Plasmodium microtubule-binding protein EB1 is critical for partitioning of nuclei in male gametogenesis

Abstract

Sexual reproduction of the malaria parasites is critical for their transmission to a mosquito vector. Several signaling molecules, such as kinases and phosphatases, are known to regulate this process. We previously demonstrated that Plasmodium falciparum (Pf) Ca²⁺-dependent protein kinase 4 (CDPK4) and serine/arginine-rich protein kinase 1 (SRPK1) are critical for axoneme formation during male gametogenesis, with genetic deletion of either gene causing a complete block in parasite transmission to the mosquito. A comparative phospho-proteome analysis of Pfcdpk4^- and RNA-seq analysis of Pfsrpk1^- gametocytes showed that these kinases regulate similar biological processes linked to both microtubule (MT) dynamics and cell motility. One of these proteins was a nuclear MT-associated End Binding protein 1 (EB1), which was hypophosphorylated in Pfcdpk4^- gametocytes. To study the functional relevance of EB1, we created gene deletion parasites for EB1. We further demonstrate that Pfeb1^- parasites like WT NF54 parasites proliferate normally as asexuals and undergo gametocytogenesis and gametogenesis. Strikingly, these parasites suffer a severe defect in nuclear segregation and partitioning of nuclei into emerging microgametes. Further genetic crosses utilizing male- and female-sterile parasites revealed that Pfeb1^- parasites only suffer a male fertility defect. Overall, our study reveals an essential function for PfEB1 in male gamete nuclear segregation and suggests a potential therapeutic avenue in the design of transmission-blocking drugs to prevent malaria transmission from humans to mosquito. IMPORTANCE Gametogenesis and subsequent gamete fusion are central to successful transmission of the malaria parasites to a female Anopheles mosquito vector and completion of the sexual phase of the parasite life cycle. Male gametogenesis involves the formation of axonemes inside male gametes from male gametocytes via active cytoskeleton remodeling. The tubulin and tubulin-binding proteins are, thus, attractive anti-malarial drug targets. In the present study, we demonstrate that a microtubule-binding protein PfEB1 is essential for male gamete fertility, specifically for the inheritance of nuclei from activated male gametocytes. Targeting PfEB1 function may provide new avenues into designing interventions to prevent malaria transmission and disease spread.

Keywords: Plasmodium; gamete; microtubules; mosquito; nucleus; transmission.

Fig 1

PfEB1 and generation of Pfeb1⁻ parasites. (A) Domain architecture of PfEB1 (upper cartoon) and HsEB1 (lower cartoon) showing the CH domains in blue and the EB1 domains in dark blue. The EB1 domain extends from 417 to 451 amino acids (aa) in Pf and from 210 to 248 aa in Hs. The linker region is shown in yellow. (B) Structural model of PfEB1 created using Alphafold (https://alphafold.ebi.ac.uk/). (C) The schematic of strategy for PfEB1 deletion. Oligonucleotides were designed from outside 5′HR and 3′HR. The arrows indicate their positions inside PfEB1 locus and outside homology arms. (D) Confirmation of Pfeb1⁻ clonal parasites by a set of diagnostic PCRs. (E) The expected sizes for different sets of PCRs are shown.

Fig 2

Pfeb1⁻ asexual stages grow normally and undergo gametocytogenesis. (A) Ring-stage synchronous cultures for WT PfNF54 and Pfeb1⁻ parasites (clones 3D4 and 11C4) were plated, and parasite growth was measured over the course of two asexual replication cycles. Data from three biological replicates were averaged and presented as mean ± standard deviation. ns, not significant. (B) Ring-stage synchronous cultures for WT PfNF54 and Pfeb1⁻ parasites (clones 3D4 and 11C4) were assessed for their ability to form gametocytes. Light microscopy images of Giemsa-stained smears at 100× magnification showed development of WT PfNF54 and Pfeb1⁻ parasites through the five (I–V) distinct morphological stages. Female and male gametocytes are indicated using sex symbols shown on top of stage V gametocytes. (C) Immunofluorescence assays were performed on thin culture smears of mature stage V gametocytes for WT PfNF54 and Pfeb1⁻ parasites and were stained using anti-PfP230p (green) and anti-Pfg377 antisera (red), markers for stage V male and female gametocytes, respectively. Representative images are shown. Parasite DNA was visualized with DAPI (blue). Scale bar = 5 µm. Merge I indicates the merged images for red and green panels. Merge II indicates the merged images for red, green, and blue panels. DAPI, 4′,6-diamidino-2-phenylindole; DIC, differential interest contrast. Male and female gametocytes are indicated using sex symbols shown on the left side of the image panels. (D) On day 15 of in vitro culture, gametocytemia were measured using thin Giemsa-stained smears. Data from three biological replicates were averaged and presented as mean ± standard deviation. ns, not significant.

Fig 3

The Pfeb1⁻ parasites exhibit defect in nuclear segregation during microgametogenesis. (A) Exflagellation centers per field were quantified at 15-minute post-activation. Data from three biological replicates were averaged and presented as mean ± standard deviation. Pfeb1⁻ male gametocytes formed similar exflagellation centers as WT NF54 parasites. (B) and (C) IFAs were performed on mature stage V microgametocyte thin culture smears. Cultures were activated for 20 minutes in vitro for WT PfNF54 or Pfeb1⁻ (clones 3D4 and 11C4) and stained for α-tubulin (green), a marker for male gametocytes in an IFA. Microgametes emerged from an exflagellating male gametocyte in the WT PfNF54 and Pfeb1⁻ male gametocytes, shown via α-tubulin staining. (D) IFAs were performed on free microgametes for WT PfNF54 and Pfeb1⁻ using α-tubulin, and parasite DNA was stained with DAPI. Quantitation of microgametes showed normal nuclear segregation for WT NF54 parasites, while the majority of Pfeb1⁻-free microgametes did not inherit nuclei. (F) IFAs were performed on free macrogametes for WT PfNF54 and Pfeb1⁻ parasites using α-Pfs25, and parasite DNA was stained with DAPI. Pfeb1⁻ female gametes formed normally like WT NF54 parasites. ns, not significant.

Fig 4

The Pfeb1⁻ parasites exhibit a robust defect in parasite transmission to the mosquitos. (A) On day 7 post-feed, A. stephensi mosquitos were dissected, and the number of oocysts per midgut were measured. The Pfeb1⁻ parasites did not transmit to the mosquitoes. Data from three biological replicates with a minimum of 50 midguts were averaged and presented as mean ± standard deviation. (B) On day 7 post-feed, A. stephensi mosquitos were dissected, and the number of oocysts per midgut were measured for WT PfNF54, Pfeb1⁻, Pfcdpk4⁻, Pfmacfet⁻, Pfeb1⁻ × Pfcdpk4⁻, Pfeb1⁻ × Pfmacfet⁻, and Pfcdpk4⁻ × Pfmacfet⁻. In vitro genetic crosses revealed a microgamete fertility defect in Pfeb1⁻ parasites. ND, not detected; ns, not significant.