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A multidomain adhesion protein family expressed in Plasmodium falciparum is essential for transmission to the mosquito.

Pradel G, Hayton K, Aravind L, Iyer LM, Abrahamsen MS, Bonawitz A, Mejia C, Templeton TJ - J. Exp. Med. (2004)

Bottom Line: Three of these identified genes, named PfCCp1, PfCCp2, and PfCCp3, have multiple adhesive modules including a common Limulus coagulation factor C domain also found in two additional Plasmodium genes.PfCCp expression markedly decreased after formation of zygotes.Our results describe members of a conserved apicomplexan protein family expressed in sexual stage Plasmodium parasites that may represent candidates for subunits of a transmission-blocking vaccine.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.

ABSTRACT
The recent sequencing of several apicomplexan genomes has provided the opportunity to characterize novel antigens essential for the parasite life cycle that might lead to the development of new diagnostic and therapeutic markers. Here we have screened the Plasmodium falciparum genome sequence for genes encoding extracellular multidomain putative adhesive proteins. Three of these identified genes, named PfCCp1, PfCCp2, and PfCCp3, have multiple adhesive modules including a common Limulus coagulation factor C domain also found in two additional Plasmodium genes. Orthologues were identified in the Cryptosporidium parvum genome sequence, indicating an evolutionary conserved function. Transcript and protein expression analysis shows sexual stage-specific expression of PfCCp1, PfCCp2, and PfCCp3, and cellular localization studies revealed plasma membrane-associated expression in mature gametocytes. During gametogenesis, PfCCps are released and localize surrounding complexes of newly emerged microgametes and macrogametes. PfCCp expression markedly decreased after formation of zygotes. To begin to address PfCCp function, the PfCCp2 and PfCCp3 gene loci were disrupted by homologous recombination, resulting in parasites capable of forming oocyst sporozoites but blocked in the salivary gland transition. Our results describe members of a conserved apicomplexan protein family expressed in sexual stage Plasmodium parasites that may represent candidates for subunits of a transmission-blocking vaccine.

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PfCCps are present within mature gametocytes and localize extracellularly after gametogenesis, but expression ceases after fertilization. (A) PfCCp protein labeling in mature gametocytes is associated with the parasite surface, whereas (B) PfCCp protein is released during the emergence of gametes from within erythrocytes. (C) PfCCp expression markedly decreases in emerged gametes and protein instead localizes extracellularly (red Alexa Fluor 594), adhering to complexes of emerged macrogametes and adhering microgametes (green Alexa Fluor 488; detected with rabbit α-tubulin II antiserum). (D) Zygotes or unfertilized macrogametes detected with rabbit Pfs25 antiserum (red Alexa Fluor 594) exhibited a low remaining expression for PfCCps (green Alexa Fluor 488) 20 h after gamete emergence in vitro. Bloodmeal preparations isolated from mosquito midguts 24 h after feeding on gametocyte cultures revealed neither PfCCp expression (green Alexa Fluor 488) in (E) retort stages nor in (F) ookinetes (red Alexa Fluor 594, detected with Pfs25 antiserum), whereas gamete complexes in the bloodmeal preparations still showed associated PfCCp labeling. Labeling shown is for PfCCp1. Similar results were obtained for PfCCp2 and PfCCp3 (not depicted). Bars, 2 μm (A and B) and 10 μm (C–F).
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fig6: PfCCps are present within mature gametocytes and localize extracellularly after gametogenesis, but expression ceases after fertilization. (A) PfCCp protein labeling in mature gametocytes is associated with the parasite surface, whereas (B) PfCCp protein is released during the emergence of gametes from within erythrocytes. (C) PfCCp expression markedly decreases in emerged gametes and protein instead localizes extracellularly (red Alexa Fluor 594), adhering to complexes of emerged macrogametes and adhering microgametes (green Alexa Fluor 488; detected with rabbit α-tubulin II antiserum). (D) Zygotes or unfertilized macrogametes detected with rabbit Pfs25 antiserum (red Alexa Fluor 594) exhibited a low remaining expression for PfCCps (green Alexa Fluor 488) 20 h after gamete emergence in vitro. Bloodmeal preparations isolated from mosquito midguts 24 h after feeding on gametocyte cultures revealed neither PfCCp expression (green Alexa Fluor 488) in (E) retort stages nor in (F) ookinetes (red Alexa Fluor 594, detected with Pfs25 antiserum), whereas gamete complexes in the bloodmeal preparations still showed associated PfCCp labeling. Labeling shown is for PfCCp1. Similar results were obtained for PfCCp2 and PfCCp3 (not depicted). Bars, 2 μm (A and B) and 10 μm (C–F).

