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Conservation of a gliding motility and cell invasion machinery in Apicomplexan parasites.

Kappe S, Bruderer T, Gantt S, Fujioka H, Nussenzweig V, Ménard R - J. Cell Biol. (1999)

Bottom Line: Most Apicomplexan parasites, including the human pathogens Plasmodium, Toxoplasma, and Cryptosporidium, actively invade host cells and display gliding motility, both actions powered by parasite microfilaments.We also demonstrate that TRAP-related proteins in other Apicomplexa fulfill the same function and that their cytoplasmic tails interact with homologous partners in the respective parasite.Therefore, a mechanism of surface redistribution of TRAP-related proteins driving gliding locomotion and cell invasion is conserved among Apicomplexan parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Kaplan Cancer Center, New York University School of Medicine, New York, New York 10016, USA.

ABSTRACT
Most Apicomplexan parasites, including the human pathogens Plasmodium, Toxoplasma, and Cryptosporidium, actively invade host cells and display gliding motility, both actions powered by parasite microfilaments. In Plasmodium sporozoites, thrombospondin-related anonymous protein (TRAP), a member of a group of Apicomplexan transmembrane proteins that have common adhesion domains, is necessary for gliding motility and infection of the vertebrate host. Here, we provide genetic evidence that TRAP is directly involved in a capping process that drives both sporozoite gliding and cell invasion. We also demonstrate that TRAP-related proteins in other Apicomplexa fulfill the same function and that their cytoplasmic tails interact with homologous partners in the respective parasite. Therefore, a mechanism of surface redistribution of TRAP-related proteins driving gliding locomotion and cell invasion is conserved among Apicomplexan parasites.

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TRAP cytoplasmic tail mutants and gene targeting strategy. (A) Schematic representation of the TRAP protein and amino acid sequences (one letter code), of the cytoplasmic tails of Plasmodium berghei TRAP and TRAP recombinants. CoTRAP indicates the residues conserved in at least five of six plasmodial TRAP sequenced to date (+ indicates E or D). In the TRAP recombinants shown below, the amino acid substitutions or heterologous exchange are underlined. Hatched boxes represent the leader sequence and the transmembrane domain. (B) Generation of the TRAP mutations by insertion mutagenesis in P. berghei. The wild-type (Wt), single-copy TRAP is targeted with an insertion plasmid whose targeting sequence contains the deletion/mutation (*) and is linearized upstream from the mutation (crossover); thin lines, TRAP untranslated region; open box, TRAP coding region; thick lines, bacterial plasmid and DHFR-TS resistance cassette. The recombinant locus (Rec. locus) expected to result from plasmid integration that preserves the mutation is shown. Below are the restriction maps of the 3′ end of the TRAP gene in the first duplicate of the recombinant clones. The nucleotide and the amino acid sequences tagging the mutations are indicated, and the corresponding restriction sites italicized in the sequence and the map. P, PstI; Pa, PacI; X, XbaI; B, BamHI; N, NheI. (C) The first TRAP duplicate of recombinant parasites were amplified by PCR using primer O1 and T7, which annealed upstream from the region of homology and to the vector sequence, respectively, and digested with restriction enzymes. See the restriction maps and mutation-tagging restriction sites in B.
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Figure 1: TRAP cytoplasmic tail mutants and gene targeting strategy. (A) Schematic representation of the TRAP protein and amino acid sequences (one letter code), of the cytoplasmic tails of Plasmodium berghei TRAP and TRAP recombinants. CoTRAP indicates the residues conserved in at least five of six plasmodial TRAP sequenced to date (+ indicates E or D). In the TRAP recombinants shown below, the amino acid substitutions or heterologous exchange are underlined. Hatched boxes represent the leader sequence and the transmembrane domain. (B) Generation of the TRAP mutations by insertion mutagenesis in P. berghei. The wild-type (Wt), single-copy TRAP is targeted with an insertion plasmid whose targeting sequence contains the deletion/mutation (*) and is linearized upstream from the mutation (crossover); thin lines, TRAP untranslated region; open box, TRAP coding region; thick lines, bacterial plasmid and DHFR-TS resistance cassette. The recombinant locus (Rec. locus) expected to result from plasmid integration that preserves the mutation is shown. Below are the restriction maps of the 3′ end of the TRAP gene in the first duplicate of the recombinant clones. The nucleotide and the amino acid sequences tagging the mutations are indicated, and the corresponding restriction sites italicized in the sequence and the map. P, PstI; Pa, PacI; X, XbaI; B, BamHI; N, NheI. (C) The first TRAP duplicate of recombinant parasites were amplified by PCR using primer O1 and T7, which annealed upstream from the region of homology and to the vector sequence, respectively, and digested with restriction enzymes. See the restriction maps and mutation-tagging restriction sites in B.

Mentions: We generated Plasmodium berghei sporozoites that produced TRAP proteins lacking the cytoplasmic tail. Sequence comparison of the cytoplasmic tails of the TRAP proteins sequenced so far (from six plasmodial species; Robson et al. 1988, Robson et al. 1997; Rogers et al. 1992b; Templeton and Kaslow 1997) reveals that the 14 carboxy-terminal residues are the most highly conserved (Fig. 1 A). We thus created sporozoites whose TRAP lacked the 14 or 37 carboxy-terminal residues, named TΔS and TΔL, respectively. For this, modifications in the single-copy TRAP gene were introduced via a single recombination event promoted by targeting insertion plasmids (Nunes et al. 1999). As shown in Fig. 1 B, homologous integration of these targeting plasmids generate two TRAP copies: the first is full-length, bears the mutation, and is flanked by expression sequences, whereas the second lacks the 5′ part of the gene and promoter sequences.


