In our search for PEBP domain-containing proteins in the Plasmodium genome, we found two proteins PF3D7_0303900 (PfPEBP) and PF3D7_1219700 (annotated as RAF kinase inhibitor, PfRKIP). Proteomic data from PlasmoDB suggests that PfPEBP is highly expressed in gametocyte stages. PfRKIP, in contrast, is majorly expressed in sporozoite stages, suggesting varying functions of the PEBP domain-containing proteins in developmental stages. Domain analysis revealed the presence of a single PEBP domain in PfPEBP and an N-terminus signal peptide while PfRKIP, in comparison, contained only a PEBP domain (Figures 1A, B). Multiple sequence alignment of PEBP protein from five human-infecting parasites reveals moderate level of conservation among Plasmodium species (Supplementary Figure 1A). However, minimal similarity was observed with its human ortholog. A broader similarity tree that included five human-infecting Plasmodium species, human PEBP, and a representative plant PEBP (e.g., Arabidopsis thaliana FT/TFL1 family) shows that plant and human PEBPs cluster closer to each other than to parasite PEBPs (Supplementary Figure 1B). This supports the concept of lineage-specific functional evolution and shows that the parasite PEBP family differs significantly from canonical metazoan/plant orthologs. Within the parasites, P. knowlesi and P. vivax PEBP proteins are most closely related to each other, while P. ovale, P. falciparum, P. malariae PEBP proteins are more distant, suggesting different selective pressures or paralog divergence across Plasmodium species.

To investigate PfPEBP expression and localization, antisera were generated using a synthetic KLH-conjugated peptide based on the amino acids corresponding to small stretch of PEBP domain. Immunofluorescence assays (IFAs) on asexual-stage parasites using antibody against BIP (marker for the endoplasmic reticulum) demonstrated PfPEBP expression in ring, trophozoite, and schizont stages (Figure 1C). IFAs performed on gametocyte stages further confirmed PfPEBP expression from stage II through stage V gametocytes (Figure 1D). Dual-label IFAs using antibodies against α-Tubulin and Pfs25 (markers for male and female gametocytes, respectively) revealed that PfPEBP is expressed in both male and female stage V gametocytes. (Figures 1E, F). PfPEBP appears to be predominantly localized in the cytoplasm. However, PfPEBP was absent in male gametes but remained detectable in the residual bodies of activated gametocytes (Figure 1E).

To analyze the expression of PfPEBP, we created gene deletion parasites (Pfpebp¯) using CRISPR/Cas9 mediated transgenesis (Figure 2A). A set of diagnostic PCRs with oligonucleotides specific to the PfPEBP locus and its upstream (5’) and downstream (3’) regions were used to confirm Pfpebp¯ parasites (Figure 2C). For phenotypic analysis, we used two clones for Pfpebp¯ parasites (clones B11 and F12) (Figure 2B). To assess the role of PfPEBP in asexual-stage development, parasite growth rates were measured in two independent clones (clone B11 and F12) alongside wild-type (WT) PfNF54 parasites. Growth was monitored over two replication cycles, with parasitemia quantified from Giemsa-stained thin smears prepared every 48 hours. The growth rate of Pfpebp¯ was comparable to WT NF54, indicating no significant defect in asexual parasite replication (Figure 3A).

To evaluate the ability of Pfpebp¯ parasites to generate gametocytes, gametocyte induction was performed in WT and Pfpebp¯ (clone B11 and F12) parasites. Gametocytemia was quantified on day 15 of in vitro culture using Giemsa-stained smears. Pfpebp¯ parasites developed into mature stage V gametocytes (Figures 3B, C), with gametocytemia comparable to WT parasites (Figure 3D). To assess any possible defect to the sex allocation, gametocytogenesis assay was performed with wild type and Pfpebp^- clones to quantitate the number of females to male gametocytes formed at day 15 of in vitro culture using Giemsa-stained smears. This revealed no difference in sex-allocation in Pfpebp^- parasites (Figure 3E). To assess gametogenesis, day 15 gametocyte cultures of WT and Pfpebp¯ parasites were activated by the addition of O⁺ human serum and a temperature shift from 37 °C to room temperature. Wet mounts were prepared from activated gametocytes, and the number of exflagellation centers, indicative of male gametogenesis was quantified across ten random fields of view via bright microscopy. Pfpebp¯ parasites exhibited a similar number of exflagellation centers as WT Pf NF54, indicating that male and female gamete formation was unaffected in Pfpebp¯ parasites (Figure 4A). IFA, performed on activated gametocytes of WT PfNF54 and Pfpebp¯ clones using α-Tubulin (male gametocyte marker) and Pfs25 (female specific marker) revealed that axoneme formation remained unaffected, indicating normal microgametogenesis (Figures 4B, C). Likewise, female gamete formation also appeared normal (Figure 4D).

