Dominant negative mutant of <scp><i>P</i></scp><i>lasmodium</i> <scp>R</scp>ad51 causes reduced parasite burden in host by abrogating <scp>DNA</scp> double‐strand break repair

Roy, Nabamita; Bhattacharyya, Sunanda; Chakrabarty, Swati; Laskar, Shyamasree; Babu, Somepalli Mastan; Bhattacharyya, Mrinal Kanti

doi:10.1111/mmi.12762

Cited by 19 publications

(33 citation statements)

References 40 publications

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“…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]. Our prior identification of This parasite instead may depend upon a synthesis-dependent microhomologymediated end-joining mechanism like that demonstrated in P. falciparum [42], but this remains to be determined.…”

Section: Discussionmentioning

confidence: 99%

“…Organisms with defective Rad51 consistently suffer reduced viability and enhanced sensitivity to environmental insult [24][25][26][27][28][29]. For example, knockout of the Rad51 gene in mice resulted in embryonic lethality [30], whereas loss of the Tbrad51 gene in the kinetoplastid parasite, Trypanosoma brucei, yielded parasites that were compromised in growth and hypersensitive to methyl methanesulfonate (MMS) [28].…”

Section: Introductionmentioning

confidence: 99%

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

Mack

Tagliamonte

Xiao

et al. 2020

Preprint

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AbstractThe 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 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 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 parasite’s capacity for HR-dependent integration of exogenous DNA, the loss of BbRad51 did not affect the parasite’s 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 parasite’s capacity for in vivo persistence is predicted.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Mack

Tagliamonte

Xiao

et al. 2020

Preprint

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“…Some reports which have shown that BER, MMR and DNA doublestrand break repair (DSBR) pathways are somehow linked to drug resistance in P. falciparum and some of the proteins involved in these pathways can be the new anti-malarial drug targets [21][22][23][24]. Several non-synonymous single nucleotide polymorphisms (SNPs) in the MMR genes of artemisnin drug resistant strains in P. falciparum have been reported [15].…”

Section: Introductionmentioning

confidence: 98%

Comparative insight into nucleotide excision repair components of Plasmodium falciparum

et al. 2015

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“…PfRad51. This study demonstrated that parasite exclusively relies on the HR repair pathways for DSBs repair (54). Although, it has been reported previously that DNA damage can induce up-regulation of PfRad51 and PfRad54, but how does HR repair machinery recognizes DSBs in the parasite has yet to be studied (52).…”

Section: Double-strand Break Repair Pathways (Dsbr)mentioning

confidence: 63%

“…It has been shown that the parasite mostly utilizes the homologous recombination repair (HR) system to repair its double-strand breaks, which are the most deleterious forms of genetic aberrations, which in turn can lead to cell death (54). It is believed that the parasite lacks the machinery for canonical NHEJ repair system (7).…”

Section: Introductionmentioning

confidence: 99%

Regulation of gene expression during DNA damage response in Plasmodium falciparum

Gupta¹

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First and foremost, I would like to express my sincere gratitude to my Ph. D. supervisor Associate Professor Zbynek Bozdech for his encouragement, support and guidance throughout my research. I thank him for providing an excellent scientific environment, stimulating discussions and for having faith in me. I would also like to thank my doctoral committee members Professor Daniela Rhodes and Dr. Eugene Makeyev for their valuable suggestions. I am thankful to SBS, NTU, for providing all the facilities and the financial support throughout my PhD. I am thankful to all the former and present lab members of Zbynek Bozdech Lab for providing the cordial research environment. I am very grateful to Dr. Archna Gupta for assisting me in the data analysis. I would also like to thank Dr Vineet Kumar and Alok Patra for their friendship and continuous help that accelerated my research and thesis progress. Lastly, I want to express my gratitude and love to my parents, sisters, and my wife Dr. Vijit Dalal for their incessant love and prayers which gave me the strength to finish this PhD thesis.

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Dominant negative mutant of Plasmodium Rad51 causes reduced parasite burden in host by abrogating DNA double‐strand break repair

Abstract: SummaryMalaria parasites survive through repairing a plethora of DNA double-stranded breaks (DSBs) experienced during their asexual growth. In Plasmodium Rad51 mediated homologous recombination (HR) mechanism and homology-independent alternative endjoining mechanism have been identified.

Cited by 19 publications

References 40 publications

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

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

Comparative insight into nucleotide excision repair components of Plasmodium falciparum

Regulation of gene expression during DNA damage response in Plasmodium falciparum

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