The online version of this article (doi:10.1186/1475-2875-11-88) contains supplementary material, which is available to authorized users.
Anna Rosa Sannella, Anna Olivieri contributed equally to this work.
The authors declare that they have no competing interests.
ARS carried out the molecular and cell biology experiments and participated in the study design. AO and PA conceived the study, participated in its coordination and drafted the manuscript. LB carried out the electron microscopy experiments. FF carried out the bioinformatic analysis. CS carried out the 5' race experiments. TP participated in the design of the study and helped to generate the plasmid. All authors read and approved the final manuscript.
Gametocytes, the blood stages responsible for Plasmodium falciparum 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 P. falciparum transgenic line in which these organelles are specifically marked by a reporter protein was produced and characterized.
A P. falciparum 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 pfg377 upstream and downstream regulatory regions, was produced.
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.
This is the first identification of a P. falciparum 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.
Additional file 1: Table S1. Primers used for plasmid construction and sequencing. (PDF 135 KB)12936_2011_2059_MOESM1_ESM.PDF
Additional file 2: Figure S1. Map of the pEpi377 plasmid. (PDF 111 KB)12936_2011_2059_MOESM2_ESM.PDF
Additional file 3: Figure S2. Immunofluorescence assay with anti-Pfg377 antibodies on a gametocyte culture 10 min after induction of gametogenesis. Parasites were fixed in 4% paraformaldehyde and permeabilized with 1% Triton X-100. Nuclei were stained with DAPI. Images A and B were collected on the same smear with the same exposure time. A. Residual mature gametocyte. B. Female gamete. (PDF 32 KB)12936_2011_2059_MOESM3_ESM.PDF
Additional file 4: Identification of sequence motifs shared by osmiophilic body-associated proteins [ 27– 33]. (PDF 44 KB)
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- Specific tagging of the egress-related osmiophilic bodies in the gametocytes of Plasmodium falciparum
Anna Rosa Sannella
- BioMed Central
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