Conservation of a gliding motility and cell invasion machinery in Apicomplexan parasites.

Kappe S, Bruderer T, Gantt S, Fujioka H, Nussenzweig V, Ménard R - J. Cell Biol. (1999)

TRAP cytoplasmic tail mutants and gene targeting strategy. (A) Schematic representation of the TRAP protein and amino acid sequences (one letter code), of the cytoplasmic tails of Plasmodium berghei TRAP and TRAP recombinants. CoTRAP indicates the residues conserved in at least five of six plasmodial TRAP sequenced to date (+ indicates E or D). In the TRAP recombinants shown below, the amino acid substitutions or heterologous exchange are underlined. Hatched boxes represent the leader sequence and the transmembrane domain. (B) Generation of the TRAP mutations by insertion mutagenesis in P. berghei. The wild-type (Wt), single-copy TRAP is targeted with an insertion plasmid whose targeting sequence contains the deletion/mutation (*) and is linearized upstream from the mutation (crossover); thin lines, TRAP untranslated region; open box, TRAP coding region; thick lines, bacterial plasmid and DHFR-TS resistance cassette. The recombinant locus (Rec. locus) expected to result from plasmid integration that preserves the mutation is shown. Below are the restriction maps of the 3′ end of the TRAP gene in the first duplicate of the recombinant clones. The nucleotide and the amino acid sequences tagging the mutations are indicated, and the corresponding restriction sites italicized in the sequence and the map. P, PstI; Pa, PacI; X, XbaI; B, BamHI; N, NheI. (C) The first TRAP duplicate of recombinant parasites were amplified by PCR using primer O1 and T7, which annealed upstream from the region of homology and to the vector sequence, respectively, and digested with restriction enzymes. See the restriction maps and mutation-tagging restriction sites in B.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169348&req=5

Figure 1: TRAP cytoplasmic tail mutants and gene targeting strategy. (A) Schematic representation of the TRAP protein and amino acid sequences (one letter code), of the cytoplasmic tails of Plasmodium berghei TRAP and TRAP recombinants. CoTRAP indicates the residues conserved in at least five of six plasmodial TRAP sequenced to date (+ indicates E or D). In the TRAP recombinants shown below, the amino acid substitutions or heterologous exchange are underlined. Hatched boxes represent the leader sequence and the transmembrane domain. (B) Generation of the TRAP mutations by insertion mutagenesis in P. berghei. The wild-type (Wt), single-copy TRAP is targeted with an insertion plasmid whose targeting sequence contains the deletion/mutation (*) and is linearized upstream from the mutation (crossover); thin lines, TRAP untranslated region; open box, TRAP coding region; thick lines, bacterial plasmid and DHFR-TS resistance cassette. The recombinant locus (Rec. locus) expected to result from plasmid integration that preserves the mutation is shown. Below are the restriction maps of the 3′ end of the TRAP gene in the first duplicate of the recombinant clones. The nucleotide and the amino acid sequences tagging the mutations are indicated, and the corresponding restriction sites italicized in the sequence and the map. P, PstI; Pa, PacI; X, XbaI; B, BamHI; N, NheI. (C) The first TRAP duplicate of recombinant parasites were amplified by PCR using primer O1 and T7, which annealed upstream from the region of homology and to the vector sequence, respectively, and digested with restriction enzymes. See the restriction maps and mutation-tagging restriction sites in B.
Mentions: We generated Plasmodium berghei sporozoites that produced TRAP proteins lacking the cytoplasmic tail. Sequence comparison of the cytoplasmic tails of the TRAP proteins sequenced so far (from six plasmodial species; Robson et al. 1988, Robson et al. 1997; Rogers et al. 1992b; Templeton and Kaslow 1997) reveals that the 14 carboxy-terminal residues are the most highly conserved (Fig. 1 A). We thus created sporozoites whose TRAP lacked the 14 or 37 carboxy-terminal residues, named TΔS and TΔL, respectively. For this, modifications in the single-copy TRAP gene were introduced via a single recombination event promoted by targeting insertion plasmids (Nunes et al. 1999). As shown in Fig. 1 B, homologous integration of these targeting plasmids generate two TRAP copies: the first is full-length, bears the mutation, and is flanked by expression sequences, whereas the second lacks the 5′ part of the gene and promoter sequences.

Bottom Line: Most Apicomplexan parasites, including the human pathogens Plasmodium, Toxoplasma, and Cryptosporidium, actively invade host cells and display gliding motility, both actions powered by parasite microfilaments.We also demonstrate that TRAP-related proteins in other Apicomplexa fulfill the same function and that their cytoplasmic tails interact with homologous partners in the respective parasite.Therefore, a mechanism of surface redistribution of TRAP-related proteins driving gliding locomotion and cell invasion is conserved among Apicomplexan parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Kaplan Cancer Center, New York University School of Medicine, New York, New York 10016, USA.

ABSTRACT
Most Apicomplexan parasites, including the human pathogens Plasmodium, Toxoplasma, and Cryptosporidium, actively invade host cells and display gliding motility, both actions powered by parasite microfilaments. In Plasmodium sporozoites, thrombospondin-related anonymous protein (TRAP), a member of a group of Apicomplexan transmembrane proteins that have common adhesion domains, is necessary for gliding motility and infection of the vertebrate host. Here, we provide genetic evidence that TRAP is directly involved in a capping process that drives both sporozoite gliding and cell invasion. We also demonstrate that TRAP-related proteins in other Apicomplexa fulfill the same function and that their cytoplasmic tails interact with homologous partners in the respective parasite. Therefore, a mechanism of surface redistribution of TRAP-related proteins driving gliding locomotion and cell invasion is conserved among Apicomplexan parasites.

Show MeSH
Related in: MedlinePlus