In P. falciparum, the commitment to gametocytogenesis occurs during the preceding asexual RBC cycle . The present study identifies a subset of schizont-stage parasites expressing Pfnek-4, a protein kinase previously thought to be gametocyte-specific. Upon sorting, Pfnek-4-GFP positive schizonts produced elevated levels of gametocytes in the subsequent red blood cell cycle. The experimental approach is based on episomal expression of Pfnek-4-GFP fusion protein driven by 1 kb-genomic DNA upstream of the Pfnek-4 coding sequence. It cannot be formally excluded that the proper expression pattern is not maintained in this conditions. However, a study by Khan et al. reported the ability of a 678 bp-Pbnek-4 promoter to drive the expression of a reporter GFP protein in female gametocytes, but not in male gametocytes or asexual blood stages. In this latter study, nine out of ten promoters (the only exception being most likely due to the short 5’ region available from the genome database), were found to drive the expression of the GFP reporter protein in agreement with the prediction from proteomic analyses, suggesting that sex-specific expression is mainly controlled by the 5’ UTR/promoter. In another instance, the episomal expression of the HA-epitope-tagged P. falciparum protease, PfROM1, placed under control of its own promoter element, revealed an expression at mature stages and localization to the mononeme, a newly described apical organelle of P. falciparum merozoites . The nucleus-associated punctuate pattern observed for Pfnek-4-GFP distribution, which is reminiscent of what is observed with the P. falciparum Aurora-related kinase Pfark-1, is consistent with the location of Nima- and Aurora-related kinases, many members of which associate with centrosomal structures (see below). Thus, it is proposed that there is a high likelihood that the localization observed with the episomally-encoded Pfnek-4-GFP fusion protein might reflect that of the endogenous enzyme.
It should be stressed that: (i) the sorted Pfnek-4-GFP positive schizont population not only generated gametocytes but also parasites undergoing asexual RBC cycles; and (ii) a proportion of the parasites positively selected for expression of the hDHFR resistance marker driven by the pfnek-4 gene was still able to undergo asexual cycles. Altogether these findings support and expand previous studies indicating that malaria parasites have quantitative sensitivity to gametocyte induction and that multiple stimuli can induce gametocytogenesis, reflecting the highly flexible mechanism underlying sexual differentiation . Noteworthy, high rate conversion to sexual forms of asexual parasites grown at high densities was shown to be reversible by dilution within a time window smaller than the time required for one asexual cycle (48 hours) . This feature might explain the relatively lower gametocyte conversion rate observed in the GFP+ sorted parasites (2.2%), as compared to normal 3D7 parasites grown under gametocyte-inducing culture conditions (~4-5%). Altogether, the data suggest that Pfnek-4 identifies a population of committed and reversibly pre-committed parasites. Since sex determination appears to occur simultaneously to commitment to sexual differentiation, the asexual subpopulation expressing Nek-4, a protein shown to be restricted to female gametocytes in the rodent malaria parasite P. berghei[7, 8], is likely to represent the progenitor of female rather than male gametocytes, although this question remains to be further investigated.
The expression of a gametocyte-specific gene product in sexually-committed asexual parasites is consistent with a developmental change in gene expression during sexual differentiation [18, 19], and extends previous studies reporting the expression of gametocyte-specific genes, such as Pfs16, in sub-populations of asexual-stage parasites [9, 20, 21]. Since some of the asexual parasite population expressing Pfnek-4 did not appear to be fully committed to sexual differentiation (being still able to undergo RBC cycles, see above), a switch to gametocyte-specific gene expression may occur before the “no-return”decision to commit to gametocytogenesis is made. Analysing genes expressed in the sexually-committed population would be of great interest to explore gene regulation in the context of commitment to gametocytogenesis, and would help to identify signatures of early sexual development of malaria parasites. Identification of Pfnek-4 as a molecular marker of sexually-committed schizonts provides a useful tool, making this parasite population amenable to purification followed by transcriptome and proteome analyses. Transcriptional regulator candidates controlling expression of subsets of genes are the ApiAP2 family of proteins. De Silva et al., reported highly coherent expression patterns of predicted downstream targets of P. falciparum AP2 transcription factors, suggesting essential roles in parasite development. Translational regulation also plays a critical role during commitment to gametocytogenesis [18, 19]. Targeted disruption of PfPuf2, a member of the Puf family of translational repressors was shown to promote the formation of gametocytes and the differentiation of male gametocytes .
In P. berghei, the Nek-4 kinase does not appear to be required for gametocytogenesis but is essential for pre-meiotic DNA replication in the zygote, consistent with cell-cycle related functions [7, 8]. That pfnek-4
P. falciparum parasites are able to undergo gametocytogenesis and produce mature stage V gametocytes, indicates that Pfnek-4 is not required for the early stages of the sexual cycle in both P. berghei and P. falciparum. This conclusion is also supported by the finding that pfnek-4
clones produce female gametocytes. It is intriguing that the timing of recruitment of the Pfnek-4 protein to schizont nuclear bodies appears to coincide with the occurrence of nuclear divisions. Noteworthy, all nuclei within a single schizont appear to be associated with punctuate Pfnek-4-GFP fluorescence from early developing to multinucleated schizont, in contrast to Pfark-1, a mitotic kinase that marks only a subset of nuclei in a given schizont as a result of transient recruitment at the spindle pole bodies, a consequence of asynchronous nuclear division in a single schizont . In contrast to Pfark-1, the Pfnek-4-GFP protein appears to associate to all nuclei, irrespective of their nuclear division status. Furthermore, cell cycle-arrested stage II gametocytes were found to express the Pfnek-4-GFP protein with a punctuate fluorescence similar to parasites undergoing schizogony. Preliminary results showing a close association of doublets of Pfnek-4-GFP fluorescence with short mitotic spindle microtubules in schizont-stage parasites (data not shown), are consistent with a recruitment of Pfnek-4 at nuclear spindle pole bodies, the centrosome equivalent of Plasmodium parasites, an observation consistent with the known centrosomal functions of Nima-related kinases. However, the sub-cellular structure to which Pfnek-4 associates remains to be better defined. Whether the Pfnek-4 enzyme has cell-cycle-related functions in RBC-stages still remains to be elucidated.