Specific tagging of the egress-related osmiophilic bodies in the gametocytes of Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Gametocytes, the blood stages responsible for <it>Plasmodium falciparum </it>transmission, contain electron dense organelles, traditionally named osmiophilic bodies, that are believed to be involved in gamete egress from the host cell. In order to provide novel tools in the cellular and molecular studies of osmiophilic body biology, a <it>P. falciparum </it>transgenic line in which these organelles are specifically marked by a reporter protein was produced and characterized.</p> <p>Methodology</p> <p>A <it>P. falciparum </it>transgenic line expressing an 80-residue N-terminal fragment of the osmiophilic body protein Pfg377 fused to the reporter protein DsRed, under the control of <it>pfg377 </it>upstream and downstream regulatory regions, was produced.</p> <p>Results</p> <p>The transgenic fusion protein is expressed at the appropriate time and stage of sexual differentiation and is trafficked to osmiophilic bodies as the endogenous Pfg377 protein. These results indicate that a relatively small N-terminal portion of Pfg377 is sufficient to target the DsRed reporter to the gametocyte osmiophilic bodies.</p> <p>Conclusions</p> <p>This is the first identification of a <it>P. falciparum </it>aminoacid sequence able to mediate trafficking to such organelles. To fluorescently tag such poorly characterized organelles opens novel avenues in cellular and imaging studies on their biogenesis and on their role in gamete egress.</p

The Plasmodium berghei serine protease PbSUB1 plays an important role in male gamete egress.

The Plasmodium subtilisin-like serine protease SUB1 is expressed in hepatic and both asexual and sexual blood parasite stages. SUB1 is required for egress of invasive forms of the parasite from both erythrocytes and hepatocytes, but its subcellular localisation, function, and potential substrates in the sexual stages are unknown. Here, we have characterised the expression profile and subcellular localisation of SUB1 in Plasmodium berghei sexual stages. We show that the protease is selectively expressed in mature male gametocytes and localises to secretory organelles known to be involved in gamete egress, called male osmiophilic bodies. We have investigated PbSUB1 function in the sexual stages by generating P. berghei transgenic lines deficient in PbSUB1 expression or enzyme activity in gametocytes. Our results demonstrate that PbSUB1 plays a role in male gamete egress. We also show for the first time that the PbSUB1 substrate PbSERA3 is expressed in gametocytes and processed by PbSUB1 upon gametocyte activation. Taken together, our results strongly suggest that PbSUB1 is not only a promising drug target for asexual stages but could also be an attractive malaria transmission-blocking target

Malaria transmission through the mosquito requires the function of the OMD protein

Ookinetes, one of the motile and invasive forms of the malaria parasite, rely on gliding motility in order to establish an infection in the mosquito host. Here we characterize the protein PBANKA_0407300 which is conserved in the Plasmodium genus but lacks significant similarity to proteins of other eukaryotes. It is expressed in gametocytes and throughout the invasive mosquito stages of P. berghei, but is absent from asexual blood stages. Mutants lacking the protein developed morphologically normal ookinetes that were devoid of productive motility although some stretching movement could be detected. We therefore named the protein Ookinete Motility Deficient (OMD). Several key factors known to be involved in motility however were normally expressed and localized in the mutant. Importantly, the mutant failed to establish an infection in the mosquito which resulted in a total malaria transmission blockade

Egress-related osmiophilic bodies

© 2014 John Wiley & Sons Ltd. Summary: Gametogenesis is the earliest event after uptake of malaria parasites by the mosquito vector, with a decisive impact on colonization of the mosquito midgut. This process is triggered by a drop in temperature and contact with mosquito molecules. In a few minutes, male and female gametocytes escape from the host erythrocyte by rupturing the parasitophorous vacuole and the erythrocyte membranes. Electron-dense, oval-shaped organelles, the osmiophilic bodies (OB), have been implicated in the egress of female gametocytes. By comparative electron microscopy and electron tomography analyses combined with immunolocalization experiments, we here define the morphological features distinctive of male secretory organelles, hereafter named MOB (male osmiophilic bodies). These organelles appear as club-shaped, electron-dense vesicles, smaller than female OB. We found that a drop in temperature triggers MOB clustering, independently of exposure to other stimuli. MDV1/PEG3, a protein associated with OB in Plasmodium berghei females, localizes to both non-clustered and clustered MOB, suggesting that clustering precedes vesicle discharge. A P.berghei mutant lacking the OB-resident female-specific protein Pbg377 displays a dramatic reduction in size of the OB, accompanied by a delay in female gamete egress efficiency, while female gamete fertility is not affected. Immunolocalization experiments indicated that MDV1/PEG3 is still recruited to OB-remnant structures

Identification and preliminary characterization of Plasmodium falciparum proteins secreted upon gamete formation

Malaria long-term elimination depends on parasite transmission control. Plasmodium sexual stage maturation in the mosquito, including egress from the host erythrocyte, is one of the prime targets for transmission-blocking interventions. This work aims to identify candidate molecules potentially involved in gamete emergence from the host erythrocyte, as novel transmission blocking targets. We analyzed by quantitative mass spectrometry the proteins released/secreted by purified Plasmodium falciparum gametocytes upon induction of gametogenesis. The proteome obtained showed a good overlap (74%) with the one previously characterized in similar conditions from gametocytes of the rodent malaria parasite P. berghei. Four candidates were selected based on comparative analysis of their abundance values in released vs total gametocyte proteome. We also characterized the P. falciparum orthologue of the microgamete surface protein (MiGS), a marker of male gametocyte secretory vesicles in murine models of malaria. The findings of this study reveal that all the selected candidate proteins are expressed in both genders and localize to vesicle-like structures that respond to gametogenesis stimuli. This result, together with the fact that the selected proteins are released during gamete emergence in both Plasmodium species, makes them interesting candidates for future functional studies to investigate their potential role in the gametogenesis process

Set regulation in asexual and sexual Plasmodium parasites reveals a novel mechanism of stage-specific expression

Transmission of the malaria parasite depends on specialized gamete precursors (gametocytes) that develop in the bloodstream of a vertebrate host. Gametocyte/gamete differentiation requires controlled patterns of gene expression and regulation not only of stage and gender-specific genes but also of genes associated with DNA replication and mitosis. Once taken up by mosquito, male gametocytes undergo three mitotic cycles within few minutes to produce eight motile gametes. Here we analysed, in two Plasmodium species, the expression of SET, a conserved nuclear protein involved in chromatin dynamics. SET is expressed in both asexual and sexual blood stages but strongly accumulates in male gametocytes. We demonstrated functionally the presence of two distinct promoters upstream of the set open reading frame, the one active in all blood stage parasites while the other active only in gametocytes and in a fraction of schizonts possibly committed to sexual differentiation. In ookinetes both promoters exhibit a basal activity, while in the oocysts the gametocyte-specific promoter is silent and the reporter gene is only transcribed from the constitutive promoter. This transcriptional control, described for the first time in Plasmodium, provides a mechanism by which single-copy genes can be differently modulated during parasite development. In male gametocytes an overexpression of SET might contribute to a prompt entry and execution of S/M phases within mosquito vector