All restriction enzymes were purchased from New England Biolabs (NEB), USA. Molecular biology reagents were obtained from Millipore-Sigma, USA, or Thermo Scientific, USA. Oligonucleotides were synthesized by Thermo Scientific, USA. Rabbit anti-PfPEBP antibodies (1:200; IFA) were generated by Biomatik Inc. (DE, US). All secondary antibodies for IFAs were procured from Thermo Fisher Scientific, USA. The following primary antibodies, antisera, and dilutions were used: mouse α-tubulin (1:400, Sigma-Aldrich, cat# T5168), α-Pfs25 (1:1), BEI Resources, NIAID, NIH supplied the Hybridoma 4B7 anti-Pfs25-kilodalton gamete surface protein, MRA-315, contributed by Louis H. Miller and Allan Saul.

Peptide corresponding to PEBP domain amino acids 95–116 of PfPEBP (DKNKGTKSYVITLTS), containing a fragment of the conjugated to carrier protein Keyhole limpet hemocyanin (KLH) - was synthesized by Biomatik Inc. (DE, US) and was used for immunization of animals. PfPEBP antisera was generated by immunizing rabbits by Biomatik Inc. (DE, US) and prescribes guidelines for animal handling were followed. IgG purification and ELISA was performed by Biomatik Inc. Pre immune sera was used as a control in ELISA assays to ensure the antisera specifically reacted with PEBP peptide.

P. falciparum parasites (WT PfNF54 and Pfpebp¯) were maintained as asexual blood-stage cultures following standard protocols, with complete RPMI medium supplemented with either 0.5% AlbuMAX™ II (Thermo Scientific) or 10% (v/v) type O⁺ human serum, refreshed every 24 hours. In vitro gametocyte production was conducted using O⁺ human RBCs (BioIVT, USA) and O⁺ human serum (BioIVT, USA) as described previously (Tripathi et al., 2020). PfPEBP (PlasmoDB identifier Gene - PF3D7_0303900) was deleted via CRISPR/Cas9, with successful gene disruption confirmed by genotyping PCR (Figure 2B). Two independent Pfpebp¯ clones (B11 and F12) were used for functional assays.

To compare the growth rates of PfNF54 and Pfpebp¯parasites, cultures were synchronized with 5% sorbitol and seeded at equal parasitemia (1%) at the ring stage in 6-well plates. Parasite growth was assessed over two life cycles by quantifying parasitemia on thin Giemsa-stained smears. Gametocytemia was measured on day 15 of in vitro culture using thin Giemsa-stained smears to evaluate sexual development.

Gametocytes of WT PfNF54 and Pfpebp¯ were cultured as described above. Gametocytes from both the cultures were analyzed for prevalence of stage V gametocytes using Giemsa smears. For assaying comparative exflagellation, equal volume of gametocytes from WT PfNF54 and Pfpebp¯ were mixed with human serum and O⁺ RBCs (50:50) % (v/v) and incubated at room temperature for 10 min. Exflagellation was scored for WT PfNF54 and Pfpebp¯ parasites via light microscopy by counting exflagellation centers in 10 random optical fields of view at 40× magnification in a bright field microscope. Identical acquisition and display settings were used where comparisons are implied.

For IFAs on gametocytes and exflagellating gametes, thin smears were prepared on Teflon coated slides and fixed with chilled methanol for 5 min (2 min for asexual stages). Slides were kept in a humidity chamber for each step. Fixed parasites were washed twice with PBS and permeabilized using 0.05%saponin/PBS solution for 1 min. Parasites were washed twice with PBS for 5 min each and blocked with 3%BSA/PBS for 45 min. Primary antisera in 3% BSA/PBS was added to the parasites and slides were incubated overnight at 4 °C. Antigens were visualized using anti-species antibodies. Images were obtained using a 100× 1.4 NA objective 90 on a Keyence BZ X800 Florescence microscope.

All data are expressed as mean ± SD. Statistical differences were determined using one-way ANOVA with post hoc Bonferroni multiple comparison test or unpaired two-tailed Student’s t test, as indicated. Values of p < 0.05 were considered statistically significant. Significances were calculated using GraphPad Prism 10 and are represented in the Figures as follows: ns, not significant, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.