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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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Schematic of the PfCCp2 locus, the disruption plasmid construct pPfCCp2-KO (using the pDT-Tg23 vector), and the organization of the PfCCp2 locus after integration by homologous recombination. The coding region of PfCCp2 (gray) is split into a region representing the portion homologous to the disruption plasmid (dark gray). The T. gondii DHFR gene and regulatory cassette are represented by a white box. Approximate location of restriction enzyme sites (B, BclI; Bg, BglII; S, SpeI; X, XbaI) and sizes of restriction digest fragments are indicated below the WT and disrupted PfCCp2 loci (refer to Southern blot analysis shown in Fig. S2). Amplification of a PCR product using the primers P1 and P2 was diagnostic of homologous integration. The PfCCp3 gene locus was similarly disrupted.
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fig7: Schematic of the PfCCp2 locus, the disruption plasmid construct pPfCCp2-KO (using the pDT-Tg23 vector), and the organization of the PfCCp2 locus after integration by homologous recombination. The coding region of PfCCp2 (gray) is split into a region representing the portion homologous to the disruption plasmid (dark gray). The T. gondii DHFR gene and regulatory cassette are represented by a white box. Approximate location of restriction enzyme sites (B, BclI; Bg, BglII; S, SpeI; X, XbaI) and sizes of restriction digest fragments are indicated below the WT and disrupted PfCCp2 loci (refer to Southern blot analysis shown in Fig. S2). Amplification of a PCR product using the primers P1 and P2 was diagnostic of homologous integration. The PfCCp3 gene locus was similarly disrupted.

Mentions: A PfCCp2 KO construct, pPfCCp2-KO, was designed such that after homologous integration a “pseudo-diploid” locus is generated in which the first copy of the disrupted gene is truncated and possesses an in-frame stop codon at the 3′ end, whereas the second copy is truncated at the 5′ end and lacks a start methionine, a signal peptide sequence, and the start of the coding region. The disruption construct contained a 523-bp amino-terminal region segment of PfCCp2 that was PCR amplified from NF54 P. falciparum genomic DNA template using gene-specific 5′ primer 5′-TAGCGGCCGCACCCTAATGGCACCGAAT-3′ and 3′ primer 5′-ATGCGGCCGCTTAAGGAGAACTAGGTAACGC-3′. Each primer introduced a NotI restriction site (underlined) to facilitate ligation of restriction enzyme–digested PCR product into a NotI-cut pDT-Tg23 vector (22, 23). The PfCCp3 KO construct, pPfCCp3-KO, was similarly designed and contained a 565-bp amino-terminal region segment that was amplified from NF54 WT DNA using gene-specific 5′ primer 5′-TAGCGGCCGCAGCTGGATAGGTGCTCCT-3′ and 3′ primer 5′-ATGCGGCCGCTTATACACAAACGGTACCCCA-3′. The resulting constructs were loaded into uninfected erythrocytes via electroporation, and the erythrocytes were then inoculated with NF54 isolate parasites (24). At the time of the first parasite passage, this process was repeated such that the parasite cultures were diluted into freshly electroporated erythrocytes. After 3 d, 10 mM pyrimethamine drug pressure was initiated to select for TgDHFR expression in parasites that have taken up and retained plasmid. After ∼90 d of pyrimethamine drug pressure a plasmid-integrant population predominated and the presence of homologous gene disruptants was demonstrated by a diagnostic PCR assay of a disrupted locus (described in Fig. 7). At this time, transformed parasite clones were isolated by dilution cloning in microtiter plates using the Malstat reagent assay (25) as an indicator of parasite-positive wells. Homologous recombination in clonal isolates was confirmed by the above PCR assay and in select isolates was further confirmed by Southern blot analysis (see Supplemental Materials and Methods, which is available at http://www.jem.org/cgi/content/full/jem.20031274/DC1).


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)

Schematic of the PfCCp2 locus, the disruption plasmid construct pPfCCp2-KO (using the pDT-Tg23 vector), and the organization of the PfCCp2 locus after integration by homologous recombination. The coding region of PfCCp2 (gray) is split into a region representing the portion homologous to the disruption plasmid (dark gray). The T. gondii DHFR gene and regulatory cassette are represented by a white box. Approximate location of restriction enzyme sites (B, BclI; Bg, BglII; S, SpeI; X, XbaI) and sizes of restriction digest fragments are indicated below the WT and disrupted PfCCp2 loci (refer to Southern blot analysis shown in Fig. S2). Amplification of a PCR product using the primers P1 and P2 was diagnostic of homologous integration. The PfCCp3 gene locus was similarly disrupted.
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Related In: Results  -  Collection

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fig7: Schematic of the PfCCp2 locus, the disruption plasmid construct pPfCCp2-KO (using the pDT-Tg23 vector), and the organization of the PfCCp2 locus after integration by homologous recombination. The coding region of PfCCp2 (gray) is split into a region representing the portion homologous to the disruption plasmid (dark gray). The T. gondii DHFR gene and regulatory cassette are represented by a white box. Approximate location of restriction enzyme sites (B, BclI; Bg, BglII; S, SpeI; X, XbaI) and sizes of restriction digest fragments are indicated below the WT and disrupted PfCCp2 loci (refer to Southern blot analysis shown in Fig. S2). Amplification of a PCR product using the primers P1 and P2 was diagnostic of homologous integration. The PfCCp3 gene locus was similarly disrupted.
Mentions: A PfCCp2 KO construct, pPfCCp2-KO, was designed such that after homologous integration a “pseudo-diploid” locus is generated in which the first copy of the disrupted gene is truncated and possesses an in-frame stop codon at the 3′ end, whereas the second copy is truncated at the 5′ end and lacks a start methionine, a signal peptide sequence, and the start of the coding region. The disruption construct contained a 523-bp amino-terminal region segment of PfCCp2 that was PCR amplified from NF54 P. falciparum genomic DNA template using gene-specific 5′ primer 5′-TAGCGGCCGCACCCTAATGGCACCGAAT-3′ and 3′ primer 5′-ATGCGGCCGCTTAAGGAGAACTAGGTAACGC-3′. Each primer introduced a NotI restriction site (underlined) to facilitate ligation of restriction enzyme–digested PCR product into a NotI-cut pDT-Tg23 vector (22, 23). The PfCCp3 KO construct, pPfCCp3-KO, was similarly designed and contained a 565-bp amino-terminal region segment that was amplified from NF54 WT DNA using gene-specific 5′ primer 5′-TAGCGGCCGCAGCTGGATAGGTGCTCCT-3′ and 3′ primer 5′-ATGCGGCCGCTTATACACAAACGGTACCCCA-3′. The resulting constructs were loaded into uninfected erythrocytes via electroporation, and the erythrocytes were then inoculated with NF54 isolate parasites (24). At the time of the first parasite passage, this process was repeated such that the parasite cultures were diluted into freshly electroporated erythrocytes. After 3 d, 10 mM pyrimethamine drug pressure was initiated to select for TgDHFR expression in parasites that have taken up and retained plasmid. After ∼90 d of pyrimethamine drug pressure a plasmid-integrant population predominated and the presence of homologous gene disruptants was demonstrated by a diagnostic PCR assay of a disrupted locus (described in Fig. 7). At this time, transformed parasite clones were isolated by dilution cloning in microtiter plates using the Malstat reagent assay (25) as an indicator of parasite-positive wells. Homologous recombination in clonal isolates was confirmed by the above PCR assay and in select isolates was further confirmed by Southern blot analysis (see Supplemental Materials and Methods, which is available at http://www.jem.org/cgi/content/full/jem.20031274/DC1).

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