<i>Plasmodium</i> mitochondrial exodeoxyribonucleases, Exo _mit1 and Exo _mit2 , are conserved within alveolates with Exo _mit1 essential for the establishment of blood‐stage infection

The 6-kb linear repeat genome of the mitochondrion (mtDNA) of the malaria parasite is among the smallest known in nature, but is well-conserved in comparison with its apicoplast and nuclear genomes. Except for the presence of base excision repair (BER) and two double-strand break repair (DSBR) proteins in mitochondria, the mechanisms for preservation of mtDNA integrity during traversal of the parasite through different cell types and environments in the mosquito vector and mammalian host are not characterized. We identified two putative organellar exonucleases in Plasmodium falciparum, PfExo_mit1 and PfExo_mit2, with homologs present only within certain alveolates. Immunofluorescence localization and chromatin immunoprecipitation experiments using antibodies generated against recombinant proteins showed that they are localized to the mitochondrion. PfExo_mit1 and PfExo_mit2 demonstrated specificity for different DNA substrates; PfExo_mit1 cleaved ssDNA in both polarities, while PfExo_mit2 was a bipolar exonuclease on dsDNA with 3'-5' exonuclease activity on ssDNA. The mismatch repair (MMR) protein PfMutS, which carries an additional endonuclease domain, was localized in the mitochondria and interacted with PfExo_mit2 in pull-down assays. PfExo_mit2 also interacted with the mitochondria-targeted DSBR protein PfRad51, suggesting that it is a component of both MMR and DSBR pathways. When Exo_mit1 expression in the rodent parasite P. berghei was silenced in sporozoites via conditional mutagenesis, PbExo_mit1 conditional knockout sporozoites invaded hepatocytes and developed in the liver, but could not transition to the blood stage. PbExo_mit1 localized to the mitochondria in liver stages as well, indicating that its ssDNA exonuclease function in mtDNA processing in the liver impacted establishment of blood-stage infection.