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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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Membrane-associated protein expression of PfCCps in mature gametocytes. (A) Western blot analysis of extracts from mixed asexual (lanes A) and mature gametocyte (lanes G) stages show gametocyte-specific PfCCp expression. As a control for protein loading, extracts were additionally screened with mouse sera recognizing Pf39, an abundant ER-resident protein (lanes marked Pf39). Lanes marked MBP indicate lack of reactivity with mouse control sera recognizing the recombinant fusion partner MBP. (B) Indirect IFA assay using mouse sera against PfCCp1, PfCCp2, and PfCCp3 revealed a punctate pattern associated to the parasite surface in mature gametocytes (green Alexa Fluor 488), whereas asexual stage parasites did not exhibit any labeling. Mouse sera directed against MBP shows no labeling of gametocytes. Erythrocytes were counterstained with Evans Blue (red). Arrows indicate asexual parasites visualized by TOTO-3 nuclear stain (blue). Bar, 5 μm.
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fig4: Membrane-associated protein expression of PfCCps in mature gametocytes. (A) Western blot analysis of extracts from mixed asexual (lanes A) and mature gametocyte (lanes G) stages show gametocyte-specific PfCCp expression. As a control for protein loading, extracts were additionally screened with mouse sera recognizing Pf39, an abundant ER-resident protein (lanes marked Pf39). Lanes marked MBP indicate lack of reactivity with mouse control sera recognizing the recombinant fusion partner MBP. (B) Indirect IFA assay using mouse sera against PfCCp1, PfCCp2, and PfCCp3 revealed a punctate pattern associated to the parasite surface in mature gametocytes (green Alexa Fluor 488), whereas asexual stage parasites did not exhibit any labeling. Mouse sera directed against MBP shows no labeling of gametocytes. Erythrocytes were counterstained with Evans Blue (red). Arrows indicate asexual parasites visualized by TOTO-3 nuclear stain (blue). Bar, 5 μm.

Mentions: Transcript expression was studied by real time RT-PCR analysis using RNA isolated from asexual and gametocyte intraerythrocytic in vitro–cultivated P. falciparum parasites. Expression was gametocyte stage specific for PfCCp1 through PfCCp4 and PfFNPA, in comparison with transcript expression controls for asexual (AMA-1) and gametocyte (Pfs48/45) stage–specific genes (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20031274/DC1). PfCCp5 showed transcript expression in asexual parasites in addition to its expression in gametocyte stages (Fig. S1). PfCCp1, PfCCp2, and PfCCp3 were chosen for further study of life cycle expression and cellular localization due to their striking multidomain architectures and the presence of orthologues in other apicomplexans. Protein expression for PfCCp1, PfCCp2, and PfCCp3 was shown to be gametocyte stage specific as assayed by Western blot analysis using mouse sera generated against PfCCp1, PfCCp2, and PfCCp3 recombinant protein (Fig. 4 A). Protein bands detected in gametocyte preparations had the expected molecular weights of roughly 185 kD for PfCCp1 and PfCCp2, and 150 kD for PfCCp3. Control mouse sera directed against the MBP recombinant protein fusion partner showed no labeling in gametocyte and asexual preparations, whereas the loading control mouse antibody Pf39 recognized a 39-kD ER protein (17, 18) abundantly expressed in both asexual and gametocyte parasites.


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)

Membrane-associated protein expression of PfCCps in mature gametocytes. (A) Western blot analysis of extracts from mixed asexual (lanes A) and mature gametocyte (lanes G) stages show gametocyte-specific PfCCp expression. As a control for protein loading, extracts were additionally screened with mouse sera recognizing Pf39, an abundant ER-resident protein (lanes marked Pf39). Lanes marked MBP indicate lack of reactivity with mouse control sera recognizing the recombinant fusion partner MBP. (B) Indirect IFA assay using mouse sera against PfCCp1, PfCCp2, and PfCCp3 revealed a punctate pattern associated to the parasite surface in mature gametocytes (green Alexa Fluor 488), whereas asexual stage parasites did not exhibit any labeling. Mouse sera directed against MBP shows no labeling of gametocytes. Erythrocytes were counterstained with Evans Blue (red). Arrows indicate asexual parasites visualized by TOTO-3 nuclear stain (blue). Bar, 5 μm.
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Related In: Results  -  Collection

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fig4: Membrane-associated protein expression of PfCCps in mature gametocytes. (A) Western blot analysis of extracts from mixed asexual (lanes A) and mature gametocyte (lanes G) stages show gametocyte-specific PfCCp expression. As a control for protein loading, extracts were additionally screened with mouse sera recognizing Pf39, an abundant ER-resident protein (lanes marked Pf39). Lanes marked MBP indicate lack of reactivity with mouse control sera recognizing the recombinant fusion partner MBP. (B) Indirect IFA assay using mouse sera against PfCCp1, PfCCp2, and PfCCp3 revealed a punctate pattern associated to the parasite surface in mature gametocytes (green Alexa Fluor 488), whereas asexual stage parasites did not exhibit any labeling. Mouse sera directed against MBP shows no labeling of gametocytes. Erythrocytes were counterstained with Evans Blue (red). Arrows indicate asexual parasites visualized by TOTO-3 nuclear stain (blue). Bar, 5 μm.
Mentions: Transcript expression was studied by real time RT-PCR analysis using RNA isolated from asexual and gametocyte intraerythrocytic in vitro–cultivated P. falciparum parasites. Expression was gametocyte stage specific for PfCCp1 through PfCCp4 and PfFNPA, in comparison with transcript expression controls for asexual (AMA-1) and gametocyte (Pfs48/45) stage–specific genes (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20031274/DC1). PfCCp5 showed transcript expression in asexual parasites in addition to its expression in gametocyte stages (Fig. S1). PfCCp1, PfCCp2, and PfCCp3 were chosen for further study of life cycle expression and cellular localization due to their striking multidomain architectures and the presence of orthologues in other apicomplexans. Protein expression for PfCCp1, PfCCp2, and PfCCp3 was shown to be gametocyte stage specific as assayed by Western blot analysis using mouse sera generated against PfCCp1, PfCCp2, and PfCCp3 recombinant protein (Fig. 4 A). Protein bands detected in gametocyte preparations had the expected molecular weights of roughly 185 kD for PfCCp1 and PfCCp2, and 150 kD for PfCCp3. Control mouse sera directed against the MBP recombinant protein fusion partner showed no labeling in gametocyte and asexual preparations, whereas the loading control mouse antibody Pf39 recognized a 39-kD ER protein (17, 18) abundantly expressed in both asexual and gametocyte parasites.

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