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 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

A Culture-Adapted Strain of Babesia bovis Has Reduced Subpopulation Complexity and Is Unable to Complete Its Natural Life Cycle in Ticks

“…Importantly, and in contrast to parasites found in circulation in infected animals, in vitro grown parasites develop in the absence of pressure of the immune system, and thus do not need to resort to activating mechanisms involved in antigenic variation, which are required to evade the immune system responses of their natural hosts. These mechanisms are known to require epigenetic modifications, including chromatin re-arrangements ( Mack et al, 2020 ). Altogether, this suggests that Babesia parasites have a great ability to adapt in order to survive in the face of important selective environmental forces ( Lau et al, 2011 ).…”

Integration of DNA Repair, Antigenic Variation, Cytoadhesion, and Chance in Babesia Survival: A Perspective

“…This finding led to identification of the ves multigene family encoding VESA1 polypeptides ( Allred et al., 2000 ; Xiao et al., 2010 ), and determination that variation is achieved through a process of segmental gene conversion (SGC) ( Al-Khedery and Allred, 2006 ). During SGC short segments (109 bp on average) of the actively transcribed ves genes are replaced with alternative sequences from silent ves genes in loci scattered about the genome ( Al-Khedery and Allred, 2006 ; Mack et al., 2020 ). The outcome of this back-and-forth between recognition and destruction versus variation and escape is long-term persistence with wide population fluctuations ( Allred et al., 1994 ; Calder et al., 1996 ).…”

“…The ves multigene family is large and scattered over all B. bovis chromosomes ( Al-Khedery and Allred, 2006 ; Brayton et al., 2007 ), yet only one ves locus (comprised of one ves1 α and one ves1 β) is transcriptionally active at a time, the locus of active ves transcription (LAT) ( Al-Khedery and Allred, 2006 ; Żupańska et al., 2009 ; Mack et al., 2020 ). Importantly, transcriptionally active chromatin is more susceptible than silent chromatin to adoption of oxidation-prone higher-order structure, damage, and mutation ( Makova and Hardison, 2015 ).…”

Recent Advances in Molecular Genetic Tools for Babesia

“…To dissect segmental gene conversion through homologous recombination in B. bovis, Mack et al, (2019 2020) disrupted rad51 gene and showed that it is not essential for parasite growth in vitro. However, these parasites lost homologous recombination-dependent gene integration and showed a reduction of in-situ transcriptional switching [9,48]. Recently, a novel multigene family encoding protein with multi-transmembrane domain (mtm) was discovered and overexpressing studies showed that their expression was linked to blasticidin S resistance [26].…”

“…can pave the way for finding pan-Babesia drug targets [8]. Several Babesia parasites are being adapted to in vitro culture [9][10][11][12][13][14][15], which facilitate high-throughput compound screens to find novel drugs [16]. However, the molecular targets of the currently available drugs are lagging mainly due to limited genetic tools for these parasites.…”