Mentions: Cellular localization was studied via indirect IFA confocal laser scanning microscopy and immunoelectron microscopy. For PfCCp1, PfCCp2, and PfCCp3, a punctate rim fluorescence pattern was observed in P. falciparum gametocytes, whereas asexual stage parasites did not show any labeling (Fig. 4 B). Labeling was first observed in gametocytes stage III and remained throughout maturation (unpublished data). Colocalization of the three PfCCp proteins was investigated using rabbit sera generated against PfCCp2 peptide sequences in combination with mouse antisera against PfCCp1 and PfCCp3, respectively. Experiments revealed colocalization of all three proteins at the gametocyte surface (unpublished data). Double labeling experiments, using rabbit sera recognizing α-tubulin II as a marker for microgametocytes (19), further indicated that PfCCp proteins are expressed in both microgametocytes and macrogametocytes (unpublished data). The ultrastructural localization of PfCCp1 was additionally investigated by immunoelectron microscopy using two different preembedding labeling techniques. Immunogold labeling revealed localization of PfCCp1 associated with the parasite surface membrane in the parasitophorous vacuole of mature gametocytes (Fig. 5, A and C). Similar results were obtained by alkaline phosphatase labeling, which results in a loose precipitation at the site of protein expression (Fig. 5, B and D). Alkaline phosphatase labeling further showed protein localization in the cytoplasm of gametocytes and attached to the outside of erythrocytes, indicating possible release of the protein. No labeling was detected in asexual parasites or erythrocytes (Fig. 5 E). We further investigated the role of PfCCps during gamete emergence, triggered by incubation at room temperature in the presence of human serum. Although mature gametocytes exhibited PfCCp labeling associated with the parasite surface (Fig. 6 A), during emergence labeling was detected outside the infected erythrocytes (Fig. 6 B). Consistent with a secreted release of PfCCps, we have observed that PfCCp expression markedly decreases in emerged gametes and protein localizes extracellularly, adhering to complexes of macrogametes (Fig. 6 C). These complexes are readily observable in vitro due to the high gametocytemia of the parasite cultures and are formed by the inherently “sticky” macrogametes associating with exflagellating microgametes, mature gametocytes, and erythrocytes.


A multidomain adhesion protein family expressed in Plasmodium falciparum is essential for transmission to the mosquito.

Pradel G, Hayton K, Aravind L, Iyer LM, Abrahamsen MS, Bonawitz A, Mejia C, Templeton TJ - J. Exp. Med. (2004)

