Specific tagging of the egress-related osmiophilic bodies in the gametocytes of Plasmodium falciparum
- Anna Rosa Sannella†1,
- Anna Olivieri†1Email author,
- Lucia Bertuccini2,
- Fabrizio Ferrè3,
- Carlo Severini1,
- Tomasino Pace1 and
- Pietro Alano1
© Sannella et al; licensee BioMed Central Ltd. 2012
Received: 22 December 2011
Accepted: 27 March 2012
Published: 27 March 2012
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.
Progression of parasite development through diverse intracellular and extracellular forms is accompanied by the biogenesis of several organelles, some of which - rhoptries, dense granules and micronemes - are specific of apicomplexan parasites. Functional role of such organelles is being elucidated in key aspects of parasite development such as invasion of and egress from the host cell, and they are increasingly attracting attention as potential targets of anti-parasitic drugs. Functional studies on protein trafficking to such organelles revealed that specific aminoacid sequences are necessary for proper organelle targeting . Some of such studies, however, indicated that appropriate timing of expression for some of these proteins can be critical for targeting, as shown in the cases of Plasmodium berghei protein AMA-1  and Plasmodium falciparum proteins RESA and RhopH2 [3, 4].
Gametocytes, the blood stages responsible for parasite transmission to the mosquito vector, contain electron dense organelles similar to dense granules, traditionally named osmiophilic bodies . In P. falciparum these oval shaped organelles appear on day 4 of sexual differentiation (stage III)  and accumulate in female gametocytes, where they progressively increase in number and reach their subcellular localization under the mature gametocyte surface. Ultrastructural observations showed that osmiophilic bodies are connected to the gametocyte surface by ducts and are virtually no longer present after transformation into female gamete, thus supporting the hypothesis of their involvement in gamete egress from the host cell [5, 7]. This notion found additional support from functional studies on the P. falciparum protein Pfg377, whose ablation by gene knock out caused defective egress of female gametes , and the P. berghei proteins Mdv-1/Peg3  and PbGEST .
In order to provide novel tools in the cellular and molecular studies of the biology of osmiophilic bodies, this work aimed to produce a P. falciparum transgenic line, in which a reporter protein fused to a portion of the pfg377 coding sequence was specifically targeted to such organelles.
Parasite culture and transfection
The gametocyte-producing P. falciparum clone 3D7 was used to derive the transgenic line 3D7/pEpi377. Parasites were cultured and synchronized using standard protocols . For transfection, ring stage parasites (~5% parasitaemia) were electroporated with 80 micrograms of purified plasmid DNA as previously described  and drug selected with 2.5 nM WR99210 48 h later. Resistant parasites appeared after approximately three weeks of selection. Gametocyte production was induced by growing parasites to high parasitaemia, while asexual stages were killed by treatment with 0.05 M N-acetylglucosamine for 72 h . Gametocyte activation was induced by exposing mature gametocytes to room temperature in incomplete medium at pH 8.2.
5' RACE analysis
5' RACE (Rapid Amplification of cDNA Ends) experiments were performed using the Boehringer Mannheim 5'/3' RACE kit, according to the manufacturer's instructions. Briefly, a first strand cDNA was generated with the pfg377 antisense primer pR (see Additional file 1: Table S1 for all primers used), whose sequence is located 30 bp downstream of the gene ATG codon, using 5 micrograms of total mRNA from 3D7 Percoll purified stage III-IV gametocytes. cDNA, purified in GlassMax columns and dCTp/TdT tailed, was used to produce double-stranded DNA by PCR, using the 3' tail specific primer provided by the kit. 5 clones of the resulting PCR products were sequenced.
