locus, concomitant with integration of a human dihydrofolate reductase (hDHFR) expression cassette, to enable selection of transformants with pyrimethamine [62, 73]. pBb rad51 wt_comp was made by PCR-amplifying Bb rad51 3´ terminator sequences from B .…”