PMID- 37212694 OWN - NLM STAT- MEDLINE DCOM- 20230619 LR - 20231120 IS - 2165-0497 (Electronic) IS - 2165-0497 (Linking) VI - 11 IP - 3 DP - 2023 Jun 15 TI - Plasmodium Topoisomerase VIB and Spo11 Constitute Functional Type IIB Topoisomerase in Malaria Parasite: Its Possible Role in Mitochondrial DNA Segregation. PG - e0498022 LID - 10.1128/spectrum.04980-22 [doi] LID - e04980-22 AB - The human malaria parasite undergoes a noncanonical cell division, namely, endoreduplication, where several rounds of nuclear, mitochondrial, and apicoplast replication occur without cytoplasmic division. Despite its importance in Plasmodium biology, the topoisomerases essential for decatenation of replicated chromosome during endoreduplication remain elusive. We hypothesize that the topoisomerase VI complex, containing Plasmodium falciparum topiosomerase VIB (PfTopoVIB) and catalytic P. falciparum Spo11 (PfSpo11), might be involved in the segregation of the Plasmodium mitochondrial genome. Here, we demonstrate that the putative PfSpo11 is the functional ortholog of yeast Spo11 that can complement the sporulation defects of the yeast Deltaspo11 strain, and the catalytic mutant Pfspo11Y65F cannot complement such defects. PfTopoVIB and PfSpo11 display a distinct expression pattern compared to the other type II topoisomerases of Plasmodium and are induced specifically at the late schizont stage of the parasite, when the mitochondrial genome segregation occurs. Furthermore, PfTopoVIB and PfSpo11 are physically associated with each other at the late schizont stage, and both subunits are localized in the mitochondria. Using PfTopoVIB- and PfSpo11-specific antibodies, we immunoprecipitated the chromatin of tightly synchronous early, mid-, and late schizont stage-specific parasites and found that both the subunits are associated with the mitochondrial genome during the late schizont stage of the parasite. Furthermore, PfTopoVIB inhibitor radicicol and atovaquone show synergistic interaction. Accordingly, atovaquone-mediated disruption of mitochondrial membrane potential reduces the import and recruitment of both subunits of PfTopoVI to mitochondrial DNA (mtDNA) in a dose-dependent manner. The structural differences between PfTopoVIB and human TopoVIB-like protein could be exploited for development of a novel antimalarial agent. IMPORTANCE This study demonstrates a likely role of topoisomerase VI in the mitochondrial genome segregation of Plasmodium falciparum during endoreduplication. We show that PfTopoVIB and PfSpo11 remain associated and form the functional holoenzyme within the parasite. The spatiotemporal expression of both subunits of PfTopoVI correlates well with their recruitment to the mitochondrial DNA at the late schizont stage of the parasite. Additionally, the synergistic interaction between PfTopoVI inhibitor and the disruptor of mitochondrial membrane potential, atovaquone, supports that topoisomerase VI is the mitochondrial topoisomerase of the malaria parasite. We propose that topoisomerase VI may act as a novel target against malaria. FAU - Singh, Priyanka AU - Singh P AD - Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, India. FAU - Tabassum, Wahida AU - Tabassum W AD - Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India. FAU - Fangaria, Nupur AU - Fangaria N AD - Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, India. FAU - Dey, Sandeep AU - Dey S AD - Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad, India. FAU - Padhi, Siladitya AU - Padhi S AD - TCS Research-Hyderabad (Life Sciences Division), Tata Consultancy Services Limited, Hyderabad, India. FAU - Bhattacharyya, Mrinal K AU - Bhattacharyya MK AUID- ORCID: 0000-0001-6640-4081 AD - Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India. FAU - Arun Kumar, Kota AU - Arun Kumar K AD - Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad, India. FAU - Roy, Arijit AU - Roy A AD - TCS Research-Hyderabad (Life Sciences Division), Tata Consultancy Services Limited, Hyderabad, India. FAU - Bhattacharyya, Sunanda AU - Bhattacharyya S AUID- ORCID: 0000-0003-2786-7299 AD - Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, India. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20230522 PL - United States TA - Microbiol Spectr JT - Microbiology spectrum JID - 101634614 RN - Y883P1Z2LT (Atovaquone) RN - 0 (DNA, Mitochondrial) RN - 0 (Protozoan Proteins) RN - EC 3.1.- (Spo11 protein, S cerevisiae) RN - EC 3.1.- (Endodeoxyribonucleases) RN - 0 (Saccharomyces cerevisiae Proteins) SB - IM MH - Animals MH - Humans MH - *Parasites/genetics/metabolism MH - Atovaquone MH - Saccharomyces cerevisiae/metabolism MH - Plasmodium falciparum/genetics MH - *Malaria, Falciparum/parasitology MH - *Malaria MH - DNA, Mitochondrial/genetics MH - Protozoan Proteins/genetics/metabolism MH - Endodeoxyribonucleases MH - *Saccharomyces cerevisiae Proteins PMC - PMC10269783 OTO - NOTNLM OT - Plasmodium mitochondria OT - Plasmodium topoisomerase VI OT - apicomplexan topoisomerase COIS- The authors declare no conflict of interest. EDAT- 2023/05/22 13:04 MHDA- 2023/06/19 13:08 PMCR- 2023/05/22 CRDT- 2023/05/22 10:13 PHST- 2023/06/19 13:08 [medline] PHST- 2023/05/22 13:04 [pubmed] PHST- 2023/05/22 10:13 [entrez] PHST- 2023/05/22 00:00 [pmc-release] AID - 04980-22 [pii] AID - spectrum.04980-22 [pii] AID - 10.1128/spectrum.04980-22 [doi] PST - ppublish SO - Microbiol Spectr. 2023 Jun 15;11(3):e0498022. doi: 10.1128/spectrum.04980-22. Epub 2023 May 22.