Malaria is an infection caused by protozoan parasites of the genus Plasmodium. In 2020, there were an estimated 229 million cases and 409,000 deaths worldwide (WHO: https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2020). Malaria is difficult to control; no efficacious vaccine has been developed. Moreover, there is continuous antigenic variation, genetic diversity, and antimalarial drug resistance in Plasmodium [1, 2]. This parasite has a unique life cycle . In humans, it is transmitted from host to host via Anopheles mosquitoes. When a mosquito takes a blood meal from a host, it injects Plasmodium sporozoites into the bloodstream. The sporozoites ultimately migrate to the host liver where they develop into merozoites which are then released into circulation and start an asexual reproduction cycle. Only a small proportion of the parasites exist as gametocytes in the blood. When a female Anopheles mosquito takes a blood meal from a host, gametocytes are ingested into the midgut and differentiate into male and female gametes. Fertilization, zygote formation, and meiosis then occur. Next, the zygotes differentiate into motile ookinetes. The latter migrate, traverse the midgut epithelium, settle under the midgut basal lamina, and differentiate into oocysts at approximately twenty-four hours after blood ingestion. The asexual reproduction cycle then resumes once again. Several thousands of sporozoites form within each oocyst and are released approximately two weeks after blood ingestion. The sporozoites then migrate, invade the mosquito salivary glands, and enable the insect to transmit the pathogen to another host.
Efficacious drugs targeting all Plasmodium life cycle stages are required to control malaria. The stages from zygote to ookinete are feasible as drug targets as their numbers are far lower than those of the other stages. Moreover, they are not enclosed in the erythrocyte membrane and are, therefore, relatively more susceptible to direct drug exposure . However, currently available anti-Plasmodium drugs only target blood-stage parasites. Sexual reproduction and meiosis occur from the zygote to ookinete stages. In mammals, Rad51 and Dmc1 DNA recombinases regulate homologous recombination (HR) during meiosis . Plasmodium Rad51 also contributes to HR [3, 6, 7]. Dmc1 knockout in Plasmodium berghei resulted in reduced numbers and altered oocyst and sporozoite development [3, 6, 7]. Thus, Rad51 and Dmc1 are required for HR during meiosis. The recombinases in mammals interact with BRCA2 and are regulated by it. This mechanism also contributes to HR during meiosis [5, 8]. Mammalian BRCA2 harbors several functional domains. In humans, Rad51 and Dmc1 interact with BRCA2 through BRC repeats. Rad51 and Dmc1 also interact with BRCA2 through the C-terminal Rad51- and DMC1-binding domains, respectively [9–11]. However, Thorslund et al. reported that DMC1 interacted with BRCA2 through the DMC1-binding domain but not through BRC repeats . In contrast, Martinez et al. demonstrated that BRCA2 does, in fact, interact with BRC repeats . Both recombinases required BRCA2 for effective recruitment to HR sites. BRCA2 also has tower and oligonucleotide/oligosaccharide-binding (OB)-fold domains that bind DNA. They facilitate Rad51 and Dmc1 recruitment to the DNA HR sites. HR also participates in DNA double-strand break (DSB) repair. Most living organisms repair DSBs via HR and/or non-homologous end joining (NHEJ). The HR-related proteins are conserved in Plasmodium whereas the NHEJ proteins are not . Thus, most DSBs are repaired by Plasmodium HR proteins .
BRCA2 orthologs have been detected in eukaryotes such as mammals, Xenopus, fish, Caenorhabditis elegans, Ustilago maydis, Trypanosoma, and Leishmania. Nevertheless, they are absent in yeast, archaea, and bacteria [13–17]. In yeast, Rad52 is the Rad51-regulating protein. Human BRCA2 cannot interact with yeast Rad51 . Human BRCA2 is a large protein (384 kDa). However, in lower eukaryotes such as Caenorhabditis elegans, Ustilago maydis, Trypanosoma, and Leishmania, Brca2 orthologs possess only the vital BRC repeats and the tower and OB-fold domains. In Plasmodium, Rad51 and Dmc1 are conserved and their functions resemble those of human Rad51 and Dmc1. However, neither the Rad51 nor the Dmc1 regulatory protein has been identified. Thus, the aims of the present study were to salvage BRC repeats and DNA binding domain-containing proteins from the Plasmodium database and clarify the functions of Plasmodium Brca2 by generating knockout (KO) P. berghei.