The P. berghei (Pb) Brca2 (PbBrca2) (PbANKA_1343400) genomic sequences used in the present study were retrieved from PlasmoDB (http://www.plasmodb.org). The online hidden Markov model structure prediction tool HHpred (https://toolkit.tuebingen.mpg.de/tools/hhpred) was used to predict the features of the PbBrca2 domain [19, 20]. The online signal peptide prediction tool PSORT II Prediction (https://psort.hgc.jp/form2.html) was used to predict the subcellular localization of PbBrca2 [21, 22].
Crystal structure modeling
The crystal structures of human Rad51 and BRC repeat 4 were retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB) at http://www.rcsb.org/ [PDB identifier (ID) 1N0W] and analyzed with Chimera software (http://www.cgl.ucsf.edu/chimera/) . The OB-fold and tower domains of PbBrca2 were predicted with AlphaFold2 , and their structures were compared via Chimera against those of human BRCA2 helical, OB, and tower domains (PDB ID 1MIU) or that of the OB domain of human replication protein A1 (PDB ID 4O0A).
Mice, parasites, and mosquitoes
ICR and BALB/c mice aged 6–8 weeks were obtained from Charles River Laboratories Japan (Yokohama, Kanagawa, Japan). The P. berghei ANKA strain constitutively expressing green fluorescent protein was obtained from Dr. M. Yuda (Mie University, Tsu, Mie Prefecture, Japan) . Anopheles stephensi (STE2 strain) mosquitoes were kept in an insectary at 27 °C, 80% relative humidity, and a 14-h/10-h light/dark cycle and fed 10% (w/v) sucrose solution. All experimental procedures were executed following a protocol approved by the Kitasato University Animal Care and Use Committee (approval nos. 16-020 and 19-087).
Mammalian two-hybrid assay
The coding regions of the PbBrca2 BRC repeats domain were cloned into pM plasmids (Clontech Laboratories, Mountain View, CA). Plasmodium berghei Rad51 and Dmc1, Saccharomyces cerevisiae Rad51, and human Rad51 were cloned into pVP16 plasmids (Clontech Laboratories). The methods used for the mammalian two-hybrid assays have been previously described . FuGENE HD transfection reagent (Promega, Madison, WI) was used to co-transfect ~ 2 × 105 cells 293 T cells with pM and pVP16 vectors, the pGluc luciferase reporter, and pRL-tk normalization plasmids (Promega). The luciferase activity of the cell extracts was measured with a Dual-Luciferase Reporter Assay System (Promega).
Generation of PbBrca2-KO parasites
PbBrca2 was knocked out by double-crossover HR technology using a pBluescript II-based plasmid containing a puromycin resistance gene inserted upstream (5′) of the PbEf1α gene and downstream (3′) of the PbDhfr-ts gene, as previously described [26, 27]. A donor plasmid with 1000-bp homology arms was cloned upstream with the primers 5′-PbBrca2 forward (F) (5′-CGGGGTACCTTTTATTGTATCCCTATAA-3′), and 5′-PbBrca2 reverse (R) (5′-GGCGGGCCCCTTTATTTAATTATTAAGATTTTTTTGTTAC-3′); and cloned downstream with the primers 3′ PbBrca2 F (5′-CCGTCTAGATATCGTTTTAAGGTTGTTTC-3′), and 3′-PbBrca2 R (5′-CCGGCGGCCGCTATGTTGTATTGTTTGTTTT-3′). To clone the homology arms, the plasmids were treated with the restriction enzymes KpnI and ApaI for upstream of the PbBrca2 gene and XbaI and NotI for downstream of the PbBrca2 gene (all restriction enzymes were purchased from New England Biolabs, Ipswich, MA). Ten micrograms of donor vector was linearized using ScaI (New England Biolabs) and electroporated with a Nucleofector 2b device (Lonza Group, Basel, Switzerland) into cultured P. berghei schizonts. Transfected parasites were selected by puromycin and cloned by limiting dilution as previously described [26, 27].
PbBrca2-KO parasite genotyping and PbBrca2 amplification in complementary DNA
Blood infected with wild type (WT) or PbBrca2-KO parasites was collected and the DNA or RNA extracted with a Gentra Puregene Blood Kit (Qiagen, Düsseldorf, Germany) or a NucleoSpin RNA Blood Kit (TaKaRa Bio, Kusatsu, Shiga, Japan) according to the manufacturers’ instructions. To genotype the PbBrca2-KO, the PbBrca2 locus was amplified by polymerase chain reaction (PCR) using 5′-PbBrca2 F and 3′-PbBrca2 R from genomic DNA. The extracted RNA was reverse-transcribed with SuperScript IV VILO Master Mix (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. PbBrca2 expression was detected by PCR using PbBrca2 exon 3 F, 5′-GGGAACCACATTTTTAAATGA-3′, and PbBrca2 exon 4 R, 5′-CCTTTGGGTATGTTCTTAGGG-3′.
