Plasmodium DEH is ER-localized and crucial for oocyst mitotic division during malaria transmission.

Cells use fatty acids (FAs) for membrane biosynthesis, energy storage, and the generation of signaling molecules. 3-hydroxyacyl-CoA dehydratase-DEH-is a key component of very long chain fatty acid synthesis. Here, we further characterized in-depth the location and function of DEH, applying in silico analysis, live cell imaging, reverse genetics, and ultrastructure analysis using the mouse malaria model Plasmodium berghei DEH is evolutionarily conserved across eukaryotes, with a single DEH in Plasmodium spp. and up to three orthologs in the other eukaryotes studied. DEH-GFP live-cell imaging showed strong GFP fluorescence throughout the life-cycle, with areas of localized expression in the cytoplasm and a circular ring pattern around the nucleus that colocalized with ER markers. Δdeh mutants showed a small but significant reduction in oocyst size compared with WT controls from day 10 postinfection onwards, and endomitotic cell division and sporogony were completely ablated, blocking parasite transmission from mosquito to vertebrate host. Ultrastructure analysis confirmed degeneration of Δdeh oocysts, and a complete lack of sporozoite budding. Overall, DEH is evolutionarily conserved, localizes to the ER, and plays a crucial role in sporogony.

A divergent cyclin/cyclin-dependent kinase complex controls the atypical replication of a malaria parasite during gametogony and transmission.

Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.

Vital role for Plasmodium berghei Kinesin8B in axoneme assembly during male gamete formation and mosquito transmission.

Sexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are expelled in a process called exflagellation. Surprisingly little is known about the actors involved in these processes. We are interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of kinesin-8B (PbKIN8B) expressed specifically in male gametocytes and gametes. By generating Pbkin8B-gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of the absence of PbKIN8B on male gametogenesis. We show that the ability to produce sexually differentiated gametocytes is not affected in ΔPbkin8B parasites and that the 3 rounds of genome replication occur normally. Nevertheless, the development to free motile microgametes is halted and the life cycle is interrupted in vivo. Ultrastructural analysis revealed that intranuclear mitoses are unaffected whereas cytoplasmic microtubules, although assembled in doublets and elongated, fail to assemble in the normal axonemal '9+2' structure and become motile. Absence of a functional axoneme prevented microgamete assembly and release from the microgametocyte, severely reducing infection of the mosquito vector. This is the first functional study of a kinesin involved in male gametogenesis. These results reveal a previously unknown role for PbKIN8B in male gametogenesis, providing new insights into Plasmodium flagellar formation.

Kinesin-8B controls basal body function and flagellum formation and is key to malaria transmission.

Eukaryotic flagella are conserved microtubule-based organelles that drive cell motility. Plasmodium, the causative agent of malaria, has a single flagellate stage: the male gamete in the mosquito. Three rounds of endomitotic division in male gametocyte together with an unusual mode of flagellum assembly rapidly produce eight motile gametes. These processes are tightly coordinated, but their regulation is poorly understood. To understand this important developmental stage, we studied the function and location of the microtubule-based motor kinesin-8B, using gene-targeting, electron microscopy, and live cell imaging. Deletion of the kinesin-8B gene showed no effect on mitosis but disrupted 9+2 axoneme assembly and flagellum formation during male gamete development and also completely ablated parasite transmission. Live cell imaging showed that kinesin-8B-GFP did not co-localise with kinetochores in the nucleus but instead revealed a dynamic, cytoplasmic localisation with the basal bodies and the assembling axoneme during flagellum formation. We, thus, uncovered an unexpected role for kinesin-8B in parasite flagellum formation that is vital for the parasite life cycle.

An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell.

Infection by Toxoplasma gondii leads to massive changes to the host cell. Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5). We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has similarity to the PEXEL-motif of Plasmodium parasites. We show that ASP5 matures substrates at both the N- and C-terminal ends of proteins and also controls trafficking of effectors without this motif. Furthermore, ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the vacuole. Assessment of host gene expression reveals that the ASP5-dependent pathway influences thousands of the transcriptional changes that Toxoplasma imparts on its host cell. All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell.

Oocyst wall formation and composition in coccidian parasites

The oocyst wall of coccidian parasites is a robust structure that is resistant to a variety of environmental and chemical insults. This resilience allows oocysts to survive for long periods, facilitating transmission from host to host. The wall is bilayered and is formed by the sequential release of the contents of two specialized organelles - wall forming body 1 and wall forming body 2 - found in the macrogametocyte stage of Coccidia. The oocyst wall is over 90 percent protein but few of these proteins have been studied. One group is cysteine-rich and may be presumed to crosslink via disulphide bridges, though this is yet to be investigated. Another group of wall proteins is rich in tyrosine. These proteins, which range in size from 8-31 kDa, are derived from larger precursors of 56 and 82 kDa found in the wall forming bodies. Proteases may catalyze processing of the precursors into tyrosine-rich peptides, which are then oxidatively crosslinked in a reaction catalyzed by peroxidases. In support of this hypothesis, the oocyst wall has high levels of dityrosine bonds. These dityrosine crosslinked proteins may provide a structural matrix for assembly of the oocyst wall and contribute to its resilience.