Fig 1.
Phylogenetic analysis of apicomplexan kinesins.
(A) Phylogenetic distribution of detected kinesin genes in alveolate genomes. Blue boxes denote the presence of genes, with the number of detected genes shown. (B) The expression levels of P. berghei genes in different developmental stages [36] are shown as circles. Note that Plasmodium spp. contain two kinesin-8 genes.
Fig 2.
Kinesin-8X shows ATPase, gliding motor and depolymerization activities.
(A) Schematic protein organisation of PBANKA_0805900 (Pbkinesin-8X) and PF3D7_0319400 (Pfkinesin-8X) showing their full-length sequence and central location of the motor domain, Pbkinesin-8X (green) and Pfkinesin-8X (blue). (B-D) Activities of Pb (top—green) and Pf (bottom–blue) kinesin-8X motor domains in three kinesin assays. (B) MT stimulated ATPase activity; data fitted to an adapted Michaelis-Menten equation with calculated Vmax, Km and V0 parameters. V0 was included as a term to aid the curve fitting and account for the non-zero basal ATPase activity of the kinesins in absence of MTs. Error bars represent the mean +/- SD for each MT concentration, n = 3. (C) MT gliding activity measured by TIRF microscopy; left, the average motility (nm/s) and individual data points are plotted. The difference between Pbkinesin-8X and Pfkinesin-8X velocity is statistically significant (t-test P <0.0001). Error bars represent the mean +/- SD; right, an exemplar kymograph demonstrates plus-end directed MT gliding using polarity-marked MTs (schematic above). (D) MT depolymerization measured using TIRF microscopy; depolymerization rate (nm/s) in the presence of ATP and AMPPNP is compared to a control in the absence of nucleotide. Error bars present the mean +/- SD, for Pbkinesin-8X control n = 77, AMPPNP n = 74, ATP n = 93, and for Pfkinesin-8X control n = 112, AMPPNP n = 116, ATP n = 117. An ordinary one-way ANOVA was performed on the depolymerisation data for Pbkinesin-8X and Pfkinesin-8X separately in Prism to establish the significance of the nucleotide-dependent differences. Significance values are displayed as asterisks, all p-values were <0.0001 (****) comparing control with the presence of AMPPNP or ATP and comparing activity in the presence of AMPPNP or ATP.
Fig 3.
Dynamics of Pbkinesin-8X show localization on spindle fibres during male gametogenesis.
(A) Live imaging of Pbkinesin-8X-GFP (green) during male gametogenesis showing an initial location at the putative microtubule organizing centre (MTOC) just after activation, and then on spindles and spindle poles in later stages. (B) Localization of Pbkinesin-8X (red) during female gametogenesis before (0 min) and after activation (15 min). (C) Indirect immunofluorescence assays showing co-localization of Pbkinesin-8X (red) and α-tubulin (green) during male gametogenesis. (D) Deconvoluted images of male gametocytes showing Pbkinesin-8X (red) with α-tubulin (green). (E) Indirect immunofluorescence assays showing location of Pbkinesin-8X (red) and α-tubulin (green) during female gametogenesis. Scale bar = 5 μm.
Fig 4.
Pbkinesin-8X localizes to a putative MTOC and spindle during ookinete development, sporogony and liver stage development.
(A) Live cell imaging showing Pbkinesin-8X-GFP location during ookinete development. A cy3-conjugated antibody, 13.1, which recognises the protein P28 on the surface of activated female gametes, zygotes and ookinetes was used to mark these stages (red). (B) Representative images showing Pbkinesin-8X, located at basal end of early-mature ookinetes as well as in the nucleus. (C) Pbkinesin-8X-GFP location in oocyst and sporozoite. (D) Location of Pbkinesin-8X in liver stages. Scale bar = 5 μm.
Fig 5.
Pbkinesin-8X is essential for oocyst development and sporogony.
(A) Male gametogenesis (exflagellation) of Δkinesin-8X line (black bar) compared with WT-GFP line (white bar) measured as the number of exflagellation centres per field. Mean ± SD. n = 5 independent experiments. (B) Ookinete conversion as a percentage for Δkinesin-8X (black bar) and WT-GFP (white bar) parasites. Ookinetes were identified using 13.1 antibody as a surface marker and defined as those cells that differentiated successfully into elongated ‘banana shaped’ ookinetes. Mean ± SD. n = 5 independent experiments. (C) Total number of GFP-positive oocysts per infected mosquito in Δkinesin-8X (black bar) compared to WT-GFP (white bar) parasites at 7, 10, 14 and 21-day post-infection (dpi). Mean ± SD. n = 3 independent experiments (>15 mosquitoes for each) *p≤0.05, **p≤0.01. (D) Mid guts at 10x magnification showing oocysts of Δkinesin-8X and WT-GFP lines at 7, 10, 14 and 21 dpi. Scale bar = 50 μm. * p ≤ 0.05 and ** p ≤ 0.01 (E) Mid guts at 63x magnification showing oocysts of Δkinesin-8X and WT-GFP lines at 7, 10, 14 and 21 dpi. Scale bar = 20 μm. (F) Total number of sporozoites in oocysts of Δkinesin-8X (black bar) and WT-GFP (white bar) parasites at 14 and 21 dpi. Mean ± SD. n = 3 independent experiments. (G) Total number of sporozoites in salivary glands of Δkinesin-8X (black bar) and WT-GFP (white bar) parasites. Bar diagram shows mean ± SD. n = 3 independent experiments (H) Bite back experiments showing no transmission of Δkinesin-8X parasites (black bar) where WT-GFP parasites (white bar) show successful transmission from mosquito to mice. Mean ± SD. n = 3 independent experiments (I) Rescue experiment showing male allele of Δkinesin-8X is affected.
Fig 6.
Ultrastructure analysis of oocyst development in Δkinesin-8X parasites.
Electron micrographs of WT (A, C) and mutant (B, C, D) oocysts located in the mid gut of the mosquito at 14 days post-infection. Bar is 10 μm in panel A, and 1μm in other micrographs. (A) Low power image through a mid-stage oocyst showing early stages in sporozoite (S) formation. (B) Low power showing the collapsed appearance of three oocysts (O). (C) Detail of an oocysts showing budding sporozoites (S) containing nucleus (N) and developing rhoptry (R). (D, E) Enlargements of oocysts in panel B showing the collapsed oocyst wall (CW) surrounding cytoplasm with degenerate organelles.
Fig 7.
Global transcript analysis of Δkinesin-8X parasites by RNAseq.
(A) RNA sequence analysis showing no transcript in Δkinesin-8X parasites. (B) Upregulated and downregulated genes in Δkinesin-8X parasites compared to WT-GFP parasites. (C) Gene ontology enrichment analysis showing most affected genes involved in various biological processes. (D) Validation of relevant and selected genes from the RNAseq data by qRT-PCR. Mean ± SD. n = 3 independent experiments. *p ≤ 5.