PfCCps are present within mature gametocytes and localize extracellularly after gametogenesis, but expression ceases after fertilization. (A) PfCCp protein labeling in mature gametocytes is associated with the parasite surface, whereas (B) PfCCp protein is released during the emergence of gametes from within erythrocytes. (C) PfCCp expression markedly decreases in emerged gametes and protein instead localizes extracellularly (red Alexa Fluor 594), adhering to complexes of emerged macrogametes and adhering microgametes (green Alexa Fluor 488; detected with rabbit α-tubulin II antiserum). (D) Zygotes or unfertilized macrogametes detected with rabbit Pfs25 antiserum (red Alexa Fluor 594) exhibited a low remaining expression for PfCCps (green Alexa Fluor 488) 20 h after gamete emergence in vitro. Bloodmeal preparations isolated from mosquito midguts 24 h after feeding on gametocyte cultures revealed neither PfCCp expression (green Alexa Fluor 488) in (E) retort stages nor in (F) ookinetes (red Alexa Fluor 594, detected with Pfs25 antiserum), whereas gamete complexes in the bloodmeal preparations still showed associated PfCCp labeling. Labeling shown is for PfCCp1. Similar results were obtained for PfCCp2 and PfCCp3 (not depicted). Bars, 2 μm (A and B) and 10 μm (C–F).
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fig6: PfCCps are present within mature gametocytes and localize extracellularly after gametogenesis, but expression ceases after fertilization. (A) PfCCp protein labeling in mature gametocytes is associated with the parasite surface, whereas (B) PfCCp protein is released during the emergence of gametes from within erythrocytes. (C) PfCCp expression markedly decreases in emerged gametes and protein instead localizes extracellularly (red Alexa Fluor 594), adhering to complexes of emerged macrogametes and adhering microgametes (green Alexa Fluor 488; detected with rabbit α-tubulin II antiserum). (D) Zygotes or unfertilized macrogametes detected with rabbit Pfs25 antiserum (red Alexa Fluor 594) exhibited a low remaining expression for PfCCps (green Alexa Fluor 488) 20 h after gamete emergence in vitro. Bloodmeal preparations isolated from mosquito midguts 24 h after feeding on gametocyte cultures revealed neither PfCCp expression (green Alexa Fluor 488) in (E) retort stages nor in (F) ookinetes (red Alexa Fluor 594, detected with Pfs25 antiserum), whereas gamete complexes in the bloodmeal preparations still showed associated PfCCp labeling. Labeling shown is for PfCCp1. Similar results were obtained for PfCCp2 and PfCCp3 (not depicted). Bars, 2 μm (A and B) and 10 μm (C–F).
Mentions: Cellular localization was studied via indirect IFA confocal laser scanning microscopy and immunoelectron microscopy. For PfCCp1, PfCCp2, and PfCCp3, a punctate rim fluorescence pattern was observed in P. falciparum gametocytes, whereas asexual stage parasites did not show any labeling (Fig. 4 B). Labeling was first observed in gametocytes stage III and remained throughout maturation (unpublished data). Colocalization of the three PfCCp proteins was investigated using rabbit sera generated against PfCCp2 peptide sequences in combination with mouse antisera against PfCCp1 and PfCCp3, respectively. Experiments revealed colocalization of all three proteins at the gametocyte surface (unpublished data). Double labeling experiments, using rabbit sera recognizing α-tubulin II as a marker for microgametocytes (19), further indicated that PfCCp proteins are expressed in both microgametocytes and macrogametocytes (unpublished data). The ultrastructural localization of PfCCp1 was additionally investigated by immunoelectron microscopy using two different preembedding labeling techniques. Immunogold labeling revealed localization of PfCCp1 associated with the parasite surface membrane in the parasitophorous vacuole of mature gametocytes (Fig. 5, A and C). Similar results were obtained by alkaline phosphatase labeling, which results in a loose precipitation at the site of protein expression (Fig. 5, B and D). Alkaline phosphatase labeling further showed protein localization in the cytoplasm of gametocytes and attached to the outside of erythrocytes, indicating possible release of the protein. No labeling was detected in asexual parasites or erythrocytes (Fig. 5 E). We further investigated the role of PfCCps during gamete emergence, triggered by incubation at room temperature in the presence of human serum. Although mature gametocytes exhibited PfCCp labeling associated with the parasite surface (Fig. 6 A), during emergence labeling was detected outside the infected erythrocytes (Fig. 6 B). Consistent with a secreted release of PfCCps, we have observed that PfCCp expression markedly decreases in emerged gametes and protein localizes extracellularly, adhering to complexes of macrogametes (Fig. 6 C). These complexes are readily observable in vitro due to the high gametocytemia of the parasite cultures and are formed by the inherently “sticky” macrogametes associating with exflagellating microgametes, mature gametocytes, and erythrocytes.

Bottom Line: Three of these identified genes, named PfCCp1, PfCCp2, and PfCCp3, have multiple adhesive modules including a common Limulus coagulation factor C domain also found in two additional Plasmodium genes.PfCCp expression markedly decreased after formation of zygotes.Our results describe members of a conserved apicomplexan protein family expressed in sexual stage Plasmodium parasites that may represent candidates for subunits of a transmission-blocking vaccine.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.

ABSTRACT
The recent sequencing of several apicomplexan genomes has provided the opportunity to characterize novel antigens essential for the parasite life cycle that might lead to the development of new diagnostic and therapeutic markers. Here we have screened the Plasmodium falciparum genome sequence for genes encoding extracellular multidomain putative adhesive proteins. Three of these identified genes, named PfCCp1, PfCCp2, and PfCCp3, have multiple adhesive modules including a common Limulus coagulation factor C domain also found in two additional Plasmodium genes. Orthologues were identified in the Cryptosporidium parvum genome sequence, indicating an evolutionary conserved function. Transcript and protein expression analysis shows sexual stage-specific expression of PfCCp1, PfCCp2, and PfCCp3, and cellular localization studies revealed plasma membrane-associated expression in mature gametocytes. During gametogenesis, PfCCps are released and localize surrounding complexes of newly emerged microgametes and macrogametes. PfCCp expression markedly decreased after formation of zygotes. To begin to address PfCCp function, the PfCCp2 and PfCCp3 gene loci were disrupted by homologous recombination, resulting in parasites capable of forming oocyst sporozoites but blocked in the salivary gland transition. Our results describe members of a conserved apicomplexan protein family expressed in sexual stage Plasmodium parasites that may represent candidates for subunits of a transmission-blocking vaccine.

Show MeSH
Related in: MedlinePlus