Southern blot analysis
Two micrograms of the extracted DNA was digested with the restriction endonuclease EcoRV (Roche Diagnostics, Basel, Switzerland), separated on a 0.8% agarose gel, transferred to a Hybond N+ membrane (GE Healthcare, Little Chalfont, UK), and hybridized with probes labeled using a PCR DIG Probe Synthesis Kit (Roche Diagnostics) with the primers PbPuro SH F2 (5′-ACCTCGAGAGATCCCGTTTT-3′) and PbPuro SH R2 (5′-TTTATGAATCATTGAAGAGACAACA-3′). The signal was detected with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Diagnostics) and CDP-Star Detection Reagent (GE Healthcare).
Phenotypic analysis of PbBrca2-KO parasites
The development of WT and PbBrca2-KO parasites in the BALB/c mice was assessed. Five mice per group were each injected with 1 × 106 WT or PbBrca2-KO parasites. Parasitemia was monitored daily using Giemsa-stained blood smears. The morphology of blood-stage parasites was observed using a phase-contrast microscope (ECLIPSE E200; Nikon, Tokyo). A survival assay was performed using nine and 10 mice infected with WT and PbBrca2-KO1 parasites, respectively, for 11 days after infection. The humane endpoints were when mice showed rapid weight loss (> 20% of body weight) or poor physical appearance (reduced mobility, rough coat, and depression). When parasitemia reached approximately 10% in four mice per group, the gametocytes per infected red blood cell were enumerated and the gametocyte sex ratios were determined by Giemsa-stained tail blood smears. Male gametocyte exflagellation was quantified as previously described . Briefly, 1 µL gametocyte-infected blood was drawn from the tail vein and immediately mixed with 1 µL heparin and 38 µL complete ookinete culture medium (OKM; RPMI 1640 medium; Gibco, Grand Island, NY) and 20% (v/v) heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO). The mixture was placed under a coverslip at room temperature. After 5 min, the exflagellation centers were counted under a phase-contrast microscope (ECLIPSE E200; Nikon) for the next 10 min. Parasite differentiation into gametocytes and exflagellation were then assessed. Anopheles stephensi mosquitoes were fed on the infected mice, and the oocysts were microscopically examined between days 13–15. At least 30 mosquitoes were dissected. Enhanced green fluorescent protein-expressing oocysts were detected by fluorescence stereoscopic microscopy (Leica M205 FA; Leica Microsystems, Wetzlar, Germany). The oocysts in each positive midgut were counted to determine infection intensity and the prevalence of infection . The sporozoites in A. stephensi mosquitoes treated as described above were examined microscopically at day 28. Enhanced green fluorescent protein-expressing sporozoites in the salivary glands were detected using fluorescence microscopy (Leica M205 FA; Leica Microsystems).
Ookinete culture and purification
Blood presenting with 10 × 104 red blood cells in which gametocytes were undergoing exflagellation was obtained from mice by heart puncture. Ten volumes of OKM was added to the blood and the suspension was cultured at 19 °C for 24 h. Ookinetes were enumerated and their structure was examined using culture suspension smears. They were purified in a MidiMACS Separator System (Miltenyi Biotec, Bergisch-Gladbach, Germany) as previously described . A total of 3 mL culture was passed through the system thrice before removing the column from the magnet. The ookinetes were recovered by passing 5 mL OKM through the column. The purified ookinetes were centrifuged at 1000 g and 4 °C for 10 min and washed thrice in phosphate-buffered saline. Ookinete morphology was observed by differential interference contrast microscopy (Olympus IX83; Olympus, Tokyo, Japan).
Transmission electron microscopy
Mosquito midguts were collected at 7, 14, and 21 days post-infection to evaluate oocyst differentiation, sporozoite development, and sporozoite maturation, respectively. The midguts were fixed in 2.5% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.03 M HEPES buffer (pH 7.4) at 4 °C for 2 h. The cells were then treated with 1% (w/v) osmium tetrachloride/1.5% (w/v) potassium ferrocyanide at 21–24 °C for 2 h to enhance cytoplasmic contrast . The midguts were then dehydrated, embedded in epoxy resin, sectioned to 70-nm thickness with an Ultracut N (Reichert–Nissei, Vienna), stained with 1% (w/v) uranyl acetate for 18 min and 2% (w/v) lead citrate for 1 min, and examined under a transmission electron microscope (H-7650; Hitachi, Tokyo) at 80 kV .
The F-test followed by Student’s t-test with or without Holm’s correction was used to compare treatments in terms of interaction activity (mammalian two-hybrid assay), parasitemia, infection intensity (number of oocysts/midgut), gametocytemia, gametocyte and exflagellation ratios, and ookinete numbers. A log-rank (Mantel–Cox) test was used to compare the survival rate in the survival assay. The total number of each blood-stage counts and the total number of female and male gametocyte counts were analyzed by a χ2-test. The number of oocysts was assessed by a Mann–Whitney U-test.