An essential role of the basal body protein SAS-6 in Plasmodium male gamete development and malaria transmission

Abstract

Gametocytes are the sole Plasmodium parasite stages that infect mosquitoes; therefore development of functional gametes is required for malaria transmission. Flagellum assembly of the Plasmodium male gamete differs from that of most other eukaryotes in that it is intracytoplasmic but retains a key conserved feature: axonemes assemble from basal bodies. The centriole/basal body protein SAS-6 normally regulates assembly and duplication of these organelles and its depletion causes severe flagellar/ciliary abnormalities in a diverse array of eukaryotes. Since basal body and flagellum assembly are intimately coupled to male gamete development in Plasmodium, we hypothesized that SAS-6 disruption may cause gametogenesis defects and perturb transmission. We show that Plasmodium berghei sas6 knockouts display severely abnormal male gametogenesis presenting reduced basal body numbers, axonemal assembly defects and abnormal nuclear allocation. The defects in gametogenesis reduce fertilization and render Pbsas6 knockouts less infectious to mosquitoes. Additionally, we show that lack of Pbsas6 blocks transmission from mosquito to vertebrate host, revealing an additional yet undefined role in ookinete to sporulating oocysts transition. These findings underscore the vulnerability of the basal body/SAS-6 to malaria transmission blocking interventions.

Fig 1

Comparison of malarial SAS-6 with other Apicomplexa and eukaryotes.A. Comparative domain organization of predicted SAS-6 orthologues in apicomplexan and other eukaryote genomes. Red box represents the PISA motif, light green box represents predicted coil-coiled domains, yellow star represents the phenylalanine residue demonstrated to be essential for dimerization in Danio rerio (van Breugel et al., 2011).B. Multiple sequence alignment of the putative N-terminal oligomerization domain of SAS-6 orthologues. Horizontal red bars indicate the possible interface residues in the N-terminus dimer previously identified in C. reinhardtii SAS-6 (Kitagawa et al., 2011) Yellow star represents the dimerization residue in D. rerio SAS-6 (van Breugel et al., 2011).C. Phylogenetic tree of SAS-6 orthologues. Asterisk * signifies bootstrap support greater than 85%. Scale bar stands for number of substitutions per site. PF, Plasmodium falciparum; PB, Plasmodium berghei; TG, Toxoplasma gondii; NC, Neospora caninum; CP, Cryptosporidium parvum; DR, Danio rerio; CR, Chlamydomonas reinhardtii; HS, Homo sapiens.

Fig 2

Distribution and location of sas6-myc prior to and during gametogenesis. (A–C). Bright-field and confocal stacks of transgenic sas6-myc gametocytes fixed at different times before and after activation. Immunofluorescence images show DAPI staining of DNA in blue, anti-MYC in green and anti-tubulin in red.A. Before activation, sas6-myc and tubulin are distributed ubiquitously in the cytoplasm of male but not female gametocytes (Fig. S1D).B. At 10 min post-activation (mpa), sas6-myc aggregates in small punctae (green arrow) in the cytoplasm.C. At 15 mpa, sas6-myc is detected in the gametocyte body and at the distal part of the male gametes (green arrows). Scale bars – 2 μm.

Fig 3

Gametogenesis analysis of Δsas6 and wt clones.A–D. Bright field and immunofluorescence images from confocal stacks projections of fixed male gametocytes at 10 and 15 min post activation (mpa). DAPI staining of DNA is in blue and anti-tubulin in red. At 10 mpa wt (A) and Δsas6 (B) are indistinguishable from each other, white arrows point out formed tubulin structures. At 15 mpa, wave-like nucleated gametes are seen outside of the gametocyte body in wt (C), as opposed to Δsas6 (D) where tubulin protrusions are thin straight and not nucleated. Blue arrows point out nuclei. Scale bars – 2 μm.E–J. Analysis of gametocyte DNA content by flow cytometry. Representative density plots of Hoechst positive cells of Nycodenz purified gametocyte samples. UNACT represents samples fixed before activation and ACT represents samples fixed at 8 mpa. Plots display intensity of DAPI staining – 405–450/50A versus SSC- sidescatter. Pink gate defines cells with highest DNA content. The percentage of cells with high DNA in unactivated wt gametocyte samples (E) is lower than in activated wt gametocyte samples (F) due to male gametocyte replication. Δcdpk4 male gametocytes do not undergo DNA replication (Billker et al., 2004) therefore unactivated and activated gametocytes display similar DNA intensity patterns (G, H). Δsas6 unactivated gametocyte samples (I) also display a lower percentage of cells with high DNA content when compared to activated gametocyte samples (J) indicating that male gametocyte replication has occurred. Presence of gated cells in Δcdpk4 and unactivated preparations most likely represents asexual contamination as observed in Giemsa staining of purified samples. Mean percentage of gated cells upon activation is 9.75 ± 3 for Δcdpk4, 23.35 ± 1 for wt and 24.75 ± 3 for Δsas6.