A 1883 bp fragment, including the putative promoter region and 240 bp of pfg377 coding region, was amplified from 3D7 genomic DNA with primers p1 and p2 and cloned into the pHHMC*/3R0.5 plasmid , using SacII and XhoI restriction sites. DsRed gene was PCR amplified from plasmid pBac(3xP3RED)AgApy , with primers p3 and p4 and cloned in the same plasmid with XhoI and KpnI. A second fragment, 385 bp long, spanning the pfg377 stop codon was amplified from 3D7 genomic DNA with primers p5 and p6 and cloned downstream the DsRed gene using KpnI and NheI restriction sites. Coding sequences were double checked by sequencing, using primers p7 to p9. The plasmid was named pEpi377 and the map is shown in Additional file 2: Figure S1.
Gametocytes were fixed in 4% paraformaldehyde and blocked in 3% bovine serum albumin overnight, and were simultaneously reacted with the above anti-Pfg377 B portion serum and a polyclonal rabbit anti-DsRed antiserum (MBL International) respectively diluted 1:500 and 1:100. Smears were then incubated with FITC and TRITC-conjugated anti-rat and anti-rabbit IgG, diluted 1:200, and 2 mg/ml DAPI for 1 h. Smears were washed in PBS, mounted and examined on a Leitz DMR fluorescent microscope using an oil immersion objective.
Immuno electron microscopy
Stage IV and V gametocytes from the 3D7/pEpi377 transgenic line were Percoll purified and processed for immuno-electron microscopy according to published protocols . Briefly, samples were fixed overnight at 4°C with 4% paraformaldehyde/0.1% glutaraldehyde in 0.1 M sodium cacodylate buffer. Next, the suspension was gently washed in sodium cacodylate buffer, dehydrated in ethanol serial dilutions and embedded in LR White, medium-grade acrylic resin (London Resin Company, UK). The samples were polymerized in a 50°C oven for 48 h and ultrathin sections, collected on gold grids, were sunk in 100% ethanol for 3 min, immersed in Tris buffer 0.05 M (pH 10.0) in PCR tubes, and then kept at 99°C for 30 min using a constant temperature box .
For immunostaining, the grids were floated on drops of PBS containing 0.1 M glycine for 10 min, washed with PBS, blocked with 5% normal goat serum/1% BSA in PBS for 30 min. For single immunogold-labelling of Ds-Red/Pfg377 fusion protein, ultrathin sections were incubated overnight at 4°C with a 1:50 dilution of rabbit polyclonal anti-DsRed serum (MBL International) in PBS/0.1% BSA/0,05% TWEEN20 buffer. Samples were then rinsed and incubated for 1 h with 10 nm gold-conjugated goat anti-rabbit IgG (SIGMA) (1:50), rinsed again in buffer followed by distilled water and finally air dried. For double immunogold labelling, ultrathin sections were incubated overnight at 4°C with a 1:100 dilution of mouse polyclonal anti-377-B serum  in PBS/0.1% BSA/0,05% TWEEN20 buffer. After washing, the grids were incubated for 1 h with 5 nm gold-conjugated goat anti-mouse IgG (Sigma Aldrich) diluted 1:50, rinsed in buffer and further incubated with the above rabbit polyclonal anti-DsRed serum (1:100) overnight at 4°C. Ultrathin sections were then rinsed and incubated for 1 h with 10 nm gold-conjugated goat anti-rabbit IgG (SIGMA) (1:50), rinsed again in buffer followed by distilled water and air dried. Finally, the samples were stained successively with uranyl acetate 2% in H20 and Reynolds lead citrate solution, and observed with an EM208 Philips transmission electron microscope. As controls, the sections were stained without any heating, without the primary antibody and with the diluted gold anti-mouse IgG, or with the diluted mouse pre-immune serum in place of the first antibody.
