Abstract
BackgroundMalaria parasites undergo, in the vertebrate host, a developmental switch from asexual replication to sexual differentiation leading to the formation of gametocytes, the only form able to survive in the mosquito vector. Regulation of the onset of the sexual phase remains largely unknown and represents an important gap in the understanding of the parasite’s complex biology.MethodsThe expression and function of the Nima-related kinase Pfnek-4 during the early sexual development of the human malaria parasite Plasmodium falciparum were investigated, using three types of transgenic Plasmodium falciparum 3D7 lines: (i) episomally expressing a Pfnek-4-GFP fusion protein under the control of its cognate pfnek-4 promoter; (ii) episomally expressing negative or positive selectable markers, yeast cytosine deaminase-uridyl phosphoribosyl transferase, or human dihydrofolate reductase, under the control of the pfnek-4 promoter; and (iii) lacking a functional pfnek-4 gene. Parasite transfectants were analysed by fluorescence microscopy and flow cytometry. In vitro growth rate and gametocyte formation were determined by Giemsa-stained blood smears.ResultsThe Pfnek-4-GFP protein was found to be expressed in stage II to V gametocytes and, unexpectedly, in a subset of asexual-stage parasites undergoing schizogony. Culture conditions stimulating gametocyte formation resulted in significant increase of this schizont subpopulation. Moreover, sorted asexual parasites expressing the Pfnek-4-GFP protein displayed elevated gametocyte formation when returned to in vitro culture in presence of fresh red blood cells, when compared to GFP- parasites from the same initial population. Negative selection of asexual parasites expressing pfnek-4 showed a marginal reduction in growth rate, whereas positive selection caused a marked reduction in parasitaemia, but was not sufficient to completely abolish proliferation. Pfnek-4- clones are not affected in their asexual growth and produced normal numbers of stage V gametocytes.ConclusionsThe results indicate that Pfnek-4 is not strictly gametocyte-specific, and is expressed in a small subset of asexual parasites displaying high rate conversion to sexual development. Pfnek-4 is not required for erythrocytic schizogony and gametocytogenesis. This is the first study to report the use of a molecular marker for the sorting of sexually-committed schizont stage P. falciparum parasites, which opens the way to molecular characterization of this pre-differentiated subpopulation.
Highlights
Malaria parasites undergo, in the vertebrate host, a developmental switch from asexual replication to sexual differentiation leading to the formation of gametocytes, the only form able to survive in the mosquito vector
Transgenic expression of a Pfnek-4-GFP fusion protein in gametocytes and a subset of asexual blood stage parasites P. falciparum 3D7 parasites were transfected with a plasmid containing the Pfnek-4 coding sequence fused
The Pfnek-4-GFP protein appeared to accumulate in the cytosol of stage III gametocytes (Figure 1C) and remained present at high levels throughout the formation of mature stage V gametocytes (Additional file 1, panel A)
Summary
In the vertebrate host, a developmental switch from asexual replication to sexual differentiation leading to the formation of gametocytes, the only form able to survive in the mosquito vector. A small subset of parasites, upon invasion of new red blood cells, do not enter schizogony, but develop into cell cycle-arrested gametocytes [1]. Gametocytes are the only forms able to survive in the mosquito vector, and their formation is essential for malaria transmission. How malaria parasites regulate the switch from erythrocytic schizogony to gametocytogenesis is still not understood. Bruce et al [2] showed that merozoites released from a single schizont become either all asexual parasites or all gametocytes, indicating that the switch to sexual differentiation is likely to occur during the preceding asexual red blood cell cycle. The mechanism of parasite commitment to sexual differentiation appears to be constitutive but may be modulated by the environment, as reviewed in [4]
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