Fig 4

Ultra-structural analysis of gametogenesis of Δsas6 and wt clones.A–C. Transmission electron microscopy images of male gametocytes fixed at 15–30 min after activation. (A) Wt and Δsas6 activated male gametocytes display an enlarged nucleus (N). The wt inset display a canonical 9 + 2 microtubule rosette (white arrowhead) and an incomplete microtubule rosette (black arrowhead). Δsas6 cytoplasmic inset displays a complete lack of canonical ‘9 + 2’ structures with formation of microtubule central pairs (red arrow) and outer microtubule doublets (blue arrow). Cross-section of wt male gamete shows a ‘9 + 2’ structure, cross-section of a Δsas6 microtubular protrusion shows lack of normal microtubule patterning. (B) In wt, basal bodies (BB) are present in the cytoplasm and nucleate axonemes (AX) that usually display 3 sets of microtubules (arrowheads), 2 peripheral and 1 central when sectioned longitudinally. BBs connect through a nuclear pore with the spindle-pole-body (SPB). In Δsas6, BBs are rarely seen and longitudinal microtubules show disorganization. Two of the BBs observed in a Δsas6 appear normal. (C) Kinetochore (K) appearance in wt and Δsas6 is indistinguishable from each other but in the Δsas6, kinetochores frequently appear detached from the SPB, in a more central position. A scale bars – 2 μm. Inset scale bars – 200 nm. B, C scale bars – 500 nm.

Fig 5

Analysis of sexual reproduction and infectivity to mosquitoes in Δsas6 and wt clones.A. Ookinete conversion rate as measured by the number of ookinetes per total number of fertilized and unfertilized females in Δsas6-gfp, Δsas6 and crosses of Δsas6 with Δcdpk4 (male deficient) and Δnek4 (female deficient) clones. Δsas6 form significantly fewer ookinetes than wt, a phenotype that can be rescued by crossing it with Δnek4. Asterisk * indicates statistically significant differences in Student's t-test with P-values lower than 0.01, Table S1.B. Ookinete invasion of An. gambiae L3-5 midguts. Mosquitoes were infected with wt and Δsas6, mosquitoes were dissected at day 6 and melanized ookinetes were counted in midguts in 3 different biological replicates. Intensity of infection is significantly decreased in all replicates, Table S2A.C. Infectivity of Δsas6 to An. stephensi mosquitoes. Mosquitoes were infected with wt, wt-gfp, Δsas6 and Δsas6-gfp, midguts were dissected at day 12 and oocysts were counted in 3 different biological replicates. Intensity and prevalence of infection are significantly different from wt, Table S2B.D. Bright-field and fluorescent oocyst images at day 9 and day 13. At both time points, Δsas6 appear smaller than wt. This size difference was quantified and is statistically significant at day 9 (Table S1). At day 13, wt oocysts display densely packed slender sporozoites that appear like striations, in contrast Δsas6 oocysts appear empty.

Fig 6

Schematic model of the impact of Pbsas6 deletion on male gametogenesis.Upon activation, the wt amorphous microtubule organizing centre (mtoc) develops into two planar tetrads of BBs (green), which later separate into 8 individual BBs. Each BB serves as a platform for axonemal microtubule (red) assembly. BB individualization is concomitant with 3 rounds of mitotic division, with BBs connecting with mitotic spindles (purple) and kinetochores (black) via the spindle pole body (grey). Haploid genomes (blue) connect with BBs and are pulled into the emerging wt microgametes, which display a ‘9 + 2’ structure. Δsas6 gametocytes display reduced numbers of BBs and lack of ‘9 + 2’ canonical axonemal structures. Axonemal defects are likely due to abnormal formation or segregation of BBs. Abnormal BB production probably disrupts the link between axonemal microtubules and genomes, therefore abnormal Δsas6 microgametes rarely contain DNA.