Results and discussion
Regulatory and coding sequences of the pfg377 gene for reporter construct
In order to produce a plasmid construct expressing a Pfg377 fragment fused to a reporter protein, for possible targeting to female gametocyte osmiophilic bodies, the following regulatory sequences were used. A 1.6 kb fragment (1643 bp) of genomic region upstream the pfg377 ATG codon and a 0.36 kb fragment (361 bp) of downstream region from the gene TAA stop codon were amplified and cloned to drive expression of the pfg377 fusion protein. A 5' RACE experiment on total RNA from Percoll purified stage IV gametocytes was preliminarily conducted to investigate length of the 5'UTR. Results showed that all cDNA clones obtained in this experiments extended to nucleotide -294 from the gene start codon, thus indicating that 1.6 kb of upstream sequences was adequate to contain the pfg377 promoter.
Stage-specificity, timing and localization of the transgenic Pfg377-DsRed fusion protein
Ultrastructural analysis of Pfg377-DsRed localization
Analysis of the transgenic Pfg377/DsRed fusion protein during gamete emergence
The N-terminal portion of Pfg377 is sufficient for targeting a fluorescent reporter to female gametocyte osmiophilic bodies
The experiments reported here indicate that the first 80 aminoacids of Pfg377 are sufficient to target the DsRed reporter to female gametocyte osmiophilic bodies. This is to our knowledge the first identification of a P. falciparum aminoacid sequence able to mediate trafficking to such organelles, which are comparatively less characterized than similar electron dense organelles such as rhoptries, micronemes and dense granules in other parasite developmental stages. Molecular composition of osmiophilic bodies is also much less characterized than that of the above organelles, as only the gametocyte proteins Pfg377 and PfMdv-1/Peg3, the latter however abundantly present also in additional membrane compartments [22, 23], have been so far localized in such organelles in P. falciparum. Virtually nothing is known on biogenesis of and protein trafficking to osmiophilic bodies. In general, trafficking to organelles of the apical complex such as rhoptries, dense granules and micronemes have been mainly investigated in asexual stages of Plasmodium and T. gondii, and requirement of specific sequences in proper organelle localization is still poorly understood. From studies on vesicular trafficking in lysosome biogenesis in higher eukaryotes it results that C-terminal portions of lysosomal proteins are recognized in the Golgi by cytoplasmic adaptor proteins that mediate their trafficking to such organelles. In T. gondii, the microneme proteins MIC2 and MIC6 and the rhoptry protein ROP2 reach their organelle destination with such a mechanism [24–26]. At odds with the picture emerging from the above studies, functional analyses of fusion protein localization indicated instead that in T. gondii the first 85 aminoacids of the rhoptry protein ROP1 are sufficient to ensure proper organelle targeting , and in P. falciparum the initial 24 residues of the rhoptry protein RhopH2 are able to drive localization of a fluorescent reporter to such organelles . The work presented here further supports the hypothesis that a signal peptide and a relatively limited N-terminal portion may be sufficient to specifically traffic a parasite protein to the gametocyte osmiophilic bodies.
As only three proteins have been so far positively localized in osmiophilic bodies (Pfg377, Pf Mdv-1/Peg3, PfGEST), a comparative sequence analysis to identify functionally conserved motifs is difficult. Nevertheless, both this approach and structural modeling analysis were undertaken to predict motifs functionally involved in trafficking to such organelles (see Additional file 4). However, the motifs predicted by computational analysis lack in specificity, thus suggesting that a higher number of osmiophilic body-associated proteins is needed to identify the determinants of osmiophilic body localization.
The positive identification presented here of a parasite sequence targeting proteins to female gametocyte osmiophilic bodies is a relevant improvement in the ongoing studies on protein trafficking in parasite sexual differentiation. In addition, the possibility 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.
Prof. G. Girelli, University of Rome "La Sapienza", is gratefully acknowledged for the gift of human erythrocytes. This work was funded by EU FP7 Projects MALSIG (Contract 223044) and NoE EVIMalaR, and the Italy-USA Collaborative Project from Italian Ministry of Health "Membrane dynamics of Plasmodium sexual stages as potential target of malaria transmission blocking intervention".
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